Biosynthesis and degradation of gamma-glutamyltranspeptidase of rat kidney

gamma-Glutamyltranspeptidase (gamma GTP) of rat kidney is an intrinsic glycoprotein bound to the plasma membrane and composed of two nonidentical subunits and an amino-terminal portion of the heavy subunit anchors the enzyme to the membrane. The mechanisms of biosynthesis, post-translational processing and degradation of the enzyme were studied using mono-specific antibody raised to gamma-glutamyltranspeptidase purified from rat kidney. The following results were obtained. Double isotope labeling in vivo showed that gamma-glutamyltranspeptidase is synthesized as a precursor form with a single polypeptide chain of 78,000 daltons, and then processed post-translationally by limited proteolysis, resulting in two subunits of 50,000 and 23,000 daltons. Incorporation of [3H]leucine or [35S]methionine into the precursor form increased until 60 min after their intravenous injection, and a pulse-chase experiment showed that the half life of the precursor form was 53 min. [3H]Fucose and [3H]glucosamine could also be incorporated into the precursor form, showing that glycosylation of the enzyme occurs at the stage of the precursor form. Rat kidney labeled with [3H]fucose was subjected to subcellular fractionation. The Golgi fraction contained the glycosylated precursor form and a small amount of subunits, and the plasma membrane fraction contained mostly subunits with a significant amount of precursor, suggesting that post-translational processing of the precursor occurs on the plasma membrane. The apparent half lives of the native enzyme and the heavy and light subunits were all estimated as 4.3 +/- 0.5 days by labeling with [3H]leucine or [3H]fucose. gamma-Glutamyltranspeptidase has a different turnover rate from aminopeptidase M, which is located in the microvillus membrane close to gamma-glutamyltranspeptidase.     

Three subunit proteins of membrane enzymes in mitochondria of Neurospora crassa contain a pantothenate derivative

Three proteins of the inner mitochondrial membrane of Neurospora crassa were found to be covalently modified with a derivative of pantothenic acid. One of these proteins is a subunit of cytochrome c oxidase and two are subunits of the ATPase-ATP synthase. Cells of a pantothenate auxotroph of N. crassa were labeled with [14C]pantothenic acid, and mitochondrial proteins containing radiolabeled pantothenate were detected by electrophoresis of detergent-solubilized mitochondria. Mitochondria from cells that were colabeled with [14C]pantothenate and [3H]leucine were reacted with specific antisera against the cytochrome c oxidase and F1-ATPase enzyme complexes. Electrophoresis of the labeled subunits of these isolated complexes showed that the [14C]pantothenate-associated peptides corresponded to [3H]leucine-labeled subunit 6 of cytochrome c oxidase and two [3H]leucine-labeled subunits (tentatively identified as subunits 8 and 11) of the ATPase-ATP synthase. Pantothenate modification of these enzyme subunits, which are synthesized on extramitochondrial ribosomes, may contribute to their transport and assembly into mitochondria, or it may participate in the catalytic activity of the assembled enzymes.     

Structure of the cytochrome c oxidase complex: labeling by hydrophilic and hydrophobic protein modifying reagents

Beef heart cytochrome c oxidase has been reacted with [35S]diazobenzenesulfonate ([35S]DABS), [35S]-N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate ([35S]NAP-taurine), and two different radioactive arylazidophospholipids. The labeling of the seven different subunits of the enzyme with these protein modifying reagents has been examined. DABS, a water-soluble, lipid-insoluble reagent, reacted with subunits II, III, IV, V, and VII but labeled I or VI only poorly. The arylazidophospholipids, probes for the bilayer-intercalated portion of cytochrome c oxidase, labeled I, III, and VII heavily and II and IV lightly but did not react with V or VI. NAP-taurine labeled all of the subunits of cytochrome c oxidase. Evidence is presented that this latter reagent reacts with the enzyme from outside the bilayer, and the pattern of labeling with the different hydrophilic and hydrophobic labeling reagents is used to derive a model for the arrangement of subunits in cytochrome c oxidase.     

