THE JOURNAL OF BIOLOGICAL CHE~~ISTRY Vol. 243, No. 7, Issue of April 10, pp. 1603-1616, 1968 Printed in U.S.A. Rapid Labeling of Mitochondrial Lipids by Labeled Orthophosphate and Adeaosine Triphosphate* (Received for publication, November 8, 1967) ANIIYA K. HAJRA, EDWARD B. SEGUIN, AND BERNARD W. AGRANOFF From the Mental Health ResearchInstitute and the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48104 SUMMARY Rapidly labeled lipids of guinea pig liver mitochondria incubated with 32Pi or Y-~~P-ATP were studied. With the use of inhibitors of oxidative phosphorylati&, it was shown that 32Pi is converted to labeled ATP, which is the more direct precursor of labeled lipid. Atractyloside blocks phosphoryla- tion of lipid by endogenous ATP under conditions in which it has little effect on oxidative phosphorylation or lipid labeling by externally added ATP. Two radioactive lipids were identified by chromatography as phosphatidylinositol phos- phate and phosphatidic acid. The properties of an unknown third lipid were studied. Its formation was selectively blocked by the addition of hydroxylamine to the incubation medium. It is postulated that the lipids are labeled by the action of kinases and phosphatases in the mitochondrial fraction. While polyphosphoinositides have been characterized fully only from biological sources in which they occur in relatively large amounts, such as brain tissue (3-5), small amounts have been identified in virtually all animal tissues examined (6-8). In 1963, Garbus et al. (9) reported rapid labeling of phospholipids of kidney and liver mitochondria by a2Pi. They suggested that the major radioactive phospholipid was a polyphosphoinositide. Galliard and Hawthorne (10) performed alkaline deacylation of a2P-labeled mitochondrial phospholipid and found that glycero- phosphorylinositol phosphate was a major radioactive product. It was concluded that phosphatidylinositol phosphate was the 32P-labeled parent lipid. In addition to phosphatidyl-IP: there was evidence for the presence of small amounts of labeled phosphatidic acid (9, 10). Other studies have shown the rapid labeling of phosphatidyl-IP by T-~~P-ATP in liver mitochondria * Preliminary reports of this work have been published (I, 2); This research was supported by Grant NB-3101 from the Nationa Institutes of Health. 1 The abbreviations used are: phosphatidyl-IP, phosphatidyl- inositol phosphat,e; Rpi, electrophoret)ic migration rate relative t0 Pi. (1, ll), brain microsomes (12), and erythrocyte membranes (13). The rapid turnover of phosphatidic acid and phosphatidyl-IP in various brain fractions has also been reported (14, 15). In the studies reported here, we have investigated the rapid turnover of phosphatidyl-IP in mitochondrial preparations from guinea pig liver. In addition to phosphatidyl-IP and phos- phatidic acid, radioactivity was found in a third, unidentified phospholipid. Optimal conditions for the labeling of these lipids and the effects of various inhibitors are reported. MATERIALS AND METHODS 32Pi, carrier free sodium salt, purchased from IsoServe, Cam- bridge, Massachusetts, or from New England Nuclear, was diluted with unlabeled carrier upon arrival. y-32P-ATP was prepared by the method of Glynn and Chappell (16) with phos- phoglyceraldehyde dehydrogenase and 3-phosphoglycerate ki- nase (Boehringer). The +Y-~~P-ATP was purified either by chromatography on an anion exchange column (16) or by absorp- tion from a trichloracetic acid supernatant fraction on charcoal and subsequent elution by 1 y0 NH3 in ethanol-water (1 :l) (17). The purity of the labeled substrate was checked with a Van- guard scanning Geiger counter following high voltage electro- phoresis at pH 1.5 (8, 18). Oligomycin and antimycin were purchased from Sigma. i2tractyloside samples were gifts of Dr. A. Lehninger and Dr. A. Bruni. The polyphosphoinositides were prepared by solvent extraction of bovine brain according to the method of Folch (3). Organic solvents were of com- mercial reagent quality and were glass distilled before use. Dowex 50-X& H+ (200 to 400 mesh), purchased from J. T. Baker Chemical Company, Philipsburg, New Jersey, was washed with 2 N NaOH and 2 N HCl prior to use. Guinea pig liver mitochondria were prepared by the method of Schneider (19) in 0.25 M sucrose. Disodium-EDTA (0.002 M) was present in the sucrose used for homogenization, but not in that used for washing or resuspending the mitochondria. The final mitochondrial suspension contained mitochondria from 1 g of liver per ml of suspension. The incubation procedure was that described by Garbus et al. (9), except that the total volume was 1.2 ml, containing mitochondria from 0.5 g of guinea pig liver, one-half the amount used with rat liver mitochondria. Incu- bations were performed in tubes, 20 x 180 mm, in air with con- 1609 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from
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THE JOURNAL OF BIOLOGICAL CHE~~ISTRY Vol. 243, No. 7, Issue of April 10, pp. 1603-1616, 1968
Printed in U.S.A.
Rapid Labeling of Mitochondrial Lipids by Labeled
Orthophosphate and Adeaosine Triphosphate*
(Received for publication, November 8, 1967)
ANIIYA K. HAJRA, EDWARD B. SEGUIN, AND BERNARD W. AGRANOFF
From the Mental Health Research Institute and the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48104
SUMMARY
Rapidly labeled lipids of guinea pig liver mitochondria incubated with 32Pi or Y-~~P-ATP were studied. With the use of inhibitors of oxidative phosphorylati&, it was shown that 32Pi is converted to labeled ATP, which is the more direct precursor of labeled lipid. Atractyloside blocks phosphoryla- tion of lipid by endogenous ATP under conditions in which it has little effect on oxidative phosphorylation or lipid labeling by externally added ATP. Two radioactive lipids were identified by chromatography as phosphatidylinositol phos- phate and phosphatidic acid. The properties of an unknown third lipid were studied. Its formation was selectively blocked by the addition of hydroxylamine to the incubation medium. It is postulated that the lipids are labeled by the action of kinases and phosphatases in the mitochondrial fraction.
