Abstraet--l. NADH-cytochrome b 5 reductase from hog gastric microsomes was studied with respect to substrate dependence, optimum pH, thermal denaturation as well as anti-cytochrome b 5 antibodies and different ions.
2. The reduction of potassium ferricyanide by the enzyme was specific for NADH. 3. Using potassium ferricyanide or trypsin-solubilized liver cytochrome b5 (Tbs) as substrates, enzyme
activity was inhibited by ADP and to a lesser extent by ATP. 4. Tb 5- (but not ferricyanide-) reductase was activated by ionic strength up to 0.05 ion equivalent per
liter and inhibited at higher strengths whatever the ion used (CI-, Na ÷, Ca 2+, Mg2+). 5. Enzyme solubilization occurred with Triton X100. The solubilization increased the Tb 5- (but not the
ferricyanide-) reductase activity up to a Triton:protein ratio of 15. 6. We therefore suggest that gastric microsomes contain a Triton soluble membrane-bound NADH
cytochrome b 5 reductase which is in many respects similar to the liver and red cell enzymes.
INTRODUCTION
Cytochrome b :dependen t oxido-reduction systems are well documented in liver (Strittmatter and Velick, 1956) and red cell (Passon and Hultquist, 1972; Abe and Sugita, 1979). Reportedly, they involve a specific reductase (EC 1,6.2.2) which is found in either a membrane-bound form (endoplasmic reticulum (Re- macle et al., 1976) and outer mitochondrial mem- brane (Sottocasa et al., 1967) of liver and plasma membrane (Choury et al., 1981) of erythrocyte) or in a soluble form [cytoplasm of erythrocyte (Passon and Hultquist, 1972)]. Tissue-dependent acceptors of elec- trons transferred by the reductase from N A D H to cytochrome b5 include fatty acid desaturases (Oshino, 1978; Enoch et al., 1976; Lee et al., 1977), elongase (Keyes et al., 1979), cytochrome P450 (Bonfils et al., 1981), methemoglobin (Passon and Hultquist) and metmyoglobin (Hagler et al., 1979).
In gastric mucosa, microsomal oxido-reduction systems were postulated to occur and to be implicated as key (Davies and Ogston, 1950) or complementary (Sachs et al., 1976) elements in the process of H ÷ secretion. We previously reported the presence of a cytochrome b5 in gastric mucosal slices (Ghesquier et al., 1980), microsomes and isolated cells (Lewin et al., 1978). In the present study we characterize a cyto- chrome b : l inked NADH-dependen t reductase in hog gastric microsomes using ferricyanide, cytochrome c and Tb5 as substrates, and furthermore, we report solubilization of this enzyme by Triton X100.
MATERIAL AND METHODS
Preparation of microsomal fraction
Homogenate was prepared from fresh hog (Sus scrofa) stomachs obtained at the slaughter-house (Olida, Levallois, France) and microsomal fraction was obtained by
differential centrifugation as previously described (Sou- marmon et al., 1980).
Preparation of trypsin-solubilized cytochrome b 5 ( Tbs) Tb~ was prepared from hog liver microsomes by trypsin
proteolysis according to the method of Omura and Takesue (1970) and purified as previously described (Ghes- quier et al., 1980), with however, the following modification added to eliminate lipids: the trypsin solubilized supernatant (500ml corresponding to 750g of hog liver) was mixed slowly with 200 ml of DE 52 resin equilibrated with 10 mM potassium phosphate buffer (pH 7.4). The mixture was decanted for 30 min at 4°C and the supernatant was discarded. The resin was then washed twice with 500 ml of 10mM potassium phosphate buffer (pH 7.4) and filtered through sintered glass. The Tb5 was released from the resin by incubating for 30 rain at 4°C with 150ml of 1M potassium chloride-50 mM potassium phosphate buffer (pH 7.4). Tb 5 was then recovered by vacuum filtration on sintered glass, lyophilized, dissolved in 30ml of distilled water and dialyzed overnight against 31 of 10 mM potas- sium phosphate buffer (pH 7.4). Purified Tb 5 was titrated by spectrophotometry at 412.5 nm using an extinction coefficient of 117 cm-t mM- 1 (Omura and Takesue, 1970).
According to Enomoto and Sato, Tb~ purified in this manner assumedly contains the catalytic site of native cyto- chrome bs, but lacks the site for membrane attachment (Enomoto and Sato, 1973).
Assay of NADH cytochrome b5 reductase activity
Three distinct substrates were used for this assay: hog liver microsomal Tb 5 prepared as described, and two artificial electron acceptors widely used in the literature, namely, potassium ferricyanide and cytochrome c.
