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PubMed1977

Levels of acetyl coenzyme A, reduced and oxidized coenzyme A, and coenzyme A in disulfide linkage to protein in dormant and germinated spores and growing and sporulating cells of Bacillus megaterium.

Dormant spores of Bacillus megaterium were found to contain approximately 850 pmol of coenzyme A (CoA) per milligram of dry weight. Of this total, less than 1.5% was acetyl-CoA, 25% was CoA-disulfide, 43% was in disulfide linkage to protein, and the remainder was the free thiol. Dormand spores of Bacillus cereus and Clostridium bifermentans contained 700 and 600 pmol of CoA per milligram of dry weight, respectively; in both species approximately 45% of the CoA 45% of the CoA was in disulfide linkage to protein. During germination of spores of all three species, greater than 75% of the CoA-protein disulfides were cleaved. In B. megaterium, cleavage of these disulfides during spore germination did not require exogenous metabolites and occurred at about the same time as the initiation of germination. Much of the CoA was converted to acetyl-CoA at this time. Dormant spores also contained reduced nicotinamide adenine dinucleotide-dependent CoA-disulfide reductase at levels higher than those in other stages of growth. The level of total CoA in the growing cells was two- to three-fold higher than in spores. This level remained constant throughout growth and sporulation, but less than 2% of the total cellular CoA was in disulfide linkage to protein until late in sporulation. The CoA-protein disulfides accumulated exclusively within the developing spore at about the time when dipicolinic acid was accumulated.

Acetyl Coenzyme A↗
PubMed1983

Levels of sulfhydryls and disulfides in proteins from Neurospora crassa conidia and mycelia.

Proteins extracted with 6 M guanidine at 90 degrees C from conidia (asexual spores) of Neurospora crassa contained ca. 25% more total protein thiol and a fivefold-higher content of disulfide bonds than proteins extracted from mycelia, as determined by labeling with iodo[14C]acetic acid. The total thiol content was 88 mumol/g of protein in conidia and 70 mumol/g of protein in mycelia. The level of protein disulfide was 18.5 mumol/g of protein in conidia and 3.5 mumol/g of protein in mycelia, by the iodo[14C]acetic acid labeling method. Confirmatory results were obtained with 5'5-dithio-bis-2-nitrobenzoic acid titration of protein thiol groups in 1% sodium dodecyl sulfate as well as by amino acid analysis of cysteic acid derivatives. Buffer-extracted proteins from conidia, but not mycelia, were found to contain enriched levels of protein thiols and disulfides per gram of protein as compared with guanidine hydrochloride extracts. It was demonstrated that the high disulfide content of crude conidial extracts was not due to measurable levels of mixed disulfides formed between protein sulfhydryl groups and cysteine. During germination of the conidia, the high disulfide levels of the conidial proteins remained constant. These data suggest that, unlike the disulfides of glutathione, the bulk of conidial protein disulfides were not reduced, excreted, or extensively degraded during germination.

Amino Acids↗
PubMed2004

Bacillus subtilis StoA Is a thiol-disulfide oxidoreductase important for spore cortex synthesis.

Bacillus subtilis is an endospore-forming bacterium. There are indications that protein disulfide linkages occur in spores, but the role of thiol-disulfide chemistry in spore synthesis is not understood. Thiol-disulfide oxidoreductases catalyze formation or breakage of disulfide bonds in proteins. CcdA is the only B. subtilis thiol-disulfide oxidoreductase that has previously been shown to play some role in endospore biogenesis. In this work we show that lack of the StoA (YkvV) protein results in spores sensitive to heat, lysozyme, and chloroform. Compared to CcdA deficiency, StoA deficiency results in a 100-fold-stronger negative effect on sporulation efficiency. StoA is a membrane-bound protein with a predicted thioredoxin-like domain probably localized in the intermembrane space of the forespore. Electron microscopy of spores of CcdA- and StoA-deficient strains showed that the spore cortex is absent in both cases. The BdbD protein catalyzes formation of disulfide bonds in proteins on the outer side of the cytoplasmic membrane but is not required for sporulation. Inactivation of bdbD was found to suppress the sporulation defect of a strain deficient in StoA. Our results indicate that StoA is a thiol-disulfide oxidoreductase that is involved in breaking disulfide bonds in cortex components or in proteins important for cortex synthesis.

Anti-Infective Agents↗
PubMed2003

Disulfide-linked integrase oligomers involving C280 residues are formed in vitro and in vivo but are not essential for human immunodeficiency virus replication.