While polyphosphoinositides have been characterized fully only from biological sources in which they occur in relatively large amounts, such as brain tissue (3-5), small amounts have been identified in virtually all animal tissues examined (6-8). In 1963, Garbus et al. (9) reported rapid labeling of phospholipids of kidney and liver mitochondria by a2Pi. They suggested that the major radioactive phospholipid was a polyphosphoinositide. Galliard and Hawthorne (10) performed alkaline deacylation of a2P-labeled mitochondrial phospholipid and found that glycero- phosphorylinositol phosphate was a major radioactive product. It was concluded that phosphatidylinositol phosphate was the 32P-labeled parent lipid. In addition to phosphatidyl-IP: there was evidence for the presence of small amounts of labeled phosphatidic acid (9, 10). Other studies have shown the rapid labeling of phosphatidyl-IP by T-~~P-ATP in liver mitochondria
* Preliminary reports of this work have been published (I, 2); This research was supported by Grant NB-3101 from the Nationa Institutes of Health.
1 The abbreviations used are: phosphatidyl-IP, phosphatidyl- inositol phosphat,e; Rpi, electrophoret)ic migration rate relative t0 Pi.
(1, ll), brain microsomes (12), and erythrocyte membranes (13). The rapid turnover of phosphatidic acid and phosphatidyl-IP in various brain fractions has also been reported (14, 15).
In the studies reported here, we have investigated the rapid turnover of phosphatidyl-IP in mitochondrial preparations from guinea pig liver. In addition to phosphatidyl-IP and phos- phatidic acid, radioactivity was found in a third, unidentified phospholipid. Optimal conditions for the labeling of these lipids and the effects of various inhibitors are reported.
MATERIALS AND METHODS
32Pi, carrier free sodium salt, purchased from IsoServe, Cam- bridge, Massachusetts, or from New England Nuclear, was diluted with unlabeled carrier upon arrival. y-32P-ATP was prepared by the method of Glynn and Chappell (16) with phos- phoglyceraldehyde dehydrogenase and 3-phosphoglycerate ki- nase (Boehringer). The +Y-~~P-ATP was purified either by chromatography on an anion exchange column (16) or by absorp- tion from a trichloracetic acid supernatant fraction on charcoal and subsequent elution by 1 y0 NH3 in ethanol-water (1 :l) (17). The purity of the labeled substrate was checked with a Van- guard scanning Geiger counter following high voltage electro- phoresis at pH 1.5 (8, 18). Oligomycin and antimycin were purchased from Sigma. i2tractyloside samples were gifts of Dr. A. Lehninger and Dr. A. Bruni. The polyphosphoinositides were prepared by solvent extraction of bovine brain according to the method of Folch (3). Organic solvents were of com- mercial reagent quality and were glass distilled before use. Dowex 50-X& H+ (200 to 400 mesh), purchased from J. T. Baker Chemical Company, Philipsburg, New Jersey, was washed with 2 N NaOH and 2 N HCl prior to use.
Guinea pig liver mitochondria were prepared by the method of Schneider (19) in 0.25 M sucrose. Disodium-EDTA (0.002 M)
was present in the sucrose used for homogenization, but not in that used for washing or resuspending the mitochondria. The final mitochondrial suspension contained mitochondria from 1 g of liver per ml of suspension. The incubation procedure was that described by Garbus et al. (9), except that the total volume was 1.2 ml, containing mitochondria from 0.5 g of guinea pig liver, one-half the amount used with rat liver mitochondria. Incu- bations were performed in tubes, 20 x 180 mm, in air with con-
1010 Rapid Labeling of JlitochondCd Lipids 1-01. 2-13, so. 7
stant shaking at 30” or 37“. Lil)ids were extracted by a modifica- tion (9) of the method of Nigh and Dyer (20). Following in- cubation, 4.5 ml of chlorofonll-lnethallol (1:2) and 0.1 ml of 6 1v I-ICI were added to the incubation tubes aud the mixture n-as tlisl)ersctl with a Vortex miser for 30 sec. An additional 1.5 ml of chloroform were added, followed by 1.5 ml of 2 31 KC1 in 0.2 $1 I-IJ’O~. Each mixture was again mixed for 30 SW, autl the two liquid phases were separated by ceutrifugation for 5 min at 1000 x g. .I whitish disc of protein lmxil)itate was l~rcsent in the
interphase. A saml)le of the clear lower layer was l)il)etted into a test tube aud dried under a stream of nitropcii. In ordrr to re- move additional water-soluble radioactive contaminants, the lipid was washed with chloroI’orm-methanol-corlcclltrated I-ICI (200 : 100 : I) (21). The mixture (4 ml) was added to the dried lipid, follow-cd by 1 .O ml of 0.05 M H3P04. Following misiug arid cent~rifugation as above, the upl,er layer was removed and the lower layer was washed with 2.5 ml of simulated upl~r layer (22)) \vhich was made 0.1 s in HCl. The lower l)hasc was dried, rc- dissolved in chloroform, and used immediately or stored at -20”.
Clrron~atograp/2~~Forinalrlcli~~I~~-Ireatctl l)al)er was prepared b\- a modification (23) of the method of HGrhammer, Wagner, and Richter (24). Treatment iu the autoclave for 2% hours instead of 4 hours rcsultcd in leapers with yuperior streu~th. An aliquot (one-fifth or one-half of 3 to 4 mp) of radioactive lil)id from a single incubation n-as al)l)licd in a 2-cm zone together with 40 to 50 pg of carrier brain polpl~l~osl~hoinosititle. In the absence of added carrier, some trailing of radioactivity was observed. Washing the liljid with CaCl,, solution as rccommt~nded 1~13 Damson and Eichbcrg (14) did uot irnlxove the sel)aration and interferctl with the subsequent dcacylation.
