An examination of the in vivo distribution of brain hexokinase between the cytosol and the outer...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 248, No. 1, July, pp. 253-271,1986

An Examination of the in Vivo Distribution of Brain Hexokinase between the Cytosol and the Outer Mitochondrial Membrane’

HAROLD T. KYRIAZI’ AND R. E. BASFORD

Department qf Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received November 8, 1985, and in revised form February 4,1986

These studies addressed the question of the in viva distribution of rat brain hexokinase (HK), and whether physiologically relevant changes in the glycolytic rate are accompanied by changes in the distribution of HK. Homogenates of fresh tissue showed only ll-15% of the overt (assayable without added detergent) HK to be soluble (found in high-speed centrifugation supernatant fractions) when homogenization was begun within 15-20 s of sacrifice. Freeze-blown rat brain tissue also was used, coupled with a new technique wherein it was homogenized as it thawed in a buffered sucrose solution containing 1 mM EDTA. In tissue sampled 15 min (anesthetized) or 60 min (waking) after ip Nembutal injection (40 mg/kg), 23% of the overt HK and 79% of the total lactate dehydrogenase were soluble. The average phosphocreatine content of these and similar homogenates had decreased only 23% from in vivo levels, while ATP had decreased by 65%, due to the combined effects of a high level of endogenous ATPase, chelation of Mgz+ by EDTA, and the greater stability of MgATP’- relative to MgADPl-. These data indicated that the tissue experienced, at most, the equivalent of 6 s of complete ischemia prior to the completion of homogenization. Synaptosomes derived from rat and chicken cerebra were incubated at 3’7°C in a physiological salt solution containing 10 mM glucose. Addition of veratridine has been shown to stimulate glycolysis and oxidative phos- phorylation two- to threefold (H. T. Kyriazi and R. E. Basford (1986) J. Neurochem., in press), but did not alter the HK distribution, as 21% was found in the supernatant fractions of both control and veratridine-stimulated synaptosomes treated with digitonin. These results indicate that in brain tissue, large net movements of HK on and off the outer mitochondrial membrane do not occur, and thus play no role in the regulation of glycolysis. 0 1986 Academic Press, Inc.

Hexokinase (HK,3 ATP:D-hexose 6-phos- brain removal, homogenization, and high- photransferase, EC 2.7.1.1) in brain occu- speed centrifugation indicate that about pies a unique position among the glycolytic 80% of it is particulate, whereas the other enzymes, in that traditional methods of

i This research was supported by Grant NS-11773

from the National Institutes of Health. Portions of the data are taken from the dissertation of Harold T. Kyriazi accepted in partial fulfillment of the require- ments for the Ph.D. degree, and portions of the data have been presented in abstract form (1).

* Currently a National Institutes of Health Post-

doctoral Fellow in the Dept. of Medicine, Endocri- nology Div., Univ. of Pittsburgh School of Medicine.

a Abbreviations used: CPK, creatine phosphokinase;

CS, citrate synthase; Glc-1,6-Pz, glucose-1,6-bisphos- phate; Glc-6-P, glucose-6-phosphate; HDT, homoge-

nize-during-thawing; Hepes, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid; HK, hexokinase; HSS, high-speed sedimentation; LDH, lactate dehydrogenase; NCR, NADPH:Cytochrome c Reductase; OMM, outer mitochondrial membrane; P2, second pellet or

crude mitochondrial pellet; PCr, phosphocreatine; PFK, phosphofructokinase; RINCR, Rotenone-Insen- sitive NADH:Cytochrome e Reductase; SET medium,

sucrose/EDTA/Tris medium; Ss, synaptosomes.

253 0003-9861186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

254 KYRIAZI AND BASFORD

glycolytic enzymes are found predominantly in the supernatant fraction (2, 3). The particulate brain HK is bound to a specific binding protein within the OMM, alternatively known as hexokinase binding protein (4), or porin (5, 6).

Physiological concentrations of Glc-6-P have been shown to solubilize greater than 90% of the bound HK from both ascites tumor cell mitochondria (7) and rat brain mitochondria (8). (ATP also solubilizes bound HK (7, S), but since it is only the uncomplexed, ATP4- form which has this capacity (9), this probably is not physiologically important.) Glc-1,6-P, also solubilized bound HK (10). Furthermore, physiological concentrations of Pi inhibit Glc- 6-P-induced solubilization (7, S), and Mga can completely prevent Glc-6-P-induced solubilization (7,8) and partly prevent Glc- 1,6-Pz-induced solubilization (10).

Kinetic studies have provided evidence that HK is more active when bound than when soluble, with an approximately threefold lower Km for ATP for the bound form, and/or a fivefold higher Ki for Glc- 6-P or Glc-1,6-Pz (for review, see Ref. (11)). This evidence, together with the phenomenon of rapid, reversible dissociation of bound tumor HK in response to physiological concentrations of Glc-6-P and Pi4 led Rose and Warms (7) and later Kosow and Rose (13) to suggest that the reversible binding might have a regulatory role. Wil- son presented a more complete version of the same idea for brain HK, proposing that “HK activity in viva may be controlled by the relative distribution between soluble and particulate forms, the latter being more active” (8). The crux of the hypothesis is that under low energy flux conditions, Glc-6-P levels will be at their highest, and Pi levels at their lowest, disposing HK to exist in its most soluble and least active state. As the energy flux increases dramatically, for example, during the propa- gation of an action potential along a neu-

4 Subsequent work showed Pi to be no more effective in antagonizing Glc-&P-induced solubilization than other salts on an ionic strength basis, although Pi

was quite specific in its ability to counteract Glc-6-P- induced inhibition of HK (12).

ron, Glc-6-P levels would drop and Pi levels would rise, resulting in a net shift of soluble HK onto the OMM, thereby activating the enzyme. Using the terminology coined by Wilson (14), this will be referred to as the “ambiquitous enzyme” hypothesis.

In support of the ambiquitous enzyme hypothesis are the findings of Knull et al. (15,16), who perturbed chick brain energy metabolism using either complete ischemia, insulin-induced hypoglycemia, or galactose feeding, all of which cause decreased in vivo Glc-6-P levels. These treatments resulted in a net shift toward particulate HK, with maximum shifts of 15% in cerebrum and 30% in cerebellum. These experiments, however, demonstrated changes in HK distribution only under drastic and nonphysiological conditions, and thus the possibility remains that no changes in HK distribution occur under physiological conditions.

Purich and Fromm (17) attempted to address this question in in vitro experiments with rat brain particulate HK, in which the various effecters were kept at physiological concentrations while the concentration of either Glc-6-P or Pi was varied, and in no instance was significant solubilization found, indicating that HK in vivo may exist predominantly bound to the OMM. These experiments are not immune to criticism, however, because their choice of “physiological” effector concentrations ignored the possibility of concentration gradients of the effecters within the cell.

The only in vivo experiments to support the ambiquitous enzyme hypothesis which make an attempt at physiological relevance are found in a series of papers from Krieglstein and colleagues (18-23). Essen- tially, they found a correlation between rat brain HK distribution and whether or not the rats were anesthetized before sacrifice. The small differences (l-8% less HK bound in rats under anesthesia) were attributed to a direct solubilizing effect of the anesthetic on bound HK (18, 19, 22, 23). They have not, however, ruled out the trivial explanation that the differences in HK distribution developed after the rats were sacrificed, since Glc-6-P levels fall more rapidly in the ischemic brain tissue from

ON THE IN VIVO DISTRIBUTION OF BRAIN HEXOKINASE 255

alert rats than in that from anesthetized rats (24,25). Thus, their interpretation that the differences in HK distribution existed in vivo may be incorrect.