Cytochrome c oxidase from bakers' yeast. Photolabeling of subunits exposed to the lipid bilayer.

Yeast mitochondria and purified yeast cytochrome c oxidase incorporated into micelles of the nonionic detergent Tween 80 were equilibrated with the hydrophobic aryl azides 5-[125I]iodonaphthyl-1-azide or S-(4-azido-2-nitrophenyl)-[35S]thiophenol. The azides were then converted to highly reactive nitrenes by flash photolysis or by illumination for 2 min and the derivatized cytochrome c oxidase subunits were identified by gel electrophoresis and radioactivity measurements. 5-[125I]Iodonaphthyl-1-azide labeled mainly the three mitochondrially made Subunits I to III and the cytoplasmically made Subunit VII. Subunits IV to VI or cytochrome c bound to the purified enzyme were labeled 9- to 90-fold less. Essentially the same result was obtained with S-(4-azido-2-nitrophenyl)-[35S]thiophenol except that Subunit V was labeled as well. In contrast, all seven subunits as well as cytochrome c were heavily labeled when the enzyme was dissociated with dodecyl sulfate prior to photolabeling with either of the two probes. These data indicate that all subunits of yeast cytochrome c oxidase except Subunits IV and VI are at least partly embedded in the lipid bilayer of the mitochondrial inner membrane.     

Nucleotide binding sites and chemical modification of the chromaffin granule proton ATPase

The purified proton ATPase of chromaffin granules contains five different polypeptides denoted as subunits I to V in the order of decreasing molecular weights of 115,000, 72,000, 57,000, 39,000, and 17,000, respectively. The purified enzyme was reconstituted as a highly active proton pump, and the binding of N-ethylmaleimide and nucleotides to individual subunits was studied. N-Ethylmaleimide binds to subunits I, II, and IV, but inhibition of both ATPase and proton pumping activity correlated with binding to subunit II. In the presence of ADP, the saturation curve of ATP changed from hyperbolic to a sigmoid shape, suggesting that the proton ATPase is an allosteric enzyme. Upon illumination of the purified enzyme in the presence of micromolar concentrations of 8-azido-ATP, alpha-[35S]ATP, or alpha-[32P]ATP subunits I, II, and IV were labeled. However, at concentrations of alpha-[32P]ATP below 0.1 microM, subunit II was exclusively labeled in both the purified and reconstituted enzyme. This labeling was absolutely dependent on the presence of divalent cations, like Mg2+ and Mn2+, while Ca2+, Co2+, and Zn2+ had little or no effect. About 0.2 mM Mg2+ was required to saturate the reaction even in the presence of 50 nM alpha-[32P]ATP, suggesting a specific and separate Mg2+ binding site on the enzyme. Nitrate, sulfate, and thiocyanate at 100 mM or N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at 100 microM prevented the binding of the nucleotide to subunit II. The labeling of this subunit was effectively prevented by micromolar concentrations of three phosphonucleotides including those that cannot serve as substrate for the enzyme. It is concluded that a tightly bound ADP on subunit II is necessary for the activity of the enzyme.     

Purification and catalytic properties of a CO-oxidizing:H2-evolving enzyme complex from Carboxydothermus hydrogenoformans.

From the membrane fraction of the Gram-positive bacterium Carboxydothermus hydrogenoformans, an enzyme complex catalyzing the conversion of CO to CO2 and H2 was purified. The enzyme complex showed maximal CO-oxidizing:H2-evolving enzyme activity with 5% CO in the headspace (450 U per mg protein). Higher CO concentrations inhibited the hydrogenase present in the enzyme complex. For maximal activity, the enzyme complex had to be activated by either CO or strong reductants. The enzyme complex also catalyzed the CO- or H2-dependent reduction of methylviologen at 5900 and 180 U per mg protein, respectively. The complex was found to be composed of six hydrophilic and two hydrophobic polypeptides. The amino-terminal sequences of the six hydrophilic subunits were determined allowing the identification of the encoding genes in the preliminary genome sequence of C. hydrogenoformans. From the sequence analysis it was deduced that the enzyme complex is formed by a Ni-containing carbon monoxide dehydrogenase (CooS), an electron transfer protein containing four [4Fe-4S] clusters (CooF) and a membrane bound [NiFe] hydrogenase composed of four hydrophilic subunits and two membrane integral subunits. The hydrogenase part of the complex shows high sequence similarity to members of a small group of [NiFe] hydrogenases with sequence similarity to energy conserving NADH:quinone oxidoreductases. The data support a model in which the enzyme complex is composed of two catalytic sites, a CO-oxidizing site and a H2-forming site, which are connected via a different iron-sulfur cluster containing electron transfer subunits. The exergonic redox reaction catalyzed by the enzyme complex in vivo has to be coupled to energy conservation, most likely via the generation of a proton motive force.     