Treatment with AU&-For alkaline mcthanolysis (25, 26), 0.1 to 5.0 mg of labeled lipid were dissolved in 1.2 ml of chloro- form, to which were added 0.2 ml of methanol and 0.4 ml of 0.5 K SaOH in anhydrous methanol. The reaction mixture was agitated and then n-as allowed to stand at room temperature. After 10 min, 0.2 ml of chloroformmethanol (2: 1) aud 0.5 ml of water were added. After agitation and centrifugation, the upl)er layer was withdrawn with a Pasteur l)il)ette and l)assed over Dowes 50, H+, in a column, 0.6 x 1.5 cm, to remove excess NaOH. l’hc lower layer was washed with 1 ml of chloroform methanol-water (3 :47 :48) (22). Following agitation and cell- trifugation, the uljlxr ljhase n-as l>assed through the ion exchange columii, follometI by 0.5 ml of n-atcr. The combined &ate was evaljorated to tlryncss at room teml)erature under reduced lxes- sure and then dissolved in a small volun~e of water. This lxo- ccdurc quantitatively convcrtcd radioactive phosphogl3-ceritles to water-soluble products. Occasionally the free acid forms of the l)hosl)hate esters were converted to their sodium salts. A1 small amount of Dowes 50, Sa+, was packed at the bottom of a Dowcx 50, II+, column through which the alkaline methanolysis lxoducts were passed. The toI) portion of the column removed the cxccss alkali and converted the r)hosphatc esters to the free acid forms, which wrre subsequently converted to sodium salts;. When Ca+f was present, relatively IOK yields of watrr-soluble radioactivity were obtained, l)robably because of the insolubility of the calcium salts of the phosl)hate esters at alkaline pH.
Rlectrophoresis of Tater-soluble Products-High voltage elec- trol)horesis was performed on Whatman Fo. 1 paper in osalate buffer, pH 1.5, for 20 min at 4000 volts (1, 8). l’hosl)hate-con taining compounds were detected by a molybdate slxay (27). For quantitative evaluation of radioactivity, peaks seen on a
strip scanucr Ivvelr cut out and l- to 3+x11 strips were counted by liquid scintillatioii iii vials coiitainin~ 10 ml of the mixture, toluene, 95 ml; ethanol, 5 ml; 2,5-tlil~l~c~~ylosazoIr, 0.5 g; p- bis[2-(4-~~~ctl~~1-5-~~he~~ylosazol~l)]bcnzc~~~~, 0.01 p. Chromato- grams on formaldehyde-treated l)al)cr autl other labeled lipid l)rel)aratiolls \vere cou~lted in a systcln n-hi& \\-a~ similar but which contained II!-amine hydrosidc (1). +%qucous solutions were counted in the latter systein or ill a tliosaii~-llictli!-1 Cello- solve s01vc11t (28).
lllit~~h~~&id ~1 TI’ Synthesis-‘l’hc convrrsion of “Pi to 321’-A1’l’ was measured hr the mcthotl of Nielscll and Lehningcr (29). \\l~cn hcsokiuax and glucnx wcrc adtlcd, decreased lipid labcliua was observed (1). l’hcxir omission did not result in an observed loss iu organic l)hosl)hatc, l)rovitlin~ the trichloracetic acid extract was assayed wit,hin 1 hour. ‘l’hc aqueous layer autl the ul)l)cr organic phase were cormted by liquid scintillation. The upper phase was dried uudcr nitrogen ill the presence of a few drol)s of l’rimcnc 81-R (30)) whirl1 lxevcutctl l)rcc.il’itation of the l’hosl,homol\-btlatr c~oml~lcs. ‘I’hc aqueous layer was also neutralized with Primeue lxior to counting. When the acid- soluble fraction was examined by l)al)er clcctrolhorcsis (18), labeled l’i, :YI’l’, and a third radioactive l)eak with the migration rate of l’l’i (npi = 2.1) was detected. The latter ileak accounted for 5 to ‘75~ of the radioactivity in the water-soluble fraction.
Other Alletkods-l’i was measured by the method of Bartlett, (31) or by the method of I~ercnblum and Chain (32, 33). Glu- cose 6-l)hosI)hatase activity was determined by the release of Pi from glucose 6-l,hosl)hate (34). I’rotcin was estimat,cd by the biuret mrthod (35). Other mcthotls ha\-c bccu tlrxribcd pre- viously (1, 8).
IL&action of Labeled Lipids-E’ivc-miilutc incubation mixtures coutaiuiug either “Pi or y-“T-.1’l’I’ were extracted by the method of 13ligh and Dyer (20) or by a modification in which the chloroformmethauol was acidified as described iu “Materials and Methods.” ‘l’wice as much radioactive product could be recovered from the acidified solvent fraction following iucubatiou with cithcr “l’i or y-“21’-hTl’ (l’ablc I). There was no evidence of ~oiltamilla.tion of the incubated sam1)k.s with nonlil)id radio- active material. Further extraction of the residue with the acidified sol!-cut did not yield addit ioual labeled material.