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication.

Cysteine↗
PubMed1985

Different susceptibility of inter- and intra-chain disulfide bonds to reductive cleavage in native fibronectin and effect of their cleavage on conformation.

The two thread-like subunits (Mr approximately equal to 250 000) of the multidomain protein fibronectin are connected by a pair of inter-chain disulfide bridges in their C-terminal regions. In addition each chain contains 29 intra-chain disulfide bonds which are located in 12 type I and 2 type II structural domains in the N-terminal and C-terminal regions of the strands. The 15 to 17 type III domains in the central portion of the strands do not contain disulfide bonds. The susceptibility of inter-chain disulfide bonds to 10mM 1,4-dithiothreitol at pH 7.8 as quantitated by the rate of reductive cleavage of fibronectin into its subunits was found to be only 8-fold larger than that of the intra-chain bonds. Consequently at 90% completion of chain separation 30% of the intra-chain disulfides are also cleaved. The rate of inter-chain disulfide cleavage was found to be identical for fibronectin and a 140-kDa fragment comprising the C-terminal portions of the two subunits. This shows that the relatively high protection of the inter-chain disulfide bonds must originate from interactions between C-terminal domains which are probably also responsible for the V shaped arrangement of the two subunit strands. Changes of circular dichroism and thermal transition profiles for fibronectin and its C-terminal 140-kDa fragment indicated that already partial reduction of the intra-chain disulfide bonds alters the conformations of type I and II domains without affecting the type III domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1995

The culture of rat lenses in high sugar media: effect on mixed disulfide levels.

Lens proteins are long lived proteins with those in the center of the lens predating the birth of the individual. As a result, they are subject to a host of modifications and damage through a variety of mechanisms. Two such modifications have been proposed as primary events which could cause conformational changes potentiating further modifications. These are non-enzymatic glycation and mixed disulfide formation. Human lenses accumulate protein-thiol mixed disulfides of three kinds throughout the lifespan. The presence of one of these, protein-glutathione (PSSG) mixed disulfide has been shown to be intimately involved in protein aggregation. We have utilized ex vivo lens culture and in vitro incubations of purified gamma-crystallin to evaluate the following hypotheses. A) Lenses cultured with a high sugar media will form higher mixed disulfide levels than controls; B) glycation of lens proteins will be dependent on initial mixed disulfide level. Xylose levels in the cultured lens rise rapidly (to 23 mM by 4 h), and the level of glycation after one week is elevated 6-7% over control values. Mixed disulfide levels are also substantially increased but not more than for lenses cultured in control media. gamma-Crystallin modified with 0, 1, or 5 equivalents of GSH was differentially glycated by radioactive fructose. The amount of fructose bound by the protein was found to be inversely related to the extent of mixed disulfide formation. These results indicate that 1) protein modification of one kind may influence further modifications of other types; 2) glycation of lens proteins has no effect on mixed disulfide formation in this system; 3) the sulfhydryl status of lens proteins can affect the potential for protein glycation.

Animals↗
PubMed2003

[The inhibitory effect of Lu-Duo-Wei on carbon disulfide-induced generation of hydroxyl radicals].

OBJECTIVE: To study the scavenging effects of Lu-Duo-Wei, thiourea, superoxide dismutase, and sodium azide on carbon disulfide-induced generation of hydroxyl radicals. METHODS: Phenanthroline-CuSO(4)-Vit C-H(2)O(2) chemiluminescence system (PHEN system) containing alcohol was established to probe the influence of various concentrations of carbon disulfide on hydroxyl radicals emission intensity and the scavenging effects of Lu-Duo-Wei and other antioxidants on carbon disulfide-induced hydroxyl radicals were observed. RESULTS: The average emission intensity of PHEN system containing alcohol appeared lower luminescence [91.03 x 10(3) (cp6s)] and longer time (75 s) to get the peak than the system without alcohol [96.11 x 10(3) (cp6s), 55 s]. The specific scavenger of hydroxyl radical, thiourea, showed clear inhibitory effect on the system. Carbon disulfide in the range of 40 - 160 mmol/L promoted the generation of hydroxyl radical, however, this effect could be efficiently inhibited by thiourea. 160 mmol/L carbon disulfide in PHEN system without copper seemed as an activator to promote the luminescence, while in PHEN system withdrawing phenanthroline appeared some weak action of luminescence agent at low concentration. Meanwhile, Lu-Duo-Wei may efficiently scavenge hydroxyl radicals induced by carbon disulfide in PHEN system but superoxide dismutase and sodium azide had little effects on the system. CONCLUSION: Carbon disulfide may induce PHEN system to generate hydroxyl radicals and Lu-Duo-Wei may efficiently scavenge these free radicals and play an important role in protection against oxidative injury induced by carbon disulfide.