Delivery of iron-sulfur clusters to the hydrogen-oxidizing [NiFe]-hydrogenases in Escherichia coli requires the A-type carrier proteins ErpA and IscA

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements.     

H(+)-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans. Studies on topology and stoichiometry of the peripheral subunits.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 subunits (NQO1-14) and is located in the cytoplasmic membrane. In the present study, topological properties and stoichiometry of the 7 subunits (NQO1-6 and NQO9) of the P. denitrificans NDH-1 in the membranes were investigated using immunological techniques. Treatments with chaotropic reagents (urea, NaI, or NaBr) or with alkaline buffer (pH 10-12) resulted in partial or complete extraction of all the subunits from the membranes. Of interest is that when NaBr or urea were used, the NQO6 and NQO9 subunits remained in the membranes, whereas the other subunits were completely extracted, suggesting their direct association with the membrane part of the enzyme complex. Both deletion study and homologous expression study of the NQO9 subunit provided a clue that its hydrophobic N-terminal stretch plays an important role in such an association. In light of this observation and others, topological properties of the subunits in the NDH-1 enzyme complex are discussed. In addition, determination of stoichiometry of the peripheral subunits of the P. denitrificans NDH-1 was completed by radioimmunological methods. All the peripheral subunits are present as one molecule each in the enzyme complex. These results estimated the total number of cofactors in the P. denitrificans NDH-1; the enzyme complex contains one molecule of FMN and up to eight iron-sulfur clusters, 2x[2Fe-2S] and 6x[4Fe-4S], provided that the NQO6 subunit bears one [4Fe-4S] cluster.     

Structure of the dissimilatory sulfite reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus

Conservation of energy based on the reduction of sulfate is of fundamental importance for the biogeochemical sulfur cycle. A key enzyme of this ancient anaerobic process is the dissimilatory sulfite reductase (dSir), which catalyzes the six-electron reduction of sulfite to hydrogen sulfide under participation of a unique magnetically coupled siroheme-[4Fe-4S] center. We determined the crystal structure of the enzyme from the sulfate-reducing archaeon Archaeoglobus fulgidus at 2-Å resolution and compared it with that of the phylogenetically related assimilatory Sir (aSir). dSir is organized as a heterotetrameric (αβ)₂ complex composed of two catalytically independent αβ heterodimers. In contrast, aSir is a monomeric protein built of two fused modules that are structurally related to subunits α and β except for a ferredoxin domain inserted only into the subunits of dSir. The [4Fe-4S] cluster of this ferredoxin domain is considered as the terminal redox site of the electron transfer pathway to the siroheme-[4Fe-4S] center in dSir. While aSir binds one siroheme-[4Fe-4S] center, dSir harbors two of them within each αβ heterodimer. Surprisingly, only one siroheme-[4Fe-4S] center in each αβ heterodimer is catalytically active, whereas access to the second one is blocked by a tryptophan residue. The spatial proximity of the functional and structural siroheme-[4Fe-4S] centers suggests that the catalytic activity at one active site was optimized during evolution at the expense of the enzymatic competence of the other. The sulfite binding mode and presumably the mechanism of sulfite reduction appear to be largely conserved between dSir and aSir. In addition, a scenario for the evolution of Sirs is proposed.     