Requirements jar Incorporation of Radioactivity into Lipid- Considerably more iucorl)oratiou iuto lil)id from =l’i was seen in the guiuca l)ig system thau was rel~ortc~l by Garbus et al. (9) for the same iucubatioll cnonditions iu rat livcxr mitochontlria. This was due only iii ljart to an iml)roved yield of liljitl uljoii estrac- tiou (Table I). WC found guinea l)ig liver mitochondria about twice as active as rat liver mitochoutlria. Liver mitochondrial preparations from c~hicken, cattle, Ijig, aud rabbit were all less activca. ‘l’here was cousidemblc variation in amount of lillid la- beled both from “l’i aud y-““I’-hT1’. Table II rcl)rescnts a typi- cal exl)erimcnt. Rcl)licatc incubations from a given batch of mi- tochondria were geucrally relmxluciblc within 5 to 10%. The amounts of lm)duct formed are based on the sl)ecific activity of the added labeled AU or Pi. Since no correction was made for misiug with endoaeuous l~~ools, the values here relnxscnt minimal iucorl)oration. Addition of unlablrd .\‘I’l’ (0.1 mar) decreased by nearly 40% the labeling from both y-“‘I’-;\‘1’1’ aud “Pi, but addi- tiou of unlabeled Pi (0.1 nix) inhibited only slightly the incor- poration of radioactivity from y-“*I’-A’l’l’ autl strongly inhibited
Issue of April 10, 1968 A. K. Hajra, E. B. Xeguin, and B. W. Agyanoff 1611
labeling from 32Pi. It can be seen that deletion of Mg++ or addition of EDTA or Ca++ inhibited labeling from either sub- strate. Deletion of glutamate had no effect. Subsequently, glutamate was used only for longer incubations. No effect on incorporation of radioactivit’y was noted when the pH was varied between 7.0 and 8.5, but labeling was sharply reduced beyond these limits. Addition of unlabeled CTP, GTP, or UTP had little effect on incorporation of labeled ATP.
A further suggestion that Pi was converted to XTP prior to labeling of lipid was obtained from comparison of mitochondria stored under different conditions (Table III). It can be seen that storage of mitochondria at 2” or frozen at -20” for 24 hours
TABLE I
Comparison of neutral and acidic extractions of lipid jyom mitochondria
Each incltbation mixture contained MgClz (10 mM), Tris-HCl (20 mM, pH 7.4)) potassium glut,amate (10 mM), sucrose (100 mM), and mitochondria from 0.5 g of guinea pig liver (7 mg of protein), in a final volume of 1.2 ml. After incubation for 3 min at 30”, 0.1 ml of either a2Pi (0.12 pmole, 5.6 X lo6 cpm) or +P-ATP (0.3 pmole, 1.5 X 10” cpm) was added and the final mixture was incu- bated at 30” as indicated. In zero time tubes, extracting solvents were added immediately after addition of the radioactive sub- strates. For neutral extraction, 4.5 ml of chloroform-methanol (1:2) were added to a 1.2.ml incubation mixture, followed by 1.5 ml of chloroform and 1.5 ml of 0.5 M potassium phosphate buffer, pT1 7.4, containing 2 M KCl. After mixing and centrifugation, a portion of the lower layer was withdrawn. The acidic solvent extraction method is described under “Materials and Methods.” Lipids were washed with acidic solvents in both cases.
Radioactive precursor Extraction method Length of Radioactivity in incubation lipid extract
12eqGrement.s for incorporalion of radioactivity from 32Pi or r-32P-ATP into mitochondrial lipid
Following preliminary incrlbation of mixtures as described in the legend to Table I, 0.1 ml of either “Pi (1.2 X lo7 cpm; 1.0 mM) or Y-~~P-ATP (7.6 X lo6 cpm; 0.1 mM) was added, followed by incubation for 5 min at 37”. Lipids were extracted, washed, and counted as described under “Materials and Methods.”
System
Labeled lipid
From “Pi From +WATP
Complete Complete (zero time). WithoutMg++..................... Withollt glutamate. _. With ?JagHz-EDTA (10 InhI). With CaC12 (10 mM)
ppWL&S
1561 8
310 1592
290 352
T.IBLE III
Effect of storage of mitochonclria on incorporation of labeled precursors into 32P-lipid
Mitochondria were prepared from guinea pig liver. One por- tion was used immediately (fresh) for incubation, another was stored at 2”, and a third portion was stored at -20” for 24 horlrs prior to incubation and extraction as described in the legend to Table II.
Labeled lipid Mitochondria
From 3:P, I’rom +“P-ATP
~pmolcs pgmoles
Fresh 450 Stored at 2” for 24 hours.. Stored at -20” for 24 hours..
3 / 1200 910
31 , 1048
TABLE IV
Eflects of inhibitors of oxidative phosphorylation on labeling of mitochondrial lipid
Conditions were as in Table II. The inhibitors, either in water or in 20 ~1 of 95y0 ethanol (dinitrophenol, oligomycin, and anti- mycin) were added to the mixtures and then incubated for 5 min at 30”. Radioactive substrates were added and the mitochondria were incrtbated further for 5 min. The total lipid was extracted and co\mted.
Addition and final concentration
None, Arsenate, 1OV x.. Azide, 5 X 1OV M.
Cyanide, 5 X 1OV M.
Atractyloside, 5 X 10dG NI Oligomycin, 2 pg/l.2 ml Antimycin, I rg/1.2 ml. Dinitrophenol, lop4 M.. _.
Labeled lipid
From “Pi From -/-=P-ATP
pl*n1ole p~moles
207 551 7 447
64 680 4 455 5 500
24 533 31 557
5 327
greatly reduced labeling from 32Pi but had relatively little effect on labeling from y?&ATP.