The experiments described herein were undertaken in order to determine both the in vivo distribution of rat brain HK, and whether physiologically relevant changes in the glycolytic rate are accompanied by changes in HK’s distribution. Two main approaches were taken. The first was to remove and homogenize the brain tissue closer and closer to the time of sacrifice, thereby approaching the in vivo HK distribution. To this end, in addition to routine decapitation and brain removal, a “rapid- sampling” device was used to remove and begin homogenizing rat brain tissue within 20 s of sacrifice. Freeze-blown brain tissue also was used, coupled with a new procedure wherein the tissue was homogenized as it thawed, which further reduced the time of ischemia.

The first approach usually measures the HK distribution only in the neuronal perikarya and astroglial cells.5 The other approximately 40-50s of the HK and LDH activity in brain homogenates is normally unavailable for assay, being contained in a “latent” form within the SS,~ which are pinched-off presynaptic nerve terminals created during the homogenization procedure. These nerve terminals in situ are, however, the sites of the most active glycolysis in brain.7 Therefore, the second major approach was to prepare structur- ally and metabolically intact rat brain Ss, alter their glycolytic rate, and rupture the Ss plasma membrane using a digitonin treatment which does not alter the HK distribution, so that the HK distribution of the synaptoplasm could be determined.

5 The exception is the homogenization of unthawed freeze-blown rat brain tissue, a process which almost completely disallows Ss formation (Table IVB).

‘Latent activity is also contained within the previously described (26) “cytosolic particles” present in Ss fractions.

’ The evidence for this is that there is an enrichment

of HK (11) and mitochondria (27) in nerve endings, and there is a three- to fourfold greater rate of glucose utilization in gray matter (synapse-rich) than in white

matter (axon-rich) (28).

The results reported herein strongly suggest that rat brain HK in vivo is predominantly bound regardless of the level of glycolytic activity, indicating that large net movements of the HK molecules on and off the OMM do not occur, and thus can play no role in the regulation of glycolysis in brain tissue.

MATERIALS AND METHODS

Note. Only those materials and methods not men- tioned in a previous article (26) are presented here.

Chemicals

Percoll was obtained from Pharmacia, aldolase (Type IV), mixed crystals of a- glycerophosphate dehydrogenase and triose phosphate isomerase (Type III), cytochrome c (Types VI and II-A), ~-C&-1,6- Pz (Grade I), D-glucose-l-phosphate (Grade V), fructose 6-phosphate, NADPH (Grade III), phosphoglucomutase, and rotenone from Sigma, and succinic acid and D-mannitol from Fisher Scientific.

Animals

Mice pups (NZB/W) were kindly donated by Dr. Bruce Rabin.

Enzyme Assays

All enzyme specific activities are expressed in units of micromoles substrate utilized per minute per milligram protein. Phosphofructokinase was assayed essentially according to Craven and Basford (29), except that the final NADH concentration was lowered to 0.25 mM. The disappearance of NADH was linear for at least 5 min in the range 0.005-0.11 AU/min at 25°C although the reaction occasionally required 30-90 s in order to achieve its maximum, linear rate. RINCR was assayed using a modification of the method of Sottocasa et al. (30). The reaction cuvette at 25°C contained the following final concentrations: 50 mM NaHzPO,, pH 7.5; 0.1 mM NADH; 0.1 mM cytochrome c (either Type VI or the less pure Type II-A); 1.0 mM KCN; and 25


PM rotenone. The assay was initiated by the addition of NADH, and the reduction of cytochrome c was followed at 550 nm. The value used for the difference between the millimolar extinction coefficients of the reduced and the oxidized forms of cytochrome c was 21.1 ml/pmol/cm (31). The reaction was linear for at least 3 min in the range 0.005-0.1 AU/min. NCR was assayed essentially as was RINCR, except that the reaction was initiated by adding NADPH. Sucrose (0.25 M) should be included (but was not) in the RINCR and NCR assay mixtures to ensure isoosmolar- ity and prevent any lysis of Ss.

Metabolite Assays

Glc-6-P was assayed by two methods, one being that of Lowry et al. (24), the other being the initial part of the ATP/PCr assay described by Lowry and Passonneau (32), without Glc-6-P dehydrogenase included in the assay mixture. Both gave excellent results, but it was more convenient to use the second assay. Glc-1,6-Pz was assayed according to Passonneau et al. (33). The concentrations of stock MgClz solutions were determined by Dr. Warren Diven, using a DuPont automatic clinical analyzer, which utilizes a modification of the spectropho- tometric methylthymol blue procedure described by Connerty et al. (34).

Calculation of the Percentage Contamination of 5’s bg Free Mitochwdria Using RINCR and NCR Assays

NCR activity is exclusively microsomal (35), and RINCR is found in both mitochondria and microsomes. The percentage contamination of Ss by free mitochondria was calculated using the following math- ematical expression, where A is the NCR specific activity of pure microsomes, B is the NCR specific activity of Ss, C is the RINCR specific activity of pure mitochondria, D is the RINCR specific activity of Ss, and E is the RINCR specific activity of microsomes:

{[D - (B/A)E]/C} x 100.

All assays were conducted in the absence of Triton X-100. (Booth and Clark (36), we believe, used an incorrect formula for this calculation, calculating the percentage of free mitochondrial contamination in their Ss as 4.2% ,whereas the figure should have been 7.4%). The small amount of NCR activity in the mitochondrial fraction (see Table V) may be due either to microsomal contamination or to the presence of pyri- dine nucleotide transhydrogenase and residual NAD in the mitochondria, as dis- cussed by Sottocasa et al. (30).

Brain Removal Techniques

Rat and Mouse Pups

A rapid and effective method for remov- ing the brain tissue from pups was found to be decapitation (just caudal to the cranium), followed by squeezing the head between thumb and fingers in a rostra1 to caudal direction, which causes the brain tissue to be expelled through the foramen magnum. This removes all of the brain tissue, and homogenization can easily be begun within 15 s following decapitation.

Adult Rats

Slow method. Following decapitation at neck level, the whole brain was removed into ice-cold homogenization medium, and, following a variable period of time (5-22 min), the cerebellum, brain stem, thala- mus, and portions of the corpus callosum were dissected away. The cerebral hemi- spheres were then chopped by razor blade prior to homogenization.

Rapid method. By decapitating at a level which cuts through the cranial cavity at a level at or just behind the cerebellum, the brain is easily accessible, and the cerebrum of an adult rat routinely was removed in 20 s.

“‘Rapid sampling. ” A “rapid-sampling” device was used (prior to the development of the above-described rapid method of brain removal) in order to remove brain tissue and begin homogenizing within 20 s. The device and its operation were described

ON THE IN VW0 DISTRIBUTION OF BRAIN HEXOKINASE 257

by Shiu and Nemoto (37). In the experiments described herein, the skin overlying the cranium was removed prior to sacrifice, and the brain tissue was removed into ice- cold homogenization medium rather than liquid nitrogen.

Brain blowing. All of these brain remov- als were conducted by Don Davis, of Dr. Richard Hawkins’ laboratory, Department of Anesthesiology and Physiology, Hershey Medical School. The procedure was performed as described by Veech et al. (38).