Properties of chloroplast F1-ATPase partially modified by 2-azido adenine nucleotides, including demonstration of three catalytic pathways

Previous investigations on the distribution of [18O]Pi isotopomers formed by hydrolysis of [gamma-18O]ATP by the chloroplast F1-ATPase (CF1) showed that a single reaction pathway is used by all participating sites and that the pathway is modulated by ATP concentration as expected for cooperative interactions between catalytic sites. Such oxygen exchange measurements have been applied to CF1 modified at a single catalytic or noncatalytic site by 2-azido adenine nucleotides. When less than one catalytic or one noncatalytic site per enzyme is modified, hydrolysis occurs in part by the pathway of the unmodified enzyme plus at least one additional pathway at 200 microM and two additional pathways at 4 microM [gamma-18O]ATP. Thus, three sites are potentially catalytically active. The two new pathways shown by the derivatized enzyme logically can arise from nonidentical interactions of the remaining two underivatized beta subunits with the derivatized beta subunit. Reversals of bound ATP cleavage before Pi is released are increased, and the amount of product formed by the new pathways is changed when the ATP concentration is lowered. These modulations must result from the behavior of two remaining active catalytic sites rather than of one catalytic and one regulatory site. When the CF1 is derivatized more extensively, the original catalytic pathway is lost, and two catalytic pathways that do not show modulation by ATP concentration are found. The remaining beta subunits now have weak but independent catalytic capacity. In addition, the enzyme is no longer activated by Ca2+, loses MgGTPase activity, and is much less sensitive to azide.     

Reaction mechanism and structure of the active site of proline racemase.

Proline racemase catalyzes the interconversion of D- and L-proline. Previous studies in this laboratory have established that the reaction proceeds by means of a two-base mechanism in which one base on the enzyme removes the substrate alpha-hydrogen as a proton and the conjugate acid of another base donates a proton to the opposite side of the alpha-carbon (Cardinale, G.J., and Abeles, R.H., (1968), Biochemistry 7, 3970. An assumption of the proposed mechanism was that no proton exchange occurs from the enzyme-substrate complex. In the present study, we have shown that the rate of 3H release from DL-[alpha-3H]proline, in the presence of proline racemase, decreases with increasing proline concentrations. These results establish that release of the substrate derived proton from the enzyme occurs largely, possibly exclusively, after release of the product. Under initial velocity conditions, the rate of 3H release from L-[alpha-3H]proline is not reduced with increasing L-proline concentrations. Thus, the enzyme-bound proton derived from one isomer can only be "captured" by the other isomer. We conclude that there are two forms of the enzyme; one binds L-proline and the other D-proline. Release of the substrate derived proton from enzyme is more rapid than the interconversion of these two forms. These results are consistent with the previously proposed mechanism. Proline racemase is composed of similar subunits of mol wt 38,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Equilibrium dialysis experiments detect only one substrate binding site for every two subunits. When the oxidized form of the enzyme, which is inactive and cannot bind substrate, is reduced by thiol to yield active enzyme, two cysteine sulfhydryl groups per dimer become available to react with iodoacetate. Inactivation of the enzyme occurs upon modification of one of these cysteines. All iodoacetate incorporation occurs at the same point in the primary sequence of the enzyme, and can be prevented by the presence of proline or pyrrole-2-carboxylate, a substrate analog. A model is proposed in which a single active site is formed by elements of two identical subunits. Although the data are consistent with this model, another interpretation, in which half of the subunits are nonfunctional, cannot be ruled out.     

Direct photoaffinity labeling of the catalytic site of mouse ribonucleotide reductase by CDP

Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.     