Effect of Inhibitors of Oxidative Phosphorylation-Addition of agents known to block oxidative phosphorylation to incubation mixtures generally blocked labeling of lipid from ‘*Pi but had little effect on incorporation from y-32P-ATP (Table IV). Only dinitrophenol significantly decreased labeling from T-~~P-ATP. The effects of various concentrations of atractyloside (Fig. 1) substantiate earlier studies (1). Low concentrations of atrac- tyloside (2.5 x 10e6 M; 0.35 mpmole per mg of protein) inhibited completely the labeling of lipid from 3’Pi with no more than 2Og, reduction in its conversion to labeled ATP. When y-32P-ATP was used as labeled precursor, very little inhibition of lipid label- ing was observed at 20 times greater concentrations of atractylo- side. The effects of various concentrations of oligomycin on both the inhibition of lipid labeling and water-soluble organic phos- phate (ATP) are similar (Fig. 2). However, while the inhibition of lipid labeling is almost complete (95%), the maximum inhibi- tion of ATP formation by oligomycin is 50%.
Characterization of Labeled Lipid-Whole lipid extracts of mitochondrial incubations were chromatographed on formalde-
1612 Rapid Labeling of Mitochondrial Lipids Vol. 243, No. 7
\ \
‘1 .----a.-, AT 32p
-----*--d .
.: & 60
c
Atractyloside concentratian~105 M
FIG. 1. Labeling of lipid and ATP from z2Pi by mitochondria in the presence of atractyloside. Incubation mixtures were as described in the legend to Table II, but glutamate was omitted and 2.6 X lo6 cpm (0.12 pmole) of s2Pi were added. Atractyloside was present during preliminary incubation. After the 5.min incubation, the reaction was stopped by the addition of 2.8 ml of 57, trichloracetic acid. Following centrifugation, lipid in the residue was extracted and counted. Radioactivity in organic phosphate (ATP) and the supernatant fract.ion was determined by the method of Nielsen and Lehninger (29). From the specific activity of added 32Pi, the amount of radioactive lipid and 32P-ATP formed was determined and compared with that in mixtures incubated without atractyloside. The control incubations contained, by calculation, 0.61 mpmole of s2P-lipid and 32.0 mpmoles of 32P-ATP.
\ ‘\ . ..0* .--A-- ----.--,,--AT+
----.
IO 3ZP-Lipid 0
I\ I I I I 0 12 4 6 13 20
,ug of ollgomycln added
FIG. 2. Labeling of lipid and ATP from “Pi by mitochondria in the presence of oligomycin (see Fig. 1).
hyde-treated paper, and the developed chromatograms were scanned for radioactivity. Three peaks were found (Fig. 3). The major radioactive peak, “A,” (60 to 70% of the total 32P)
corresponded in RF to authentic phosphatidyl-IP (RF, 0.35).
A rapidly moving peak, “C,” containing 10 to 15% of the total
radioactivity, corresponded in migration rate to many phos- pholipids which are not separated in this system, including phos- phatidic acid, cardiolipin, lecithin, and phosphatidylethanola- mine (RF, 0.85). A third radioactive spot, “B,” (RR, 0.65) represented 10 to 20% of the total radioactivity and migrated near carrier phosphatidylinositol (RF, 0.60) and phosphatidyl-
serine (RF, 0.64). Generally, 1 to 3% of the total radioactivity remained at the origin. Occasionally a shoulder was detected on the trailing edge of the phosphatidyl-IP peak. Fig. 4 shows the distribution of radioactivity following high voltage paper elec- trophoresis of the alkaline deacylation product of the total lipid obtained after incubation with yJ2P-ATP or 32Pi+ A small amount of radioactivity (10 to 1570) corresponded to carrier Pi and traveled 5 to 7 cm in 20 min. The major radioactive peak (RPi, 2.2 to 2.3) corresponded to 60 to 70% of the total radio- activity and migrated at the same rate as glycerophosphorylinos- itol phosphate obtained from hydrolysis of phosphatidyl-IP. The other major peak corresponded to glycerol 3-phosphate (RPi, 1.70), and represented 10 to 15% of the total radioactivity in the aqueous fraction. In order to see whether the deacylated
FIG. 3. Scan of a paper chromatogram after separation of mitochondrial phospholipid labeled by ra2P-ATP. The three radioactive peaks, “8, ” “B,” and “C,” are seen. The positions of various marker lipids are also shown: 1, phosphatidylinositol diphosphate; 2, phosphatidyl-IP; S, phosphatidylinositol; 4, phosphatidylserine; 5, phosphatidic acid; 6, phosphatidylcholine and phosphatidylethanolamine; 7, sphingomyelin and cardiolipin; and 8, fatty acids. 0, origin; SF, solvent front.
FIG. 4. Separation of labeled alkaline methanolysis product of lipids from a total s2P-labeled lipid extract by high voltage paper electrophoresis. Scans of radioactivity show three principal peaks, I, 11, and 111. When more than 5 mg of lipid were sub- jected to methanolysis, a minor radioactive peak (shown here as a dolled line) was occasionally seen. It has not been identified. The positions of methanolyzed products of glycerophosphatides are shown: 1, glycerophosphorylcholine; 8, glycerophosphoryl- serine; 3, Pi; 4, glycerophosphorylinositol; 5, glycerol 3-phos- phate; 6, glycerophosphorylinositol phosphate; and 7, glvcero- phosphorylinositol diphosphate.
Issue of April 10, 1968 A. K. Hajra, E. B. Seguin, and B. W. Agmnqf 1613
900
rA From Y-3’P-ATP . . P * From 32P. TABLE T7
E$ects of various agents on labeling of individual lipids by yd=P-ATP
The incubation mixtures contained MgClz (10 mu), Tris-HCl (20 mM, pH 7.4), sucrose (100 IBM), mitochondria from 0.5 g of guinea pig liver (7 mg of protein), and inhibitors. Each inhibitor was neutralized to pH 7.4 with NaOH and was 5 rnM in the incuba- tion mixture. The mixture was incubated for 3 min at, 37” and then -,J-~~P-ATP (5 X 10” cpm; 0.2 pmole) was added and the final contents (1.2 ml) were incubated for 10 more min at 37”. Lipids were extracted, washed, and separated by paper chromatography.