High-Speed Sedimentation Procedure (f~ Separating Soluble and Bound HK)

The homogenization medium consisted of either 0.35 M sucrose/O.5 lllM EGTA/lO InM Hepes buffer, pH 7.4, or 0.32 M sucrose/ 1 mM EDTA/lO mM Tris/HCl, pH 7.0 (SET medium). After thorough homogenization, the homogenates were vortexed, and l.O- ml volumes were placed in microcentrifuge tubes and spun at 38,000 rpm (13O,OOOg, rmax) in a Beckman Type 40 rotor (equipped with plastic adaptors made in this laboratory (26)) for 10 min. The soluble activity is expressed as a percentage of the total, which was measured either as the soluble plus pellet activity, or as the activity in the original homogenate (both methods gave the same total activity).

Homogenize-during-Thawing Procedure

The disk of freeze-blown tissue was bro- ken into smallerpieces under liquid Nz immediately after being removed from the rat, and stored at -70°C. A small amount (0.2-0.4 g) of the frozen tissue was added to SET medium (3-4 ml) in a 15-ml capacity Dounce homogenizer (Wheaton) kept on ice, and hand homogenization was begun immediately, using the loose pestle. A great deal of force was exerted in the downward stroke, crushing the tissue pieces together into a compact, frozen pellet. A strong up- stroke was then begun, and the process was continued until homogenization was complete (l-2 min). Each combined upward and downward stroke was accomplished in about 1 s.

Percoll Gradient Procedure for Mitochcmdrial Isolation

The P2 fraction was obtained as described for the modified Booth and Clark method of Ss preparation (26). The P2 fraction from one-half of a rat or chicken cerebrum was suspended in 10 ml of SET medium, and gently homogenized (7 ml Dounce, loose pestle). An isoosmotic Per- co11 stock solution was made by mixing 87.2 ml of Percoll (after shaking the bottle vig- orously) with 12.8 ml of a solution consist- ing of 2.5 M sucrose and 100 mM Tris base, brought to pH 7.0 (at room temperature) with HCl. This Percoll stock solution had a pH (at O-4%) of about 8.1. Four millili- ters of this solution (after vigorous shaking) was placed in each of two thick-walled centrifuge tubes, and 5 ml of the P2 suspension was added, whereupon the tubes were capped and the contents mixed. They were then centrifuged at 38,000 rpm (99,OOOg, rave) in a Beckman Type 40 rotor for 20 min, and decelerated with the brake off. The myelin and Ss banded together, and were removed by pipet. The broad mitochondrial “band” was underneath the Ss/ myelin band, and was removed and diluted with SET medium to about 30 ml, mixed, and centrifuged at 12,000 rpm (17,3009, rmax) in a Sorvall SS-34 rotor for 10 min. The mitochondria did not form a tight pellet at this point, due to the presence of Per- ~011. The bulk of the supernatant was removed, whereupon an additional 30 ml of SET medium was added, followed by mixing and recentrifugation. This produced tight pellets, and these mitochondria were then suspended in a small volume of SET medium, and gently homogenized.

Preparation of Solubilixed HK and HK-Depleted Mitochondria

Mitochondria isolated from the brain of one rat were suspended in 1.0 ml of SET medium. From this, 100 ~1 was removed for protein and enzyme assay, and to the remaining 0.9 ml was added 50 ~11 each of 0.1 M Glc-6-P and ATP (each at pH 7.0). The suspension was then incubated at 30°C for 20 min, and centrifuged at 100,OOOg for


10 min (Type 40 rotor with plastic adaptors, 38,000 rpm). The supernatant was removed and saved, and 0.9 ml of SET medium was added to the pellet, which was then resuspended and gently homogenized. Another 50 ~1 each of 0.1 M Glc-6-P and ATP was added, and the suspensions were incubated and centrifuged as before. The supernatants were combined (-2.0 ml) and di- alyzed (with rapid stirring) against 100 vol of cold SET medium for 1 h, with three changes (1 h each). This procedure produced a solubilized HK fraction essentially free of Glc-6-P and ATP, and with a HK sp act of 7.6 + 1.7 (n = 7).

The mitochondrial pellet was washed once and then resuspended in one of several media. The overt HK sp act of such HK- depleted mitochondria, isolated using the Ficoll/sucrose flotation gradient method, was 0.036 f 0.019 (n = 6), whereas that of the originally isolated mitochondria was 0.56 + 0.08 (n = 11). A similar preparation, using mitochondria isolated from Percoll gradients, had an overt HK sp act of 0.024, whereas that of the originally isolated mitochondria was 0.77 f 0.14 (n = 5). (These latter mitochondria appeared visually to have much less synaptosomal contamination than those isolated by the Ficoll/sucrose flotation gradient procedure.)

Statistical Treatment of the Data

The data are expressed as means -I- sample standard deviation. Where indicated, statistical significance was assessed using Student’s paired comparison t test.

RESULTS

If brain HK undergoes changes in its in vivo distribution with normal fluctuations in the glycolytic rate, then one would expect HK in vivo to be particulate to a lesser extent than 80%, the routine 80% figure being due to the ischemia (and the atten- dant decrease in Glc-6-P levels and increase in Pi levels) which accompanies routine decapitation and brain removal. Thus, if a technique could be devised to ex- amine the in vivo HK distribution, one might expect much less than 80% of the HK to be particulate. Moreover, one would

expect that in the brain of an anesthetized rat, which has a significantly lower rate of glycolysis (39), one would find HK in its most solubilized state.

Choice of Homogenization Medium

The homogenization media used (0.32 M

sucrose/l.0 mM EDTA/lO mM Tris-HCl or 0.35 M sucrose/O.5 mM EGTA/lO InM

Hepes) were similar to those used in most other published studies, and it seems likely that they act to maintain the HK distribution at the level existing within the cells at the time of homogenization. EDTA and EGTA chelate free M$+, preventing binding of soluble HK, but do not act to solubilize bound HK (7, 40). Vallejo et al. (41) suggested that the use of a salt solution approximating the intracellular milieu may yield a HK distribution more closely approximating that which exists in vivo. Crane and Sols (42) also addressed the possibility of the use of a physiological salt solution as a homogenization medium, and concluded that not enough was known about intracellular ion concentration gradients to be able to decide what actually is a physiological solution. Vallejo et al. (41) and Felgner and Wilson (12) have shown that high salt concentrations solubilize bound HK. Vallejo et al. (41) interpreted this to mean that low ionic strength media may promote artifactual binding of HK. However, it has been a consistent finding that binding of solubilized HK to HK-depleted mitochondria in low ionic strength media does not occur in the absence of Mg?- (7,40,43), and thus would not be expected to occur artifactually during homogenization of brain tissue in the media used in the present study.

Enzyme Distribution as Revealed by Routine Homogenization Procedures

Figure IA shows the percentages of LDH, PFK, and HK which were found to be soluble when homogenization occurred at various times after complete ischemia was introduced. Longer periods of complete ischemia resulted in small increases in the percentage of soluble HK, just the opposite

ON THE IN VW0 DISTRIBUTION OF BRAIN HEXOKINASE 259

00 0 2 4 6 8 10 12 14 16 18 20 22 24

Time (minutes)

FIG. 1. Percentages of HK, LDH, and PFK in HSS supernatants. In Part A, adult rats were used, and the timepoints refer to when homogenization began, after the time of sacrifice. The LDH, HK, and PFK

values fell within the indicated boundaries. High- speed centrifugation always was begun within 9 min

after homogenization occurred. Unless otherwise indicated, the timepoints of 3 min and less (the quickest time was 20 s) were obtained by using the “rapid-

sampling” device, those greater than 3 min were obtained by using the “slow method,” the rats were anesthetized by ip injection of Nembutal (40 mg/kg)