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1)N-4-Azido-2-nitrophenyl-gamma-[3H]aminobutyryl-AdoPP[NH] P(NAP4-AdoPP[NH]P) a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of [3H]NAP4-AdoPP[NH]P to soluble ATPase from beef heart mitochondria (F1) was studied in the absence of photoirradiation, and compared to that of [3H]AdoPP[NH]P. The photoactivable derivative of AdoPP[NH]P was found to bind to F1 with high affinity, like AdoPP[NH]P. Once [3H]NAP4-AdoPP[NH]P had bound to F1 in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP4 or AMP. Furthermore, preincubation of F1 with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by [3H]NAP4-AdoPP[NH]P upon photoirradiation. (3) Photoirradiation of F1 by [3H]NAP4-AdoPP[NH]P resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol [3H]NAP4-AdoPP[NH]P/mol F1. Photolabeling by NAP4-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl2. (4) Bound [3H]NAP4-AdoPP[NH]P was localized on the alpha- and beta- subunits of F1. At low concentrations (less than 10 microM), bound [3H]NAP4-AdoPP[NH]P was predominantly localized on the alpha-subunit; at concentrations equal to, or greater than 75 microM, both alpha- and beta-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (alpha or beta), suggesting that each of the two subunits, alpha and beta, is required for the activity of F1. (6) The covalently photolabeled F1 was able to form a complex with aurovertin, as does native F1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The precentage of inactivation of F1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggestion that fluorescence quenching is related to the binding of ATP to the catalytic site of F1.     

Tryptophan 2,3-dioxygenase: a review of the roles of the heme and copper cofactors in catalysis.

L-Tryptophan, 2,3-dioxygenase (EC 1.13.11.11) has been purified to homogenity from L-tryptophan induced Pseudomonas acidovorans (ATCC 11299b) and from L-tryptophan and cortisone induced rat liver. The enzyme from both sources is composed of four subunits and contains two g-atoms copper and two moles heme per mole tetramer. The proteins from the two sources are not identical. Three oxidation states of tryptophan oxygenase have been isolated: (1) fully oxidized, [Cu(II)]2[Ferriheme]2; (2) half reduced, [Cu(i)]2[ferriheme]2; and (3) fully reduced, [Cu(I)]2[ferroheme]2. Catalytic activity is dependent solely on the presence of Cu(I) in the enzyme, the heme may be either ferro or ferri. The presence of Cu(II) in the enzyme results in a requirement for an exogenous reductant, such as ascorbate, in order to elicit enzymic activity. Ligands, such as cyanide and carbon monoxide, can inhibit catalysis by binding to either or to both the copper and heme moieties. Metal complexing agents, such as bathocuproinesulfonate and bathophenanthrolinesulfonate, can inhibit catalysis by binding to Cu(I) resent only in catalytically active enzyme molecules. During catalysis by the fully reduced form of the enzyme, molecular oxygen binds to the heme moieties, while during catalysis by the half reduced form of the enzyme it does not, presumably binding instead to the Cu(I) moieties. Enzymes that catalyze similar reactions have been purified from other sources. Indoleamine 2,3-dioxygenase appears to be a heme protein, but its copper content is unknown. Pyrrolooxygenases appear to be completely different enzymes, although they have not yet been purified to homegeneity.     

Molecular organization of subunits of electroplax (sodium plus potassium)--activated adenosine triphosphatase.

Antisera against each of the two major subunits of detergent-solubilized electroplax (sodium plus potassium)-activated adenosine triphosphatase from Electrophorus electricus were prepared. Antiserum against the small subunit (a glycoprotein, Mr = 58,000) partially inhibits [3H]ouabain binding to the enzyme, but does not interfere with the phosphorylation of enzyme. Conversely, antiserum against the large subunit (the catalytic subunit Mr = 96,000) partially inhibits phosphorylation of the enzyme, but does not interfere with the binding of [3H]ouabain to the enzyme. Since ouabain only interacts with enzyme from the outer surface of the membrane and phosphorylation of enzyme takes place on the inner surface of the membrane, the results suggest that the small subunits are exposed on the outer surface of the membrane, whereas the large subunits are oriented predominantely facing the cytoplasmic side.     