1 +P-ATP incorporated into
Addition Phosphatidyl-IP
ppn7oles
None
NHsOII. CN- . F-.......... Semicarhazide .’ Hgdrazine Iodoacetate. Bisulfite.. I
537 400 GO4 033 701 G65 985 660
- Lipid B Phosphatidic
acid
~fiflzoles ~p??dfX
286 220 50 110
115 88 403 161 210 191
86 123 49 75
92. 78
FIG. 5. Time course of labcling of phosphatidyl-TP, Lipid R, and phosphatidic acid from rl, r”‘-P-A’TP (0.2 pmole; 3 X 107 cpm) and B, 3’Pi (0.12 @mole; 4.2 X lo7 cpnl) in mitochondtia. The inc\lbation conditions and lipid extraction were as described in the legend to Table II.
products of alkaline hydrolysis corresponded to the specific lipid peaks seen on chromatography, individual labeled lipids sepa- rated on formaldehyde-trcatcd paper were isolated. After the radioactive spots were located by scanning, each labeled lipid was eluted with chloroform-methanol-water (86 : 14: 1) (22) which was made 0.1 K with HCl. The acid was removed from the eluate by washing with methanol-water (I: I). Each radioactive lipid was subjected to alkaline methanolysis followed by electrophore- sis. The radioactivity in lipid A could bc recovered (90 to 100%) in a wat#er-soluble product which migrat,ed with glyccrophos- phorylinosibol phosphate. The water-soluble labeled product of A also migrated with glycerophosphorylinositol phosphate on paper chromatography with isopropyl alcohol-ammonia-water (6:3: 1) as the developing agent (36, 23). These results sup- ported the evidence that lipid -4 was phosphatidyl-II’. Simi- larly, on alkaline methanol+, lipid C became completely water soluble and the hydrolysis product migrated with glycerol 3- phosphate on elcctrophoresis and on paper chromatography. It was apparent that lipid C was phosphatidic acid. Wheat germ phosphatase (phosphomonoesterase) completely converted the water-soluble product of lipid C, as well as that of lipid A, to “Pi, indicating that, in each instance, phosphate was attached to an organic moiety by a monoester bond. On alkaline methanolysis, the radioactivity in lipid B was converted to “Pi. The properties of this lipid did not correspond to those of any known phospho- lipid. Its subsequent identification as acyl dihydroxyacetone phosphate is dealt with in the following paper (37).
Rflects of Other Inhibitors-When the lipid products following incubation with the various inhibitors (Table IV) in the pres- ence of T-~~P-ATP were examined, there was no marked diminu- tion in any of the three lipids. In contrast, several agents showed differential effects (Table V). Agents which react with carbonyl groups inhibited the formation of lipids B and C (phos- phatidic acid), with little effect on the labeling of lipid A (phos- phatidyl-IP) Fluoride appeared to stimulate labeling, pre- sumably because of the inhibition of mit,ochondrial adenosine triphosphatase (38).
Because of t,he unusual susceptibility of lipid B to dilute alkali,
T.\BLE VI
Eflect of hytlroxUla?nine on labeling of mitoctzondrial lipid
Experimental conditions were as in Table C. NHzOH-HCl (neutralized to pH 7.4 with Tris) was added to incubation mix- tures either 3 min before or immediately after the 5.min incuba- t,ion with labeled precursors. When NH2013 was added after the incubation, the reaction was allowed to proceed for 10 min more before solvent was added. -
Labeled lipid
From JZP i From +P-ATP NHzOII addition _I-
Phos- phatidi,
acid
Phos-
phai%dy’- Lipid B
Phos- phatidic
acid P
Lq.Lmoles
Experiment 1 None........... 1 mM, 3 min be-
fore 32P 5 m&f, 3 min be-
fore azP. 10 InM, 3 min be-
fore 32P. 10 mM, 5 min
after 32P Experiment 2
None........... 1 mu, 3 min be- fore 32P. 1 mM, 5 min after
3ZPG 5 mM, 5 min afler
=Pa None&.
408 92 48
240 15 25
128 8 16
110 7 13
168
19
20
14
41
190
38
167
185 181
71
60
52
32
98
131
104
136
118 128
598
680
1071
1122
532
523
531
521
506 511
- - a After 5 min of incubation with Y-~~P-ATP, the mixtures were
heated at 100” for 10 min, cooled, and incubated with or without NHzOII at 37” for 10 min more.
1614 Rapid Labeling of Mitochondrial Lipids Vol. 243, I\To. 7
TIRLF VII
Phosphorylation of lipid substrates !I$J y32P-A?‘P in mifochondria
Mixtures were incubated as described in the legend to Table Jr. After preliminary incltbation with added lipid substrates for 5 min, y-32P-ATP (0.12 rmole; 2.5 X 10” cpm) was added and the samples were incubated further for 10 min at 37”. Lipids were extracted, washed, and separated by paper chromatography.
Lipid added
Xone Phosphatidylinositol
In ethanolQ. In aqueous emulsion”
Palmitoyldihydroxyacetone In ethanola. _. In aqueous emnlsiollh
a Lipid substrate (0.5 mg) was added in 0.05 ml of ethanol. b Lipids were emulsified in water by sonic oscillation (“Son-
fier,” Branson Instrlunents); 0.5 mg of lipid sltbstrate in aqueous emulsion was added to each incubation mixture.
c FZach lipid (0.15 mg) was added in aq\leous emrdsion.
Labeling of lipid in different jraction,s of liver homoge,naie
I Labeled lipid !