Carbon Disulfide↗
PubMed1992

Protein disulfide isomerase activity is released by activated platelets.

The release of protein disulfide isomerase by activated platelets was hypothesized on the basis of reported intermolecular and intramolecular thiol-disulfide exchange and disulfide reduction involving released thrombospondin in the supernatant solution of activated platelets (Danishefsky, Alexander, Detwiler: Biochemistry, 23:4984, 1984; Speziale, Detwiler: J Biol Chem, 265:17859, 1990; Speziale, Detwiler: Arch Biochem Biophys 286:546, 1991). Protein disulfide isomerase activity, measured by catalysis of the renaturation of ribonuclease inactivated by randomization of disulfide bonds, was detected in the supernatant solution after platelet activation. The activity was inhibited by peptides known to inhibit protein disulfide isomerase; the peptides also inhibited formation of disulfide-linked thrombospondin-thrombin complexes. The reaction catalyzed by the supernatant solution showed a pH dependence distinct from that of the uncatalyzed reaction. The activity was excluded by a 50-Kd dialysis membrane, and it was eluted in the void volume of a gel-filtration column, indicating that it was associated with a macromolecule. The activity was not removed by centrifugation at 100,000 g for 150 minutes indicating that it was not associated with membrane microvesicles. Possible functions for the release of protein disulfide isomerase by activated platelets are discussed.

Blood Platelets↗
PubMed1992

Partially folded state of the disulfide-reduced form of human serum albumin as an intermediate for reversible denaturation.

The conformation of the fully disulfide-reduced state of human serum albumin was investigated by tryptophan fluorescence spectrum, CD analyses, and size-exclusion chromatography. Both the reduction of the native disulfide-bonded form under nondenaturing conditions and the refolding of the urea-denatured disulfide-reduced form under reduced conditions yielded almost exactly the same disulfide-reduced state with partially folded unique conformation that was clearly distinguished from either the native or fully denatured state. In addition, the interconversion between the urea-denatured reduced form and the partially folded reduced form was reversible with each other; by reoxidation, the partially folded reduced form was converted to the disulfide-bonded form. The conformation of disulfide-reduced serum albumin was highly variable depending on pH and ionic strength conditions. Thus, we concluded that the disulfide-reduced state with partially folded variable conformation is involved in the reversible interconversion between the denatured reduced form and the native disulfide-bonded form of human serum albumin.

Chromatography, Gel↗
PubMed1986

An extra disulfide bridge in the constant domain of Rana catesbeiana immunoglobulin light chains.

The immunoglobulins of the bullfrog Rana catesbeiana are unusual in that, in all classes, the light chains are not disulfide bonded to heavy chains or to other light chains. Moreover, the light chains contain six, rather than the usual five, residues of half-cystine. As none of these half-cystines is in the sulfhydryl form or is alkylated after mild reduction, we suggested that the light chains probably contain three intrachain disulfide bridges. We have now carried out experiments to confirm the existence of an extra intrachain disulfide bridge in Rana catesbeiana light chains and to determine its location. Disulfide bridge assignments were based on 1) isolation and sequence analysis of S-(carboxymethyl)cysteine-containing peptides and 2) isolation, from unreduced light chains, of peptides containing a disulfide bridge. Half-cystine residues were found at positions 134 and 194, and these were shown to be joined in the conserved intradomain disulfide bridge. In addition, we found that a residue of half-cystine, located at the third position from the carboxy-terminus, forms a disulfide bridge with a half-cystine at position 119, near the amino-terminus of the domain, the latter residue replacing a proline that has been found at this position in all other light chains. An intrachain disulfide bridge has not been found at this location in any other light chain.

Amino Acid Sequence↗
PubMed1987

Location of disulfide bonds within the sequence of human serum cholinesterase.

Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.

Acetylcholinesterase↗
PubMed1988

Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Identification of a specific interchain disulfide bond.