30 min prior to sacrifice, and brain tissue was removed into ice-cold Hepes-buffered 0.35 M sucrose within 1 min of sacrifice (A). The brain tissues of the 40-s 70-

s, and 2-min timepoints (X) were removed into warm (37”) Hepes/sucrose medium immediately upon sacrifice, and were transferred to ice-cold Hepes/sucrose medium 10 s before homogenization began. In Part

B, the brain tissue of pups (both mouse and rat) of the indicated ages was removed and homogenized in SET medium. The rats were ages 4.5, 6, 7.5, 9, and 14.5 days (the 14.5-day-old rat is represented by the

square), and mice were of ages 2, 4.5, 6.25, 6.5, ‘7, 9.5, and 14.5 days. The tissue which was homogenized

of the findings with chick brain reported by Knull et a2: (16). Results similar to those shown in Fig. 1A are found in a report by Hofeler and Krieglstein (44), where rat brain tissue was homogenized at 15 or 30 s after sacrifice, and a small increase in the percent of soluble HK was observed (19.6 and 22.3% for the 15-s and 30-s samples, respectively). More early time points would be required in order to substantiate the trend toward less soluble HK at the earliest timepoints shown in Fig. lA, but the data clearly do not show the trend predicted by the ambiquitous enzyme hypothesis, of more soluble HK with lesser time of ischemia.

Rat pups do not reach the adult level of brain HK activity and distribution until about 24 days of age (45), but do have bound HK, and might be expected to display changes in HK distribution, especially since their brain tissue can be removed more rapidly than that of adult rats, and because their brain metabolic rate is much lower (9-10% of adult levels in 7-day-old pups) and the tissue undergoes changes in effector metabolite levels much more slowly after decapitation than does adult tissue (46). Figure 1B shows the results of 14 experiments with mouse and rat pups, aged 2-14 days, where the tissue was homogenized at either 15-40 s or about 2.5 min after sacrifice. The percentage of HK which was soluble varied only between 10 and 20%, and LDH between 78 and 90% soluble. (These findings appear to contra- dict the report of Land et al. (47) that in rat pups, about 60% of brain HK was soluble. The explanation is that they calculated the amount of “soluble” HK by iso- lating mitochondria, determining the amount of HK bound, and defining the rest to be “soluble.” Such a procedure does increase the apparent amount of HK “soluble,” due to solubilization of mitochondri-

within 15 to 40 s from the time of sacrifice (0) was removed as described in Materials and Methods, and

the tissue homogenized after about 2.5 min was left in the skull until about 10 s before homogenization began (0), and the whole brain was removed intact

into ice-cold SET medium.


ally bound HK during the mitochondrial isolation procedure (40).) Thus, in the brain tissue from adult rats and mouse and rat pups, HK appeared to undergo, at most, only small changes in its distribution, even though the brain tissue was subjected to complete ischemia for lengths of time ranging from 15 s to 22 min.

Evidence for Partially Particulate PFK

The percentage of LDH in the supernatant was always about 80% ,while PFK was about 65% soluble when measured by this procedure (Fig. 1). Consistent with this apparent partially particulate nature of PFK was the finding that HK and PFK in the HSS pellets were both about 42% overt (assayable before Triton X-100 addition), whereas only 23% of LDH was overt (LDH, 23.0 f 3.5% (n = 12); HK, 42.3 f 3.4% (n = 13); PFK, 40.7 + 7.1% (n = 7); p < 0.001 for both HK and PFK with respect to LDH, for n = 7 paired samples). This indicates that both PFK and HK were attached to the external surface of some component(s) of the HSS pellet. We and others have reported an association of PFK with isolated mitochondria (29,48) and with high speed pellets of crude homogenates (29, 49). However, very little PFK activity was found associated with isolated mitochondria (Table IA), even when MgClz was present throughout the mitochondrial isolation procedure (Table IB). This mito- chondrially bound PFK amounted to about 1% of the PFK activity present in the original homogenate, compared to 69% for HK, determined by calculations based on citrate synthase recovery like those employed by Land et al. (47). Vallejo et al. (41) also found less PFK in the supernatant fraction than a cytosolic marker, phosphoglucomutase (71 vs 90%); the “missing” soluble PFK activity was found in the nuclear pellet fraction, not the mitochondrial fraction, which is consistent with the present findings.

Effector Levels

The levels of some relevant effecters were followed during the period of ischemia after sacrifice, in order to assess the

TABLE I

HK, LDH, AND PFK SPECIFIC ACTIVITY IN

RAT BRAIN MITOCHONDRIA

Part A

Specific activities

Sample description HK LDH PFK

Na Diatrizoate mito 0.322 0.030 0.005 + Triton X-100 0.340 0.034 0.008

Na Diatrizoate mito 0.417 -0 0.017

Part B

Total specific activity (+Triton)

Sample description HK LDH PFK CS

Percoll mito 0.93 0.061 0.031 2.26 Percoll mito + Mg” 0.84 <O.Ol 0.044 1.98

Note. Rat brain mitochondria were isolated either

by using the sodium diatrizoate gradient method of Tamir et al (79), or by the Percoll method described in Materials and Methods. The data are the results

of single experiments. In Part B, “Percoll mitochondria + Mg’+” refers to mitochondria isolated with 1 mM free Mg2+ present throughout the entire isolation

procedure.

possible cause of the observed small shift in HK distribution. In Table IIA it can be seen that ATP, Glc-6-P, and Glc-1,6-Pz levels dropped, and presumably Pi levels rose (since PCr levels fell dramatically). The decrease in Glc-6-P and Glc-1,6-Ps levels occurring subsequent to the 19-s timepoint were modest, even after 10 min in ice-cold homogenization medium (Table IIB). These changes in Glc-6-P, Glc-1,6-Ps, and Pi would lead one to predict an increase in bound HK, whereas a slight decrease was observed. Increased free fatty acid levels (50) and increased acidity (51), resulting from hypoxia, have been reported to cause solubilization of bound HK in viva (these authors did not, however, consider that hypoxia also results in increased Glc-6-P levels in brain (52)), and there is a rapid accumulation of lactic acid in ischemic brain (Table IIA, and see p. 359 of Ref. (53)). In vitro, increased acidity promotes solu-


TABLE II

METABOLITE CONTENT OF ISCHEMIC FREEZE-CLAMPED RAT CEREBRA

Part A: Anesthetized rats

Time

(s)

19 40

60 150

G6P

0.40 0.40

0.36 0.22

GBP PCr ATP ADP AMP TAN EC Lact

0.54 8.41 13.42 6.10 1.55 21.1 0.78 13.7 0.60 3.59 11.58 6.49 2.74 20.8 0.71 30.0

0.60 2.90 11.44 8.54 3.24 21.9 0.68 39.1 0.42 0.59 3.24 7.80 9.23 20.3 0.35 64.2

Part B: Unanesthetized rats

Sample description G6P GBP

2.5 min (left in cranium) 0.31 0.40 10 min in ice-cold homogenization medium 0.19 0.41

Note. The metabolite contents are expressed as nmoI/mg protein. In Part A, the rats were anesthetized 30 min prior to sacrifice. The tissue was processed as previously described (26), and metabolite assays were

performed in duplicate, and the average values are shown. The abbreviations are as follows: G6P, Glc-6-P; GBP, Glc-1,6-P*; TAN, total adenine nucleotide; EC, adenylate energy charge ([ATP + 0.5 ADP]/[ATP + ADP

+ AMP]); Lact, lactic acid.

bilization of bound HK and allows Glc-6- P-induced solubilization in high ionic strength medium (0.2 M NaCl) (7,12). Thus, the failure of the small shift in HK distribution to correlate with levels of the usual effecters may be explained by the opposing action of increased acidity and free fatty acid levels (rat brain free fatty acid levels increase linearly following decapitation (54)). Other unexamined, but potentially important variables are the levels of the polyamines spermine and spermidine, which have been shown to function like M8+, both promoting binding of soluble ascites tumor HK to mitochondria, and counteracting the releasing action of Glc- 6-P (55). The concentrations of spermine and spermidine in rat brain have been reported to be 0.2 and 0.7 pmol/g wet weight, respectively (56), and we are unaware of any investigations into whether these concentrations are affected by ischemia. In any case, this question about changes in HK distribution with increasing periods of complete ischemia, although intriguing, is moot, since complete ischemia is a totally nonphysiological phenomenon.