Fraction Glucose
I Specific 6.phosphstase
Total activitya activity
1,SOOXy.. ........... 1 550 36 1 3.50 II, 12,000 x g .......... 1300 260 0.01 III, 100,000 X Q ...... ./ 800 271 13.00
y Calcldated from fractions obtained from 1.0 g of liver, wet weight.
* Micromoles of Pi liberated in 15 min per mg of protein.
studies on its formation and stability in the presence of hydroq-I- amine were performed. When low concentrations of hydrosyl- amine were present in the incubation mixtures, the formation of B was depressed with little effect on phosphatidyl-II? or phos- phatidic acid (Table VI). Higher concentrations appeared to stimulate phosphatidyl-II’ labeling from y-32P-ATl’. Hydrosyl- amine added following incubation also resulted in decreased amounts of labeled B. This was always accompanied by an in- crease in phosphatidic acid. In order to establish whether hydroxylamine reacted directly wit’h t’he B produced in the in- cubation, experiments were performed in which the incubation mixture was boiled prior to the addition of hydrosylamine (Table VI, Experiment 2). Heating did not destroy B and, further, prevented the decrease in B seen when hydroxylamine was added to unheat,ed enzyme preparations. These results
indicate that hydrosylaminc does not destroy B OIKC it is formed
under the conditiorls of the incubation. The possibility that B was au acyl phosphate was investigated by the addition to incu- bation mixtures of a nonspecific acyl phosl)hatase (39) and, in the other experiments, by the addition of acctyl phosphate (I mu) as a possible competitor of endogenous acyl phosphatase. Neither
of these additions, however, affected the yield of B. Tzrne Course of Lipid For?~atio?z-In an attempt to further
elucidate a possible interrelatiollshil) among the three labeled lipids, mitochondria were incubated with ‘*Pi or y-R2P-AT1’ for varying times (Fig. 5). All t,hree lipids were more rapidly labeled with ATP, but the greatest diffcrcncc was seen with 1)hosl)hatidic acid. Although considerable variability was seen, Fig. 5 rq- resents typical results. Lipid B reacshes maximal labeling from either precursor in 5 to 10 min.
Ej’ect of Addition of Possible Lipid Substrates jar Plzosphoryla- tion-Experiments were undertaken to see whether the dephos- phorylated Forms of the three labeled lipids would stimulat,e incorporation of radioactivit,y from y-32P-;ZTl’ (Table VII). Addition of phosphatidglinositol, palmit~oyldihydroxyacrtone (37), and diolein increased the labeling of phosphatidyl-IP, lipid B, and phosphatidic a&i, rcspertively, over the endogenous rate.
Subcellular Localization oj Lipid-labeling &tivity-Guinea pig liver was homogenized and then scparatctl by ccntrifugatiou into three fractions: the residue aedimenting at 800 X g, a frac?ion sedimenting at 12,000 X g (mitorhondrial fraction), and one sedimenting at 100,000 X g. Each frackion was washed twice with 0.25 M sucrose and iucubated as described under “1Iattrials and Methods” with y-““l’-A\‘l’P. Jlicrosomal contamination of mit’ochondria was checked by determination of glucose 6-l)hos- phat’asc (40, 41). The results (Table VIII) indicate that t,he mitochondrial fraction containttl most of the lipid-labeling ac- tivity, although the micsrosomal frac+ion had a similar specific activity. The major labeled lipid in each of these experiments was phosphatidyl-II’.
Lipzd Labeling in Xitochondria J?OII~ Various Organs-The nature of the radioactive lipids formed following 5-n& incsuba- tions with mitochondria from different organs with y-:‘“I’-hTP was determined. Mitochondria from guinea pig heart, kidney, and brain were prepared as described for liver and incubated under the Sam? conditions. Each incubation colrt,aincd mito- chondria from 0.5 g of tissue. Coinl)arcd t,o lirrr initocholidria,
brain incorporated 63 74 as much 321’, kidne\- incorl)orated 46 y0 and heart mitochondria only 8 qi,. On chromatographie scpara- tion, the major radioactive l~hoq~holil~itl from brain and kidney mit,ochondria was phosl)hatidyl-11’. I’hosphatidic arid was dctccted in kidney and brain, but labc,led lipid H was not dc- tccted in any of t,he three other organs.
‘*l’i Compared with y”“F.4 TP as I’hosphorylating Jgent- From t’hc degree of labcling as well as from tlilut,ion cspcrimcnts with unlabeled Pi, it is apl)arent that .Yl’l’ is the more tlirect phosphorylaCng agent for the labcling of all three lipids. The conclusion that “‘Pi is converted t,o r-:“I’-i\Tl’ by mitochondria and is then used for phosl)hor?laticln is supported by (a) the higher perccutage incoq)oratioll of 7.“‘P-=\‘l’P in brief incuba- tions, (b) the finding t,hat st,oretl mitochondria lose the ability to incorporate a2Pi into lipid without affecting labcling from Y-“~P- A1’IY~ (Table II), and (c) the obsc,rvation that addition of an
Issue of April 10, 1968 A. K. Hajra, E. B. Xequin, and B. MT. Agranqf 1615
ATP trap, such as hexokinase plus glucose, inhibits the formation of labeled lipid (1). Inhibitors of oxidative phosphorylation at concentrations which inhibit the incorporation of 3ZPi into ATP also inhibit the incorporation of 32Pi into lipid (Table IV). ht these concentrations, these inhibitors do not significantly decrease the incorporation of radioactivity into lipid from y-32P-ATP.