The catalytic (C) subunit and the type II regulatory (RII) subunit of cAMP-dependent protein kinase can be cross-linked by interchain disulfide bonding. This disulfide bond can be catalyzed by cupric phenanthroline and also can be generated by a disulfide interchange using either RII-subunit or C-subunit that has been modified with either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-4(azidophenylthio)phthalimide (APTP). When the 2 cysteine residues of the C-subunit are reacted with DTNB prior to incubation with the RII-subunit, interchain disulfide bonding occurs. Similar observations are seen with C-subunit that had been modified with APTP. Interchain disulfide bonds also form when the RII-subunit is modified with DTNB prior to incubation with the C-subunit. The presence of cAMP facilitates this cross-linking while autophosphorylation of the RII-subunit retards the rate at which the interchain disulfide bond forms. Interchain disulfide bonds also form spontaneously when the RII-subunit and the C-subunit are dialyzed at pH 8.0 in the absence of reducing agents. The specific amino acid residues that participate in intersubunit disulfide bonding have been identified as Cys-97 in the RII-subunit and Cys-199 in the C-subunit. Based on the sequence homologies of the RII-subunit with other kinase substrates and on the proximity of Cys-97 to the catalytic site, a model is proposed in which the autophosphorylation site of the RII-subunit occupies the substrate-binding site in the holoenzyme. The model also proposes that this same site may be occupied by the region flanking Cys-199 in the C-subunit when the C-subunit is dissociated.

Amino Acid Sequence↗
PubMed1987

Inactivation of rat pineal hydroxyindole-O-methyltransferase by disulfide-containing compounds.

Rat pineal hydroxyindole-O-methyltransferase activity in crude homogenates is reduced by treatment with disulfides. Cystamine (IC50 = 128 microM) and selenocystamine (IC50 = 13 microM) are the most potent compounds tested. Reduced cystamine (cysteamine) and diaminohexane are inactive. N,N'-Diacetylcystamine, penicillamine disulfide, and glutathione disulfide are less potent or inactive; but several peptides (oxytocin, vasopressin, and arginine vasotocin) are active. Inactivation by cystamine is time- and temperature-dependent and is accelerated at higher pH. Disulfide treatment of intact pinealocytes also inactivates the enzyme. Addition of dithiothreitol during the enzyme assay completely reactivates inactivated enzyme formed by disulfide treatment of homogenates or intact cells. Rat hydroxyindole-O-methyltransferase is also inactivated in the absence of added disulfides and dissolved O2. This spontaneous inactivation is time-, temperature-, and pH-dependent and can be completely prevented, but not reversed, by dithiothreitol. In contrast to the inhibitory effects of cystamine on the rat enzyme, cystamine does not alter bovine hydroxyindole-O-methyltransferase and increases ovine hydroxyindole-O-methyltransferase activity. The bovine and ovine enzymes do not become inactive in the absence of added disulfides. Together these observations indicate that rat pineal hydroxyindole-O-methyltransferase can be inactivated by a protein thiol:disulfide exchange mechanism. This mechanism may contribute to the physiological regulation of this enzyme in the rat pineal gland but does not appear to be a common feature of pineal hydroxyindole-O-methyltransferase regulation in all species.

Acetylserotonin O-Methyltransferase↗
PubMed1986

Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues.

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.

Acetylcholine↗
PubMed1979

The reaction of choline acetyltransferase with sulfhydryl reagents. Methoxycarbonyl-CoA disulfide as an active site-directed reagent.

The reaction of choline acetyltransferase with methoxycarbonyl alkyl disulfides leads to a progressive loss in enzyme activity as the size of the alkyl group increases from methyl to n-butyl. Reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or methoxycarbonyl coenzyme A (CoA) disulfide, leads to a total loss of enzyme activity. DTNB inactivation is biphasic (k1 = approximately 9 x 10(2) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1) with the slow phase being diminished by acetyl-CoA. Methoxycarbonyl-CoA disulfide inactivation is also biphasic (k1 = approximately 2.1 x 10(3) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1), with the rapid phase being diminished in the presence of acetyl-CoA. Inactivation by methoxycarbonyl methyl disulfide, ethyl disulfide, or hydroxyethyl disulfide, or by methyl methanethiosulfonate is not biphasic. Pretreatment of the enzyme with methyl methanethiosulfonate, which leads to a 25% loss in enzyme activity, abolishes the fast phase of DTNB inactivation, the slow phase of methoxycarbonyl-CoA disulfide inactivation, and any further inactivation by methoxycarbonyl ethyl disulfide. These results are interpreted to suggest that choline acetyltransferase contains two classes of reactive sulfhydryl groups, neither of which are required for enzyme activity.

Acyl Coenzyme A↗
PubMed1980

Reversible inactivation of horse liver aldehyde dehydrogenase by 2-hydroxyethyl disulfide.