A cursory glance at Fig. 1A and extrap- olation of the HK data might lead one to

believe that HK in vivo is -90% bound. However, it is well known that Glc-6-P levels decrease rapidly with ischemia (24, 38, 57), and it seemed possible that the higher in vivo Glc-6-P levels might act to maintain HK in a predominantly soluble state, and drop so rapidly with complete ischemia that even a 20-s delay before homogenization allows a large net movement of HK onto the OMM, with the increase in intracellular acidity and free fatty acid levels perhaps resulting in the observed later (ir- relevant) increase in the amount of soluble HK. A faster method of brain removal therefore was required in order to ascer- tain the in vivo HK distribution.

Freeze-Blown Tissue Experiments

The brain-blowing device developed by Veech et al. (38) removes and deposits a rat’s forebrain into an aluminum disk- shaped chamber within a fraction of a second. The aluminum chamber normally is precooled in liquid Nz in order to freeze the tissue instantly, but we originally intended simply to use the device to remove the tissue directly into a mortar so as to begin homogenization within a few seconds. This


proved to be technically infeasible. Also, “cold-blowing” the tissue into the aluminum chamber precooled on ice rather than in liquid Na, and then scraping the tissue into the mortar proved to take as long as with the rapid-sampling device (20 s), and thus offered no advantage. However, a procedure was developed which yielded reproducible and potentially significant results. It involved freeze-blowing the tissue, stor- ing the tissue at -7O”C, and then homogenizing it as it thawed in a buffered sucrose solution containing 1 mM EDTA. In one experiment, two adult rats were anesthetized by ip injection of Nembutal (40 mg/kg). The brain from one was freeze-blown 15 min after administration of anesthetic, whereas the other rat was positioned in the brain-blower and allowed to recover from anesthesia (responsive but groggy) for 60 min before freeze-blowing. Both tissues had identical adenylate energy charge (58) ([ATP + 0.5ADP]/[ATP + ADP + AMP]) values of 0.907, and the metabolite contents were almost identical. Both gave 23% of HK soluble. Subsequently, only animals sacrificed after 20 min of anesthesia were used, and the metabolite levels in the tissue, and various treatments thereof, from five freeze-blown rat brains are presented in Table III. The energy charge of the freeze-blown tissue was about 0.90, indicating that the tissue was sampled, stored,

and extracted properly. The extent of tissue disruption achieved by this technique is il- lustrated in Table IV, which compares the effect of routine homogenization of fresh tissue (Part A) with that of the HDT procedure (Part B) on the extent of release of enzyme activity. It can be seen that almost all of the cytoplasm was released, since about 94% of the total LDH activity was rendered overt, versus a normal figure of 58%) while the extent of disruption of the mitochondrial matrix was much less (36% of citrate synthase rendered overt).

An anomaly existed in the metabolite profile of the HDT samples (Table III), this being that the ATP levels were much lower than those originally present in the tissue, while the PCr levels were not very much reduced. This is the inverse of the pattern seen with ischemia, since with ischemia the PCr levels fall more rapidly than ATP levels (compare the freeze-blown tissue levels in Table III with the freeze-clamped tissue levels in Table II-A). Nor does one see these high PCr levels if the tissue is allowed to thaw completely before being homogenized. Six and ten min of thawing of freeze- blown tissue in a large volume of 2°C SET medium prior to homogenization and perchloric acid extraction produced large de- creases in ATP and PCr, a greatly increased content of AMP (6-min thaw), and a fall in TAN levels (Table III). Thus, the

TABLE III

METABOLITE CONTENT IN FREEZE-BLOWN RAT BRAIN TISSUE AND HOMOGENATES

Sample description PCr ATP ADP AMP TAN EC

Freeze-blown (n = 5) 33.1 f 2.9 20.7 + 3.0 3.77 + 0.29 0.51 + 0.16 25.0 f 3.4 0.904 + 0.006

HDT (n = 4) 25.6 f 2.2 7.31 f 0.82 16.1 k 1.8 3.7 + 2.8 27.1 + 4.3 0.57 -c 0.060 HDT + Mg2+ (n = 3) 1.38 + 0.49 1.84 + 0.24 6.81 2 0.92 11.3 f 1.6 19.9 + 1.8 0.26 +_ 0.023

6-min thaw 5.28 1.77 6.38 8.88 17.03 0.291 lo-min thaw 1.07 1.86 3.00 2.90 7.76 0.433

Note. Anesthetized adult Long-Evans rats were freeze-blown at 20 min (one at 15 min) after administration of anesthetic, except for one waking rat (60 min). The freeze-blown tissue was extracted as described previously (26). The freeze-blown samples which underwent the HDT procedure were left on ice for 10 min before perchloric acid extraction, which was conducted as previously described (26). The “HDT + Mg’+” samples were identical to the HDT samples except for the inclusion of 5 mM MgClz. and were kept on ice for 10 min prior to acid extraction. The “6-min thaw” and “10-min thaw” samples were freeze-blown tissue samples which were allowed to thaw at 2°C for 6 and 10 min, respectively, before homogenization and perchlorie acid extraction. Abbreviations are: TAN, total adenine nucleotide; EC, adenylate energy charge.

ON THE IN WV0 DISTRIBUTION OF BRAIN HEXOKINASE 263

TABLE IV

ENZYME AGTIVITIES, PERCENTAGE OF OVERT ENZYME, AND PERCENTAGE OF ENZYME IN HSS SUPERNATANTS IN

HOMOGENATES OF FRESH AND FROZEN RAT CEREBRA

Part A: Fresh rat cerebra

LDH HK cs

Total homogenate sp. act. 1.519 f 0.241 0.224 f 0.037 0.362 2 0.041 (n = 14) (n = 24) (n = 6)

Percentage overt 58.4 f 4.8% 48.5 k 5.4% 4.7% (n = 14) (n = 24) (n = 1)

Percentage in HSS supernatant

With respect to overt 84.7 * 6.0% 21.5 + 4.9% -

With respect to total 48.5 rfI 4.7% 10.0 + 2.5% -

(n = 10) (n = 16)

Part B: Homogenize-during-thawing of freeze-blown and freeze-clamped rat cerebra

Total homogenate sp act

Percentage overt

Percentage in HSS supernatant With respect to overt

With respect to total

1.427 f 0.216 0.247 f 0.007 0.392 + 0.061 (n = 6) (n = 7) (n = 6)

94.3 + 7.6% 69.5 + 3.2% 36.0 k 7.9%

(n = 6) (n = 6) (n = 6)

82.9 f 2.3% 22.8 + 5.0% 46.7 2 3.4%

77.5 f 7.5% 15.7 + 2.8% 18.8 + 5.9%

(n = 5) (n = 5) (72 = 5)

tissue behaves like normal ischemic tissue if allowed to thaw completely before homogenization, but dramatically unlike ischemic tissue when it undergoes the HDT procedure.