NADH-Tb 5 reductase activity was estimated as the reduc- tion rate of Tb 5 in the presence of NADH from the NADH-reduced minus oxidized difference spectrum. The reaction was run in a double cuvette. Each compartment of the cuvette contained 700#1 of the following mixture: 10#M Tb5, 4#M rotenone, 100mM Tris-acetate buffer
165
166 D. GHESQUIER et al.
(pH 6.5), and 10pl of gastric microsomes. After equi- libration at 25°C, the reaction was started by adding 0.1 mM NADH in one compartment and followed by recording the differential absorption from 405 to 440 nm. The specific absorption of reduced Tb 5 was calculated as the difference of absorption at 424 and 409nm with an extinction coefficient of 100cm -~ mM -~ (Strittmatter and Velick, 1956; Strittmatter and Velick, 1957).
NADH-ferricyanide reductase activity was assayed ac- cording to Takesue and Omura (1970) by measuring the absorption decrease rate of potassium ferricyanide at 420 nm. The cuvette contained 2 ml of 1 mM potassium ferricyanide, 2/~M rotenone, 100 mM Tris-acetate buffer (pH 6.5), and 10pl of gastric microsomes. After equi- libration at 25°C, the reaction was started by addition of 0.3 mM NADH. Controls for spontaneous NADH reduc- tion of ferricyanide were performed under similar conditions but without microsomes. The extinction coefficient for ferricyanide was taken as 1.02cm ~ mM -~ (Takesue and Omura, 1970).
NADH--cytochrome c reductase activity was determined as described (Ghesquier et al., 1980) by measuring the reduction rate of cytochrome c at 550 nm.
Proteins
Proteins were measured either by the method of Lowry et al. (1951) or using the Coomassie blue reagent (Bradford, 1976) with bovine serum albumin as standard.
Immunological studies
Tb 5 antibodies were raised in rabbit, purified according to Fowler et al. (1976) and titrated by the Farr's method (1958) as previously reported (Ghesquier et al., 1980). To study the effect of antibodies on the NADH-cytochrome c reductase activity, microsomes were pre-incubated 15 min at 25°C with purified Tb s antibodies.
Chemicals
NADPH, NADH, rotenone, cytochrome c, Triton X100, ADP and ATP were purchased from Sigma Chemical Co. (St. Louis, USA); pronase and potassium ferricyanide from Merck A.B. (Darmstadt, FRG). Sephadex Gl00 was ob- tained from Pharmacia (Uppsala, Sweden), DE 52 from Whatman Ltd. (Maidstone, UK) and Coomassie blue from Bio-Rad Lab. (Richmond, Ca. USA).
RESULTS
Character i za t ion
Substrate dependence of gastric N A D H cyto- chrome b5 reductase was studied using purified Tb 5, potassium ferricyanide and cytochrome c as electron acceptors. With these three substrates, maximal specific activities were found to be, respectively, 103.8 + 5.1, 2,680 + 300 and 54 .4_ 2.5 nmoles per rain per mg of microsomal proteins (mean values _-t- SEM from 15 experiments).
Freezing and thawing the microsomal preparation notably increased enzyme activity (2.2 fold) in the presence of Tb5 ("Tbs-reductase") activity but had relatively little effect (1.2 fold) in the presence of ferricyanide ("ferricyanide-reductase" activity) (data not shown).
Tb5 antibodies inhibited cytochrome c reductase activity (46~o) but did not affect the ferricyanide reductase activity (Fig. 1).
pH opt imum for maximum activity were between 5.5 and 8.5, and between 5.5 and 7 pH units for, respectively, ferricyanide and Tb5 (Fig. 2).
Fig. 1. Effect of increased concentration of Tb 5 antibodies on ferricyanide ((3--(3) and cytochrome c reductase (O- -O) activities of hog gastric microsomes (see Materials
and Methods). Mean values from three experiments.
Thermal denaturat ion of enzyme occurred beyond 37°C regardless of which substrate was used (fer- ricyanide or Tbs) (Fig. 3).
Enzyme reaction was apparently specific for NADH. At 0.1 mM concentration, N A D P H substi- tuted for N A D H was unable to promote reduction of ferricyanide or Tbs; l0 times higher concentrations caused a 50~o reduction of both substrates. Tb 5 and ferricyanide reductase activities were inhibited by 20-30~ in the presence of 2 mM ADP and by 7-90.o in the presence of 2 mM ATP.