Incubation of horse liver aldehyde dehydrogenase (aldehyde:NAD oxidoreductase, EC 1.2.1.3) with 2-hydroxyethyl disulfide formed mixed-disulfides between protein sulfhydryl groups and beta-mercaptoethanol. Reduction of aldehyde dehydrogenase activity may be associated with formation of one, or at most two, mixed-disulfides per dehydrogenase subunit. Characteristically in the case of a mixed-disulfide, inactivation was was reversed by addition of thiols. Other disulfides also inactivated aldehyde dehydrogenase. The pseudo first-order rate constants for the forward and reverse reactions (aldehyde dehydrogenase + 2-hydroxyethyl disulfide in equilibrium or formed from modified aldehyde dehydrogenase + beta-mercaptoethanol) were 0.70 and 2 liter mole-1 sec-1, respectively. The equilibrium constant was approximately 0.4. After extended incubation under conditions expected to result in complete modification of aldehyde dehydrogenase, 30% of the initial catalytic activity remained. This suggests that 2-hydroxyethyl disulfide-treated aldehyde dehydrogenase retains catalytic activity and that the sulfhydryl group modified by 2-hydroxyethyl disulfide is not essential for aldehyde dehydrogenase activity.

Aldehyde Dehydrogenase↗
PubMed1994

Disulfide bonds required to assemble functional von Willebrand factor multimers.

The hemostatic functions of human von Willebrand Factor (vWF) depend on the normal assembly of disulfide-linked multimers from approximately 250-kDa subunits. Subunits initially form dimers through disulfide bonds near the COOH terminus. Dimers then form multimers through disulfide bonds near the NH2 terminus of each subunit. Previous studies of plasma vWF and recombinant vWF fragments indicate that 1 or more of the Cys residues at position 459, 462, and 464 form intersubunit disulfide bonds. No evidence has been reported that vWF multimer formation involves additional intersubunit bonds. To probe the disulfide bond requirements for multimer formation, mutant vWF proteins were expressed in which all 3 Cys residues at positions 459, 462, and 464 were changed to either Gly or Ala. Surprisingly, none of these cysteines appears to be necessary for efficient multimer assembly. Furthermore, recombinant vWF with Gly or Ala at all three positions induces platelet aggregation in the presence of ristocetin and binds to platelet glycoprotein Ib, factor VIII, and collagen in a manner similar to wild-type recombinant vWF. These results suggest that other intersubunit disulfide bonds must exist. Direct evidence for such a bond was obtained by characterization of tryptic fragments of vWF. By Edman degradation, amino acid composition, and mass spectrometry, a disulfide bond was demonstrated between Cys379 residues of adjacent vWF subunits. Thus, intersubunit disulfide bonds involving Cys379 and 1 or more of the Cys residues at positions 459, 462, and 464 connect the NH2-terminal ends of the vWF subunits in a parallel orientation.

Amino Acid Sequence↗
PubMed1994

Inhibition of vacuolar H(+)-ATPase by disulfide bond formation between cysteine 254 and cysteine 532 in subunit A.

We have previously demonstrated that the coated vesicle vacuolar H(+)-ATPase (V-ATPase) can be inactivated by formation of intramolecular disulfide bonds (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 19769-19772). The disulfide bond responsible for inactivation can be distinguished from other disulfide bonds that form by the fact that formation of the inactivating disulfide bond is blocked by ATP or high ionic strength. By taking advantage of these properties, we selectively labeled the ATPase at the relevant cysteine residues with fluorescein maleimide. After analyzing the proteolytic fragments that contain the labeled cysteine residues, we found that cysteine 254 and cysteine 532 in subunit A of the bovine V-ATPase are the residues that form the disulfide bond resulting in inactivation of the enzyme. Cysteine 254 and cysteine 532 correspond to 2 of the 3 cysteine residues that are conserved in all available V-ATPase A subunit sequences. Cysteine 254 is located in the consensus motif, G(X)4GKT, corresponding to residues 250-257, which is conserved in many nucleotide binding proteins. Cysteine 532 is located in a region not previously shown to be in proximity to the nucleotide binding site. Modification of cysteine 254 by disulfide bond formation with cysteine 532 or thio-disulfide exchange with cystine does not impair binding of 2-azido-[32P]ATP to the A subunit. The inhibition is therefore likely caused by disruption of the catalytic function of the ATPase on formation of the disulfide bond. A possible role in regulating intracellular acidification by reversible sulfhydryl oxidation and reduction is discussed.

Amino Acid Sequence↗