The only reasonable explanation for the anomalously high PCr to ATP ratio in the HDT samples is that the cell membranes are disrupted by homogenization before endogenous ATPase and CPK activity can greatly deplete PCr levels. As mixing between the cytoplasm and the SET medium (i.e., homogenization) occurs, the endogenous ATPases continue operating, gener- ating Mg-ADP’- from Mg-ATP’-, thus continuing to create substrate for the CPK reaction. The EDTA in the SET medium, however, chelates essentially all of the free MS+, causing essentially all of the ADP to exist in the free ADP3- form, which is not a substrate for the CPK reaction. The ATP levels fall much further than the PCr levels because of the combination of the endogenous ATPases and the greater stability of

the Mg-ATP’- complex wis a ti Mg-ADP1-. The absence of Mg-ADP1- also stops the myokinase reaction, i.e., the formation of Mg-ATP’- and AMP’- from Mg-ADP1- and ADP3-. To test this hypothesis, in three separate trials, freeze-blown tissue was homogenized during thawing and split into two portions. To one portion was added 5 mM MgCl, while the other received no addition, and both were incubated on ice for 10 min and then perchloric acid extracted. The results of these experiments are given in Table III (HDT and HDT + Mgz+). The addition of MgClz resulted in a drop in ATP and ADP levels,* a precipitous fall in PCr levels, and an increase in AMP levels, indicating a restoration of the substrates for the ATPase, CPK, and myokinase activities, and thus substantiating the hypothesis.

* In an abstract (l), we mistakenly reported the ADP

levels as having increased following MgCla addition.


The mean percentages (&SD) of overt enzyme activities found in the HSS supernatants of the freeze-blown tissue homogenates are as follows: for HK, 21.3 rfr 1.9% (n = 5); for LDH, 85.9 f 4.9% (n = 6); and for PFK, 89.8 f 8.2% (n = 3) (note that this level of soluble PFK is higher than the average value of -65% soluble PFK shown in Fig. 1; the much harsher conditions of homogenization in the HDT procedure may be responsible for this difference).

The question of the validity of the HDT procedure is addressed at length in the Appendix, and from those considerations it is concluded that the frozen tissue thaws only very briefly before homogenization occurs, allowing an extent of change in metabolite levels equivalent to that which would occur in 3 to 6 s of complete ischemia at 39°C (rat body temperature). This is a much shorter time interval than that af- forded by traditional brain removal and homogenization techniques, and thus the data gained by this method may be more indicative of the in viva HK distribution. However, the method is subject to the possibility of a rapid, massive artifactual movement of soluble HK onto the OMM (although there is no indication that such occurs-see the Appendix), and thus the data gained by it cannot be considered con- clusive.

Synaptosome Experiments

The second major approach to the question of the existence of any dynamic behavior of brain HK distribution was to utilize Ss as an in vitro system which is easy to manipulate with respect to the glycolytic rate, and amenable to rapid plasma membrane disruption and centrifugation, in order to measure the intrasynaptosomal HK distribution. Also, the synapse represents the site of the most active glycolysis in brain, and therefore Ss might be expected to display a large degree of ambiquitous behavior, if such exists at all for HK in brain tissue.

The structural and metabolic integrity of the Ss preparation has been adequately documented (26). The amount of free mitochondrial contamination also is an im-

portant consideration, because a large amount could present a large background problem, since the free mitochondria have bound HK, which cannot participate in a change in the glycolytic rate occurring in- side the Ss. The Ficoll/sucrose flotation gradient procedure of Booth and Clark (36), with minimal modifications (previously described in detail (26)), was chosen because of its rapidity (1.5 hours), its use of an isotonic Ficoll/sucrose gradient (which has been reported to avoid the damage caused by the use of hypertonic sucrose density gradients (59)), and its comparably low degree of free mitochondrial contamination. The amount of free mitochondrial contamination was examined using two independent methods. The first method was similar to one used by Booth and Clark (36); it is described in Materials and Methods and involves the measurement of RINCR and NCR activities, Table V compares the specific activities of both of these enzymes in microsomes and mitochondria obtained in the present study with values reported in the literature, and it can be seen that the measured activities are subject to some variation (note both the differences between literature values, and the large standard deviations in the data of the present study). Using this method, the percentage (by weight of protein) of free mitochondrial contamination of several Ficoll/sucrose flotation gradient Ss preparations ranged from 2 to 11% .The second method employs an O2 electrode, and is based upon the reasonable assumption that Ss do not take up succinate. Thus, the increase in respiration in a Ss preparation upon succinate addition is considered to be due entirely to contaminating free mitochondria. A standard curve (Fig. 2) was constructed by measuring succinate-supported respiration with varying amounts of pure mitochondria, and from this, a mg protein amount of free mitochondrial contamination can be assigned to any given level of succinate-supported respiration in Ss (for details, see Fig. 2). This method gave an estimation of the percent of free mitochondrial contamination in Ficoll/sucrose flotation gradient Ss preparations of between 8 and 11%.

265 ON THE IN VIVO DISTRIBUTION OF BRAIN HEXOKINASE

TABLE V

RINCR AND NCR ACTIVITIES IN PURIFIED MITOCHONDRIA AND MICROSOMES

Data source Fraction RINCR NCR

Gurd et al. (80) Microsomes 0.055 0.0202 Mitochondria 0.0617 0.0012

Booth and Clark (36) Microsomes 0.0193 0.0117 Mitochondria 0.0313 N.D.

Present study Microsomes 0.068 k 0.026 0.0158 + 0.0064

(n = 6) (n = 6)

Mitochondria 0.125 + 0.020 0.0031 +- 0.0018

(n = 5) (n = 7)

Note. The microsomal NCR value from Gurd et aL (80) was the average of four values they reported. In the present study, purified mitochondria were obtained either from sodium diatrizoate or Ficoll/sucrose density gradients. Purified microsomes were obtained as follows: the S2 fraction (from a lo-min, 1’7,300~ spin) was recentrifuged at 17,300g for 10 min, the supernatant was removed and sedimented at 110,000~ (36,000 rpm in

a Beckman SW 50L rotor) for 1 h, and the surface of the pellet was rinsed before the pellet was resuspended by gentle homogenization. N.D. = none detected.

Stimulation of Glycolysis in Ss Fractions

Veratridine is a plant alkaloid which increases the sodium permeability of the plasma membrane (60), and a two- to threefold stimulatory effect of veratridine addition on Ss lactate accumulation, 14COz output from [U-‘4C]Glc, and oxygen uptake has already been demonstrated (26). A change from an oxygen to a nitrogen atmosphere has been shown to result in a 2.3-fold stimulation of glycolysis in Ss (61).

Digitonin Treatment to Lyse the Synaptosomal Plasma Membrane

In order to determine the HK distribution within the Ss, it was necessary to lyse the Ss plasma membrane, while at the same time not affect the enzyme distribution. Homogenization was ineffective, son- ication in our hands was neither very effective nor reproducible, hypotonic lysis was somewhat effective but threatened to affect the distribution, and freeze-thawing was slow. A good means of lysis was found to be a 30-s digitonin treatment, at a concentration of 0.40 mg digitonin/mg Ss protein. Figure 3 shows the release of LDH, myokinase, and citrate synthase from Ss treated with increasing ratios of digitonin to Ss protein. (Myokinase was originally

chosen because we thought it to be located in the mitochondrial intermembrane space as is the liver enzyme. Brain myokinase, however, is predominantly cytoplasmic (62).) A ratio of 0.40 mg digitonin/mg Ss protein was chosen, because it was the lowest ratio which reliably would give maximal LDH and myokinase release.