Ionic s trength. Stimulation of Tb5 reductase was obtained by increasing ionic strength to an optimum of 0.05 ion Equiv./1. Inhibit ion occurred at ionic strength higher than optimum. These effects showed no apparent ion specificity since K ÷, Na ÷, Mg 2÷ and Ca 2+ behaved similarly (Fig, 4). In contrast, variation of ionic strength did not affect ferricyanide reductase activity.
Solubi l i za t ion by Tri ton X l00
Addit ion of Tri ton X100 in the reaction mixture slightly increased ferricyanide reductase activity ( × 1.4), but markedly stimulated Tb5 reductase activ- ity ( × 2-3). This stimulatory effect was a function of
lOO
\
6 8 10 pH
Fig. 2. pH dependence of ferricyanide-reductase (O- -O) and Tbs-reductase (0 - - (3 ) activities of hog gastric micro- somes. Kinetic of assays were recorded as described in
Material and Methods, in 100 mM Tris-acetate buffer.
Fig. 4. Effect of ionic strength on gastric Tbs-reductase activity. Different ionic strengths were obtained using vari- ous concentrations of KC1 and MgC12 in the presence of 10mM or 100raM Tris-acetate buffer (pH 6.5). Filled and open symbols represent Triton-solubilized and membrane- bound enzyme, respectively. (,~., 'A') 10raM "Iris; ((>, @) 10raM Tris + KCI; ([~, m) 100raM Tris; (A, A) 100raM
Tris + KCI; (O, @) 100 mM Tris + MgCI 2.
i
1
2oc
leo
5 10 ?5 2O 25 Triton : ~ e i n ratio
Fig. 5. Effect of Triton XI00 on gastric Tbs-reductase activity. Hog microsomes were diluted in 100 mM Tris-HCl buffer (pH 7.4) at concentrations ranging from 0.024 to 0.28mg protein/ml and preincubated before assay for
30min at 0°C in the presence of I% Triton X100.
the Triton: protein ratio, a maximum occurring for a ratio around 15 (w/w) (Fig. 5).
High speed centrifugation of microsomes treated by 1% Tri ton X100 demonstrated that N A D H cyto- chrome bs reductase was at least partly solubilized by the detergent. Thus, the 105,000g x 1 hr supernatant contained 32% of the ferricyanide reductase activity of Tri ton treated microsomes, with an enrichment of about 1.1, and 56% of Tb s reductase activity with a 2.7 enrichment (Table 1). In the absence of Tri ton X 100, no detectable amount of either reductase activ- ity was recovered in the supernatant.
Solubilization yield was a function of the Tri ton:protein ratio. For ratio of 0.5, 1 and 25 (w/w), percents of microsomal ferricyanide reductase solu- bilized by Triton treatment were, respectively, 32, 41 and 90%.
When microsomal Tb 5 reductase was solubilized (as 105,000g x l hr supernatant) using a low Tri ton:prote in ratio such as 0.5 (w/w), solubilized enzyme could still be activated upon further addition of Triton, with maximal activation corresponding to the same optimal Tr i ton:prote in as above (i.e. 15) (Fig. 5). When, however, solubilization was carried
Hog microsomes ( 16 to 22 mg protein ml- J) were incubated for 30 rain at 4°C in 100 mM Tris-HCl buffer (pH 7.4) containing 1% Triton XI00. Aliquots were then centrifuged at 105,000g for 1 hr. Results are mean values + 1 SEM from three experiments (nmol min/mg protein).
168 D. GHESQUIER et al.
out at a high Triton:protein ratio, such as 25 (w/w), no further activation of Tb 5 reductase was observed. Increasing ionic strength from 0.005 to 0.05 did not activate solubilized enzyme, whereas at higher con- centration it inhibited the activity as for native membrane-bound enzyme (Fig. 4).
DISCUSSION
At the concentrations used, potassium ferricyanide was the most effective of the three substrates tested. This is in agreement with previous reports on liver (Strittmatter and Velick, 1957; Mihara and Sato, 1972) and red cell (Choury et aL, 1981). The particu- lar efficacy of ferricyanide could be explained by its relatively small size (329 daltons) favoring its access to the enzyme. In contrast, Tb5 which is a larger molecule (12,500 daltons) and furthermore lacks the hydrophobic part of the native cytochrome bs, may have restricted accessibility. This would be consistent with the observation that Tb5 reductase activity was increased after freezing and thawing, a procedure known to disrupt microsomal vesicles, and after Triton treatment (as discussed below).