Ej’ect of I7~hibitors-~~t,ractyloside is a potent inhibitor of phoaphorylation by “I’i (Fig. I). At a concentration which completely inhibits the labeling of the lipid from 32Pi, little inhibition of conversion of 3zPi to “VATP was found. A similar phenomenon was observed by Noret et al. (42) in studies on the effects of atractyloside on the phosphorylation of protein by mitochondria. In the present, experiments, atractyloside did not inhibit labeling of lipid by y-“YATP. Acccrding to current hypotheses, atractyloside either inhibits the transport of adenine nucleotides across bhe mitochondrial membrane (43, 44) or blocks the exchange of free nucleotides with a bound form in the inner mitochondrial membrane (45). In eit,her event, it would appear that the lipid phosphorylation system described here is at a different, site than the one in the inner membrane for oxidative phosphorylation (46), most probably in the outer membrane of the mitochondria. Atractyloside then inhibits access of endogenous hTP to the site of lipid phosphorylation without affecting the labeling of lipid or oxidative phosphorylation. Recently, Richer and Royer (47) confirmed our report, on the effect of atractyloside on lipid labeling (1) and reached a similar conclusion. The finding that’ various lipid substrates are phosphorylated by intact mitochondria (Table VII) supports the idea that the labeling system is readily accessible, as would be expected for an outer membrane site.
In contrast t,o the report, of Garbus et al. (9), and in agreement with the observation of Galliard, Michell, and Hawthorne (ll), oligomycin inhibited the incorI)oration of “Pi into lipid. This result suggests that a high energy intermediate of oxidative phosphorylation is not involved in the lipid labeling. At very low concentrations, oligomycin stimulates the labeling of lipid by 32Pi. Low concentrations of oligomycin have also been shown to stimulate oxidative phosphorylation (48). This effect was also seen in the present study (Fig. 2). The bimodal nature of the effects of oligomycin may explain the reported variable effects of oligomycin on the incorporation of 32Pi into lipid. IJnlike the effect of atractyloside, the results with oligomycin cannot be explained on the basis of compartmentalizat,ion of labeled pre- cursor (1, 49).
Uinitrophenol affected most markedly the “Pi incorporation into lipid, but, a decrease in incorporation from y-rLP-ATP was also seen, as might be espect’ed from stimulation of mitochondrial adenosine triphosphatase activity. The effect of hydroxylamine is complex. It low concentrations it markedly reduced forma- tion of lipid B without decreasing labeling of phosphatidyl-IP or phosphatidic acid. Further, it did not affect B chemically, but appeared t)o affect its enzymatic synthesis. B might be formed by acylation of a 321’-labeled precursor with fatty acyl cocnzyme A. It would be expected that hydroxylamine would decompose the thioester at the pH of the incubation. The latter hypothesis does not appear to be supported by the effect of hydrosylamine on phosphatidic acid formation. Labeling of phosphatidic acid should also be blocked by the lowering of fatty acyl coenzyme -4 concentration. In fact, at higher concentra- tions of hydroxylamine, labeling of phosphatidic acid by Y-~~P- ATP appears to be stimulated. If, however, phosphatidic acid
were labeled by the combined action of phosphatidic acid phos- phatase and diglyceride kinase, we would not expect’ hydroxyl- amine t,o have an effect. .Uternatively, hydroxylamine could block the synthesis of lipid B by making a precursor unavailable for phosphorylation. These studies suggested to us that lipid B might, have a carbonyl group. Other carbonyl reagents also appeared to block the formation of B (Table V).
Xubcellular Site of Phosphatidyl-IP Labeling-Although we have proposed that phosphatidyl-IP labeling occurs in the outer mitochondrial membrane, it is also possible that a contaminant, of the mitochondrial fraction is responsible for the labeling. Michell and Hawthorne (50) showed that when exogenous phos- phatidylinositol was added to incubation mixtures of subcellular components, more phosphatidyl-II’ was formed in microsomal and in cell membrane fractions than in mitochondria, although the latter were also labeled t,o a considerable extent. It’ should be mentioned, however, that the addition of an insoluble or poorly soluble substrate to a particulate enzyme is quite a different matter t,han t,he reaction bet,ween an enzyme and a substrate reacting within the same membrane or particle. Colodzin and Kennedy (12) found that as the concentration of added particu- late enzyme was increased, microsomal fractions were increas- ingly active in the phosphorylation of added phosphatidyl- inositol, while mitochondrial fractions became less effective. The present study (Table VIII) indicates that, in guinea pig liver, most of the endogenous activity for labeling phosphatidgl- IP from y-32P-A’I’I’ is in the mitochondrial fraction. A similar finding in brain mitochoudria has been reported (51), although phosphatidyl-IP labeling has been reported in cell membranes (13, 50), myelin (51), and microsomes (la), as well as in mito- chondria (51, 47, 50). It is possible that the phosphatidyl-IP labeling system is a common property of membranes of higher animals in general.
Mechanism of Rapid Labeling of Mitochondrial Lipids and T&r Possible Ifunction-The three lipid fractions which are rapidly labeled in mit,ochondria have in common t,hat they are present in only trace amounts. They therefore do not reflect membrane synthesis de nouo during this period of incubation. In addition, on the basis of inhibitor studies, none of them ap- pears to be involved in oxidative phosphorylation. Phospha- tidyl-II’ and phosphat,idic acid have in common that the labeled moiety is a phosphomonoester, in contrast to the phosphodiester seen in most phospholipids. WC have learned that lipid B is also a phosphomonoester (37). Thus the rapid labeling could reflect a combination of phosphomonoesterases and kinases which to- gether cause a rapid turnover of all the lipid monoester phos- phates. Evidence for t,he presence of kinase activity is shown in Table VII. Phosphatases in various cell fractions have been described for phosphat,idyl-IP (12, 52) and for phosphatidic acid (53-55).
This report is further evidence for a system which labels phoaphatidyl-II’ as well as ot)her lipids. This labeling appears to be a property of all membranes t’hus far studied. Present studies are directed at its possible physiological significance.
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