Previous work indicated that this amount of digitonin does not solubilize bound HK, but does inhibit the rebinding of solubilized HK (63). Control experiments confirmed these earlier findings under the conditions used in the present experiments (40).

Enzyme Distribution in Synaptosomes

Control and veratridine- or N2 atmosphere-treated rat and chicken brain Ss were subjected to digitonin treatment, and the percentages of HK and LDH which were found in the supernatants are shown in Table VI. The experiments were conducted as described in the Table VI legend. The metabolite levels are not reported, but were essentially the same as those reported in Table 4 of Ref. (26). In all of these experiments, no differences in HK distribution were found between the Ss with differing glycolytic rates, as HK always was


mg mitochondrial protein

(to eliminate the possibility of any sodium gradient dependent uptake of succinate by contaminating Ss)

FIG. 2. Standard curve of succinate-supported mi-

before the mitochondria were added. Succinate was

tochondrial respiration. Rat brain mitochondria iso-

lated on Pereoll gradients were incubated at 37°C in

added to a concentration of 10 mM at time zero. The

a medium essentially the same as that described by

Lai and Clark (76), and consisted of 75 mM mannitol,

mitochondria exhibited a gradual and continual in-

25 mM sucrose, 5 mM KHzPOI, 50 pM EDTA(Kz), 100

mM KCl, and 15 mM Tris/HCl, pH 7.4. This medium

crease in the rate of respiration for at least 10 min.

was supplemented with 0.12% BSA (to help prevent uncoupling of the mitochondria) and 100 uM ouabain

This was probably due to the “reenergization” of the mitochondria, with the subsequent active uptake of

Pi and additional succinate. Ss were incubated under the same conditions, and rates of Oz uptake were determined during the 2.5 to 5.5-min time interval (0)

and the 6.5- to 9.5-min interval (m). The lines were derived by linear regression analysis.

within the mitochondria-containing particles.

DISCUSSION

Wilson has postulated that since in the presence of 1 mM Me HK will remain bound to mitochondria even in the presence

100

of Glc-6-P, and since the intracellular concentration of Me has been estimated to be about 1 mM, HK may exist predomi-

z

nantly bound in vivo (11,64). The data pre-

so-

sented in this article are consistent with this suggestion. Figure 1 shows only small

f

changes in distribution, and HK was predominantly bound even when homogenization was begun 15 s after sacrifice. Sim- ilar results (80% bound) were obtained by Hofeler and Krieglstein (44) using unfrozen brain-blown tissue which had been isch- emit for only 15 s. The data obtained from freeze-blown rat brain tissue using the HDT procedure also indicated that HK is

found to be predominantly bound, and LDH predominantly soluble. Data presented in Table 1 of Ref. (26) showed that 24% of the total HK in Ss fractions may be bound to free mitochondria. If this is subtracted from the total, then the observed 20-25% soluble HK would correspond to 26-33% soluble intraparticle HK. However, half of the soluble HK probably exists in the “cytosolic particles” (26), which are devoid of mitochondria. Thus, the amount of soluble HK in the mitochondria-containing particles (mostly Ss), where HK has an opportunity to change its distribution, may be closer to lo-12.5% of total, both before and after veratridine addition, which corresponds to 15-20% {[lO/(lOO-24-lo)] X 100, [12.5/(100-24-12.5)] X lOO} soluble HK

ol .,l,~(l,l,l,. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 c

mg Digitonin/mg Ss protein

FIG. 3. Lysis of the synaptosomal plasma membrane

by treatment with digitonin. Ss were treated with digitonin as described previously (26), at the indicated

weight ratios of digitonin to Ss protein. The percentage release into the supernatant fraction of total myokinase (MK), LDH and citrate synthase (CS) are plotted. (Unlike LDH and MK, the CS activity in the

supernatant was mostly (8085%)latent, and therefore was assayed in the presence of Triton X-100. Centrif- ugation at 100,OOOg for 10 min reduced the supernatant CS level to 1-Z%.)The data points from each of three experiments are grouped as follows (in terms of the

weight ratios): 0.1,0.15,0.20, 0.25, and 0.30; 0.27, 0.32, 0.38,0.43, and 0.49; and 0.48,0.58, and 0.68. The control (0.0 mg digitonin/mg Ss protein) values for LDH and MK are difficult to see on the graph, and are as follows: LDH = 7.8, 9.7, and 7.3%;MK = 10.2, 8.0, and 8.0%.


TABLE VI

HK AND LDH PARTITIONING IN SYNAPTOSOMES

Part A: Veratridine experiments

Expt 1 (Rat)

Expt 2 (Chick) Expt 3 (Chicken)

Mean + SD (n = 3)

% HK in supernatant % LDH in supernatant

Control Veratridine Control Veratridine

19.2 21.3 84.6 83.7 17.9 14.2 76.6 76.0 22.0 22.5 73.1 76.8

19.7 * 2.1 19.3 + 4.5 78.1 f 5.9 78.8 + 4.2

Part B: Nitrogen atmosphere experiments -

% HK in supernatant % LDH in supernatant

Expt 1 24.0 28.1 78.2 85.2 Expt 2 21.3 18.6 74.9 75.2 Expt 3 25.6 26.1 74.2 79.8

Mean f SD (n = 3) 23.6 -t 2.2 24.3 f 5.0 75.8 +- 2.1 80.1 + 5.0

Note. Ss were prepared by a modification of the method of Booth and Clark (36) as described in Ref. (26). The incubated SS were lysed with digitonin as previously described (26). In Part A, Expt 1, rat brain Ss were

incubated under 100% O2 at 37°C for 15 min in a bicarbonate-buffered physiological salt solution essentially the same as that described in Ref. (81) (but also contained 0.5 mM glutamine and 10 mM glucose), whereupon aliquots were removed both for perchloric acid extraction and digitonin lysis. Veratridine (final concentration

of 200 pM) was then added (in a small volume of 100% ethanol) to the remaining Ss, and after 5 min of further incubation, aliquots were removed for perchloric acid extraction and digitonin lysis. In Expt 2, the Ss were prepared from ll-day-old chick brain, and incubated and treated as in Expt 1, except that the incubation

medium was that described by Ksiejak and Gibson (61) containing 1 mM glucose, and only 80 pM veratridine was added. In Expt 3, Ss from an adult chicken were incubated as in Expt 2, with and without 58 pM veratridine

for 25 min, before aliquots were taken for perchloric acid extraction and digitonin lysis. In Part B, Expts 1, 2, and 3 were rat brain Ss incubations at 37°C in the bicarbonate-buffered physiological salt solution described

in Ref. (81) (also containing 10 mM glucose) under either an oxygen atmosphere (95% 02/5% COP in Expts 1 and 2, and 100% O2 in Expt 3) or 100% Nz atmosphere, for 15, 20-25 or 60 min, respectively, before aliquots were removed for perchloric acid extraction and digitonin lysis.

predominantly (79%) bound, even though homogenization was accomplished with the equivalent period of only 3-6 s of complete ischemia. The HDT data may seem to rest on only one experiment, since the freeze- blown tissue from only one waking rat was used. This criticism is allayed by the fact that anesthetized rats showed -80% of HK bound by this technique, and brain tissue from alert rats would be expected, according to the ambiquitous enzyme hypothesis, to have more HK bound than that from anesthetized rats; thus, there is not much room left for additional binding, and no great differences between anesthetized and

alert rats are possible. The fact that the HDT procedure ruptures presynaptic nerve endings as well as neuronal perikarya and glial cells (94% of LDH rendered overt, Ta- ble IVB) indicates that the HK of the presynaptic nerve endings in situ also is predominantly particulate, a finding which is corroborated by the Ss experiments.