Activity of hog gastric microsomal enzyme was found equivalent to that of hog liver microsomes. The latter corresponded to 117.3 4- 7.2 and 2,930 + 300 nmoles per min per mg of protein, re- spectively, with Tb5 and ferricyanide as substrates (unpublished observations). Since we previously re- ported that gastric microsomes contain 8 times less cytochrome b5 than liver microsomes (Ghesquier et al., 1980), the present results might suggest that the gastric reductase could also function with other elec- tron acceptors. That Tb5 antibodies inhibited NADH cytochrome c reductase activity but did not inhibit NADH ferricyanide activity is consistent with the following scheme:
In this scheme, X represents a hypothetical electron acceptor in parallel with cytochrome b5 and X' a so far unidentified intermediate between cytochrome b5 and molecular oxygen.
The pH activation profile for gastric ferricyanide reduetase is similar to that of liver enzyme (Stritt- matter and Velick, 1956). It is not superimposable on that of NADH Tb5 reductase which shows a nar- rower peak as already reported for red cell soluble enzyme (Abe and Sugita, 1979). Like liver enzyme, gastric enzyme is specific for NADH and is inhibited in the presence of ADP, probably because of the relative similarity in the nucleotides' conformation.
Inhibition of Tbs- reductase at high ionic strength was previously described for red cell soluble enzyme (Passon and Hultquist, 1972). In the present study, we found that such an inhibition occurred on Triton-
solubilized enzyme and on membrane-bound enzyme as well. Activation of membrane-bound enzyme by low ionic strength, up to 0.05 ion equiv./l, is a new observation. This activation is likely not a con- sequence of enzyme solubilization because in gastric microsomes, as in red cell (Choury et al., 1981), ionic strength was unable per se to release any detectable activity in the 105,000g × 1 hr supernatant. Since the activation was no longer observed on Triton solu- bilized Tbs-reductase, it could be due to release from the membrane of extrinsic or adsorbed proteins, e.g. hemoglobin, serving as electron acceptor for cyto- chrome bs. The stimulatory effect of Triton XI00 on Tb5 reductase activity of gastric microsomes might be partly accounted for by the same phenomenon. How- ever, since addition of Triton XI00 to microsomes already activated by optimal ionic strength caused further enhancement of enzyme activity, it appears that solubilization of the enzyme (or of its catalytic moiety) results in increased efficacy of the reductase- Tb5 reaction. In this respect, Triton activation of gastric Tbs-reductase could be compared to the acti- vation of liver Tbs-reductase by lysosome cathepsin (Takesue and Omura, 1970) that is suggested to separate and release catalytic moiety of the enzyme from the membrane-bound hydrophobic tail. The finding that addition of Triton XI00 on solubilized enzyme caused further activation of Tbs-reductase (up to a Triton:protein ration of 15, suggests that enzyme is at first solubilized as a complex which can afterwards dissociate to a greater extent.
In conclusion, the above results support that gas- tric microsomes contain a NADH-cytochrome b~ reductase that is comparable in many respects to the NADH~zytochrome b~ reductases of liver and red cells. In particular, like liver (Mihara and Sato, 1972) and red cell (Choury et al., 1981) enzymes, gastric NADH cytochrome-b5 reductase is readily solu- bilized by Triton XI00 and the solubilization appears to increase its activity. Further studies are now required to characterize the subcellular origin of the microsomal membranes associated with gastric NADH-cytochrome b5 reductase. The possibility that such an enzyme system could also occur in soluble form, as recently suggested in red cell, placenta and liver (Passon and Hultquist, 1972; Leroux et al., 1977: Lostanlen et al., 1978), will be also examined. More- over, the nature of the cytochrome b~ oxidases func- tionally linked to the reductase in the gastric mucosa deserves further investigation in view of their possible role in the general process of acid secretion.
Very recently, when this paper was finished, Me- gias et al. (1983) reported the properties of the NADH-cytochrome b~ reductase from Cerat i t is cap- itata. These properties showed similarities between this enzyme and the enzyme from liver, red cell and gastric mucosa.
Acknowledgements--This study was supported in part by Grant ATP 74 79 106 from Institut National de la Sant~ et de la Recherche M~dicale. The authors gratefully acknowl- edge Dr. D. Blangy from the Institut de Recherche sur le Cancer (CNRS, Inst. Gustave Roussy, Villejuif, France) tbr helpful discussions during this work and Mrs. F. Pamart and M. Sauvadet for their help with the manuscript.
Microsomal NADH--cytochrome b5 reductase 169
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