Phosphorylation of small subunit plays a crucial role in the regulation of RuBPCase in moss and spinach

RuBPCase has been purified to electrophoretic homogeneity from moss and spinach. On denaturing SDS-polyacrylamide gels the purified enzyme revealed two discrete bands, thereby indicating the presence of large and small subunits. The phosphoprotein nature of RuBPCase was proved by in vivo labelling of enzyme with [³²P]orthophosphate. Autoradiographic analysis of ³²P-labelled RuBPCase on SDS-PAG demonstrated that phosphorylation was restricted to the small subunit. Dephosphorylation of purified RuBPCase with alkaline phosphatase resulted in a dramatic decline (70% decrease) in the biological activity of the enzyme. Fractionation of the dephosphorylated enzyme on denaturing gels revealed only the presence of large subunits of RuBPCase. Thus it became evident that dephosphorylation of RuBPCase brings about the dissociation of small subunits from the catalytic large subunits (octamer). The dephosphorylated small subunits were isolated as dimers. These results clearly indicate that phosphorylation of small subunits is mandatory for the reconstitution of holoenzyme and hence crucial for the activation of RuBPCase.     

Chemical labeling studies on bovine heart mitochondrial cytochrome c oxidase dispersed in nonionic detergents

In order to investigate the structural interactions of nonionic detergents with bovine heart mitochondrial cytochrome c oxidase (COX), a series of hydrophilic chemical modification reagents were used to map regions on COX which are not shielded by dodecyl beta-D-maltoside (DM), Triton X-100 (TX-100), and Tween 80 (TW-80). Low levels of incorporation of the chemical reagents [35S]benzenediazoniumsulfonate (DABS) and N-succinimidyl [3H]propionate (SP) into COX dispersed in TW-80 indicate that the bulky headgroup and hydrophobic moiety of this detergent effectively shield the enzyme from the aqueous environment. Subunits II and Va/Vb [nomenclature of Merle, P., & Kadenbach, B. (1982) Eur. J. Biochem. 125, 239-244] show an increased reactivity to [35S]DABS and [3H]SP in TW-80 and may reflect an increased exposure of these subunits to the aqueous phase in comparison to COX dispersed in TX-100 or DM. More [35S]DABS is incorporated into COX in DM than TX-100-dispersed enzyme; DABS heavily labels subunits III, VIa, and VIb in DM. While COX in TX-100 is more reactive with [3H]SP than DM-dispersed enzyme, there is no difference in the distribution of label (either DABS or SP) within the subunits of COX in DM or TX-100. Increased surface exposure of COX in TX-100 is indicated by an enhanced sensitivity of COX electron-transfer activity in enzyme chemically modified by the cross-linking reagent N-succinimidyl 3-[(4-azidophenyl)dithio]propionate (SADP) in TX-100 as compared to enzyme chemically cross-linked in the other detergents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa.

The rotenone sensitive NADH:ubiquinone was isolated from mitochondria of Neurospora crassa as a monodisperse preparation with the apparent mol. wt. in Triton solution of 0.9 X 10(6). The enzyme is composed of at least 22 subunits with apparent mol. wts. in SDS between 70 and 11 kd. Six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35S]methionine in the presence of cycloheximide. These subunits are synthesized in the mitochondria. Eleven subunits were radioactively labelled in the enzyme from cells which had incorporated [35S]methionine in the presence of chloramphenicol. These subunits are synthesized in the cytoplasm. The site of translation of the other subunits could not be established by the pulse-labelling technique. The assignment of the mitochondrially synthesized subunits to unidentified reading frames on the mitochondrial DNA is discussed.     

The organization of formate dehydrogenase in the cytoplasmic membrane of Escherichia coli.

The arrangement of the proton-translocating formate dehydrogenase of the anaerobic respiratory chain of Escherichia coli within the cytoplasmic membrane was examined by direct covalent modification with non-membrane-permeant reagents. Three methods were employed, lactoperoxidase-catalysed radioiodination, labelling with diazotized [125I] di-iodosulphanilic acid and labelling with diazobenzene [35S] sulphonate. All three procedures yield consistent with the view that the two larger subunits of the enzyme, Mr 110000 and 32000, both occupy transmembranous locations within the membrane. In each case the modification of the Ca2+ or Mg2+-activated F1-ATPase was monitored, and all reagents employed correctly located this enzyme at the cytoplasmic face of the membrane. A procedure involving agglutination with specific antibodies is described which appears to fractionate membrane vesicles of mixed orientation into two populations, one with the same membrane orientation as that of spheroplasts and the other opposite orientation.