Regarding the Ss experiments, artifactual changes in HK distribution are not likely to occur during the 30-s period between the addition of digitonin and com- mencement of centrifugation, since the digitonin treatment causes a rapid cooling of the Ss and a rapid dilution of the syn-


aptoplasm with a medium containing 3 mM EDTA, which would inhibit binding of soluble HK. Thus, the data obtained from the Ss experiments indicate that HK is predominantly bound (perhaps 80-85% in the mitochondria-containing particles) regardless of the level of glycolytic activity. Similar results have been reported by Nel- son et al. (65) in experiments where the HK distribution in ascites tumor cells was examined (using a digitonin method similar to that used in the present study) under conditions where the cells were allowed to utilize either endogenous substrate, or added glucose, in which latter case glycolysis was greatly stimulated. No difference in HK distribution accompanied the change in glycolytic rate, as about 75% always was bound,

Regarding the interpretation of this constancy of the HK distribution in rat brain, it is of interest to note that the soluble HK of cat brain may exist predominantly in astroglial cells (66), and the HK of cultured rat brain astrocytes has been shown to be 80% soluble (67). Thus, it may be that essentially all of the HK in neurons is bound to the OMM.

Why Is Brain Hexokinase Bound to the Outer Mitochondrial Membrane?

This question has been addressed by many (for example, see Refs. (11, 48, 68- 70)), but it seems to us that two pertinent questions first need to be answered.

1. Is the rate of Glc transport from blood into brain tissue in any way limiting the rate of glycolysis? Siesjo (p. 120 of Ref. (53)) and Lund-Andersen (71) believe not, whereas Bachelard (72, 73) considers the question unresolved, the differences of opinion being due to differing estimates of the intracellular Glc concentration in brain. This is an important consideration when trying to explain the 97% inhibition of mouse brain HK which Lowry and Pas- sonneau (74) calculated to exist in viva.

2. What is the nature of the two com- ponents of the HK-OMM binding inter-

action (7, 12), and do the different states of HK binding (11, 64) correlate with any functional differences in the orientation of HK with respect to porin?

Porin-bound HK has recently been impli- cated in the modulation of Ca2’ movements in brain mitochondria (75). One may won- der whether porin-bound HK, in its inter- actions with Pi, plays a role in the transport of Pi across the OMM, a process which is critical to the cell’s energy metabolism. Thus, there may be reasons for HK’s OMM location other than those involving control of the rate of the HK reaction.

APPENDIX

Efforts to Assess the Validity of the Homogenize-during-Thawing Procedure

This question was addressed at length in a doctoral dissertation (40), and it was concluded that the procedure did not dis- lodge bound HK from mitochondria, but the possibility could not be ruled out that the tissue thawed briefly before homogenization occurred. The main concern was that while the tissue exists as a frozen clump in the mortar as it is being homogenized, it is warming up slowly from -70°C to something less than 0°C. The micro- crystals of water present in the tissue may begin to grow, creating areas of concentrated cytoplasmic protein and metabolites, whose melting point will be below 0°C. If a liquid state were to exist at any time before homogenization occurs, there then would exist the opportunity for soluble HK to become bound. Experiments have since been conducted which indicate that the outer layer of the tissue clump does thaw briefly before homogenization is accomplished. The experiments attempted to address directly the question of at what stage during the HDT procedure the decrease in PCr levels occurred. Freeze- clamped rat brain tissue was homogenized during thawing in a buffered sucrose medium not containing EDTA, in order to as- sure the near total destruction of endogenous ATP and PCr. After 15-20 min, EDTA


was added to a final concentration of 2 mM.

An ice-cold mixture of PCr, ATP, ADP, and MgClz (in about the same ratios and concentrations as are found in freeze-blown brain tissue) was added with constant mixing, and at various times aliquots were removed and extracted with perchloric acid. The time courses of changes in PCr, ATP, ADP, AMP, and TAN levels are shown in Fig. 4A. After 15 min, PCr decreased by 21%, and ATP by 68%. This compares to the 23% drop in PCr, and 67% drop in ATP in the freeze-blown HDT samples (Table III), which seems to be a very good agreement. This indicates that if the levels of EDTA, M$+, ATP, and PCr are similar in the two cases, then in the freeze-blown HDT samples, most of the drop in PCr may have occurred subsequent to cell breakage, arguing against the existence of the putative concentrated liquid state. However, the ratio of EDTA to Mg+ is critical to the changes in the metabolites. The EDTA concentration in Fig. 4B was twice that of Fig. 4A, and while the adenine nucleotides still change, although less rapidly than in Fig. 4A, PCr changes much less. Therefore, the ratio of EDTA to Mgz+ which existed in the freeze-blown HDT samples is an important consideration, as is the amount of time which elapsed before the HDT samples were extracted with perchloric acid.

In Fig. 4, the ratios of EDTA to total M$+ were 1.2 (Part A) and 2.4 (Part B), assuming a value of 6.92 pmol total M$+/ g wet weight of brain tissue (77), and a value of 0.105 g protein/g wet weight of mammalian cerebral cortex (78). However, ratios of 2-2.3 prevailed in the HDT experiments (given the same assumptions), and the time between completing homogenization and beginning perchloric acid extraction was 10 min, so it seems likely that only a small portion of the PCr drop occurred after completing homogenization (-4% based on the data of Fig. 4B). A 23% drop corresponds to 5.7 s of ischemia at 39°C (assuming a linear fall in PCr levels from zero to 19 s of ischemia; see Tables IIA and III), and if one allows that a 6% drop could result from the presence of some

0 I , . , ~ , , , . , . , . , 1

0 2 4 6 a 10 12 l4

Time (minutes)

o2 Time (minutes)

FIG. 4. Time course of changes in PCr and adenine

nucleotide levels in vitro in the presence of EDTA. For the zero timepoints, perchloric acid was added to an aliquot of the homogenate prior to the addition of the mixture of ATP, PCr, ADP, and MgC&. The me-

tabolite levels in the homogenate (following the addition of EDTA) and the ATP/PCr/ADP/MgCl, mix-

ture were determined separately, as controls. EDTA added: (A) 2 mM, (B) 4 mM.

residual intact cytoplasm-containing particles (Table IVB), and another 4% to loss after homogenization, this would leave 13% of the PCr drop occurring before homogenization, which would correspond to 3.2 s of ischemia at 39°C. This -3-6 s period of ischemia is equivalent to that which would occur at 39”C, but at -0°C was undoubt- edly longer. However, the fact that in the four measured trials of the HDT procedure, metabolism was arrested at slightly different times (the evidence for this is that


if one normalizes to 25 nmol TAN content/ mg, then the individual values for the AMP increase were 3.45-, 3.5-, 6.6-, and 11.3-fold), and yet all showed about the same HK partitioning, is consistent with no change in HK partitioning, or else a very rapid artifactual binding of soluble HK. In any case, it seems likely that the constantly changing outer layer of frozen tissue re- turns briefly to a liquid state prior to homogenization, and the possibility that a massive artifactual binding of soluble HK occurs during this period cannot be ruled out.

ACKNOWLEDGMENTS

Special thanks are extended to Don Davis and Dr. Richard Hawkins for providing freeze-blown rat brain

tissue on several occasions, and to Dr. Warren Diven for performing Me determinations.

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An examination of the in vivo distribution of brain hexokinase between the cytosol and the outer...

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