Anabolic five subunit-type pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

The thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene cluster, porEDABG, encoding pyruvate:ferredoxin oxidoreductase (POR), which plays a key role in this cycle, was cloned and sequenced. The nucleotide sequence and the gene organization were similar to those of the five subunit-type 2-oxoglutarate:ferredoxin oxidoreductase from this strain, although the anabolic POR had been previously reported to consist of four subunits. A small protein (8 kDa) encoded by porE, which had not been detected in the previous work, was identified in the purified recombinant POR expressed in Escherichia coli, indicating that the enzyme is also a five-subunit type. Incorporation of PorE in the wild-type POR enzyme was confirmed by immunological analysis. PorA, PorB, PorG, and PorE were similar to the alpha, beta, gamma, and delta subunits of the four subunit-type 2-oxoacid oxidoreductases, respectively, and had conserved specific motifs. PorD had no specific motifs but was essential for the expression of the active enzyme.     

Specific Vinca alkaloid-binding polypeptides identified in calf brain by photoaffinity labeling

A radioactive, photoactive Vinca alkaloid, N-(p-azido-[3,5-3H]-benzoyl)-N'-beta-aminoethylvindesine [( 3H]NABV) with pharmacological and biological activities similar to vinblastine was synthesized and used to identify specific Vinca alkaloid macromolecular interactions in calf brain homogenate by photoaffinity labeling. The most prominent photolabeled species were 54.3- and 21.5-kDa polypeptides. The Vinca alkaloid-binding specificity of these polypeptides was confirmed by competitive blocking of specific photolabeling by vinblastine but not by colchicine or daunorubicin. The 54.3- and 21.5-kDa polypeptides exhibited specific half-maximum saturable photolabeling at 2.1 and 0.95 X 10(-7) M [3H]NABV, respectively. Relative vinblastine and NABV association constants (Ka vinblastine/Ka NABV) for the 54.3- and 21.5-kDa polypeptides were estimated to be 0.86 and 1.4, respectively. The 54.3-kDa component was found in both high speed (100,000 X g; 1 h) pellet and supernatant fractions, whereas the 21.5-kDa component was located primarily in the high speed pellet. Photolabeling of both components was maximal after 12-min UV light exposure, linear up to 120 micrograms of homogenate protein and only slightly affected by the nitrene scavenger p-aminobenzoic acid. The 54.3-kDa polypeptides of [3H]NABV-photolabeled calf brain high speed supernatant and detergent-solubilized high speed pellet fractions were identified as tubulin subunits by immunoprecipitation with monoclonal antibodies to alpha- or beta-tubulin subunits. Although the identity and function of the 21.5-kDa polypeptide is not known, this polypeptide may have a role in membrane-related effects of the Vinca alkaloids. These results demonstrate that [3H]NABV is an attractive tool for identifying and characterizing specific high affinity vinblastine cellular polypeptide acceptors which may initiate or mediate known and unknown mechanisms of Vinca alkaloid action.     

Isolation and analysis of the gene encoding the pyruvate-ferredoxin oxidoreductase of Desulfovibrio africanus, production of the recombinant enzyme in Escherichia coli, and effect of carboxy-terminal deletions on its stability.

Previous studies have shown that the pyruvate-ferredoxin oxidoreductase (POR) of the sulfate-reducing bacterium Desulfovibrio africanus is a homodimer that contains one thiamine pyrophosphate and three [4Fe-4S]2+/1+ centers/subunit. Interestingly, the enzyme isolated from a strictly anaerobic bacterium is highly stable in the presence of oxygen, in contrast to the other PORs characterized in anaerobic organisms (L. Pieulle, B. Guigliarelli, M. Asso, F. Dole, A. Bernadac, and E. C. Hatchikian, Biochim. Biophys. Acta 1250:49-59, 1995). We report here the determination of the nucleotide sequence of the por gene encoding the D. africanus POR. The amino acid sequence deduced from this nucleotide sequence corresponds to the first primary structure of a homodimeric POR from strictly anaerobic bacteria. The subunit of the D. africanus POR contains two ferredoxin-type [4Fe-4S] cluster binding motifs (CX2CX2CX3CP) and four additional highly conserved cysteines belonging to a nontypical motif. These 12 cysteine residues may coordinate the three Fe-S centers present in D. africanus POR. The thiamine pyrophosphate binding domain is located in the C-terminal part of the protein close to the four conserved cysteine residues. The D. africanus enzyme sequence appears homologous to the other POR sequences. However, the enzyme differs from all other PORs by a C-terminal extension of about 60 residues of its polypeptide chain. The two cysteine residues located in this additional region may be involved in the formation of a disulfide bridge associated with the activation process of the catalytic activity. The por gene has been expressed, for the first time, in anaerobically grown Escherichia coli behind the isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, resulting in the production of POR in its active form. The recombinant enzyme is stable toward oxygen during several days, and initial characterization of the recombinant POR showed that its activity increased in the presence of dithioerythritol. These properties indicate that the recombinant POR behaves like the native D. africanus enzyme. The study of carboxy-terminal deletion mutants strongly suggests that deletions in the C-terminal region of D. africanus enzyme can have dramatic effects on the stability of the enzyme toward oxygen.     

Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf

1. Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5-day-old germinating endosperm of the castor oil plant, Ricinus communis. An earlier study demonstrated that the purified enzyme is putatively heterotetrameric, composed of two subunits which migrate as 57-kDa and 56-kDa proteins upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis [Plaxton, W. C. (1988) Plant Physiol. (Bethesda) 86, 1065-1069]. Both proteins were detected on Western blots of extracts prepared under denaturing conditions from 4-8-day-old, but not 0-3-day-old, germinating-endosperm tissue. This suggests that both subunits exist in vivo, and that the large increase in pyruvate kinase activity which occurs around the fourth day of germination is due to an increase in pyruvate kinase concentration. 2. The cytosolic and plastidic pyruvate kinase isozymes (termed PKc and PKp, respectively) from castor-oil-plant developing endosperm and expanding leaf tissue were separated by anion-exchange chromatography on Q-Sepharose. The antigenic reaction of the partially purified enzyme preparations to rabbit polyclonal antibodies raised against homogeneous germinating-castor-bean PKc was tested by immunoprecipitation and Western blotting. Although developing-endosperm and leaf PKc appeared to be antigenically very similar to germinating-endosperm PKc, they differed from the heterotetrameric germinating-endosperm enzyme by being composed of a single type of subunit with a molecular mass of about 56 kDa. No cross-reactivity of the PKc antibodies was observed with either developing-endosperm or leaf PKp, nor with rabbit muscle or Bacillus stearothermophilus pyruvate kinase. Conversely, none of the castor-oil-plant pyruvate kinase preparations showed significant cross-reactivity with antibodies raised against purified yeast or rabbit muscle pyruvate kinases. 3. To investigate the structural relationship between the two germinating-endosperm-PKc subunits, each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation. The amino acid compositions and CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. Mild tryptic attack of native enzyme led to an approximate 6-kDa reduction in the apparent molecular mass of both subunits, further indicating sequence similarity between the two polypeptides. 4. Native molecular masses of the various castor-oil-plant pyruvate kinases were estimated by Superose-6 gel-filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme.

NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.     

IscA, an alternate scaffold for Fe-S cluster biosynthesis.

An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, M?ssbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and M?ssbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The M?ssbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.     

Characterization of the subunit structure of the maize tonoplast ATPase. Immunological and inhibitor binding studies

Gradient purified preparations of the maize 400-kDa tonoplast ATPase are enriched in two major polypeptides, 72 and 62 kDa. Polyclonal antibodies were prepared against these two putative subunits after elution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel slices and against the solubilized native enzyme. Antibodies to both the 72- and 62-kDa polypeptides cross-reacted with similar bands on immunoblots of a tonoplast-enriched fraction from barley, while only the 72-kDa antibodies cross-reacted with tonoplast and tonoplast ATPase preparations from Neurospora. Antibodies to the 72-kDa polypeptide and the native enzyme both strongly inhibited enzyme activity, but the 62-kDa antibody was without effect. The identity and function of the subunits was further probed using radiolabeled covalent inhibitors of the tonoplast ATPase, 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole ([14C]NBD-Cl) and N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-Cl preferentially labeled the 72-kDa polypeptide, and labeling was prevented by ATP. [14C]DCCD, an inhibitor of the proton channel portion of the mitochondrial ATPase, bound to a 16-kDa polypeptide. Venturicidin blocked binding to the mitochondrial 8-kDa polypeptide but did not affect binding to the tonoplast 16-kDa polypeptide. Taken together, the results implicate the 72-kDa polypeptide as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa polypeptide may comprise the proton channel. The presence of nucleotide-binding sites on the 62-kDa polypeptide suggests that it may function as a regulatory subunit.     

Purification and characterization of calmodulin-dependent protein kinase II from rat spleen: a new type of calmodulin-dependent protein kinase II

A calmodulin-dependent protein kinase has been purified from rat spleen. The enzyme showed a remarkably similar substrate specificity and kinetic parameters to those of rat brain calmodulin-dependent protein kinase II, and exhibited cross-reactivity to a monoclonal antibody against rat brain calmodulin-dependent protein kinase II, indicating that the enzyme might be a calmodulin-dependent protein kinase II isozyme. The sedimentation coefficient was 13.9S, the Stokes radius was 67 A, and the molecular weight was calculated to be 380,000. The purified enzyme gave five polypeptides bands, corresponding to molecular weights of 51,000, 50,000, 21,000, 20,000, and 18,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the purified enzyme with Ca2+, calmodulin, and ATP under phosphorylating conditions induced the phosphorylation of all five polypeptides. When the logarithm of the velocity of the phosphorylation was plotted against the logarithm of the enzyme concentration (van't Hoff plot), slopes of 0.89, 0.94, and 1.1 were obtained for the phosphorylation of the 50/51-kDa doublet, 20/21-kDa doublet, and 18-kDa polypeptide, respectively. These results indicate that the phosphorylation of the five polypeptides is an intramolecular process, and further indicate that all five polypeptides are subunits of this enzyme. Of the five polypeptides, only the 50- and 51-kDa polypeptides bound to [125I]calmodulin, the other polypeptides not binding to it. A number of isozymic forms of calmodulin-dependent protein kinase II so far demonstrated in various tissues are known to be composed of subunits with molecular weights of 50,000 to 60,000 which can bind to calmodulin. Thus a new type of calmodulin-dependent protein kinase II was demonstrated in the present study.     

Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota.

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.     

Particulate methane monooxygenase genes in methanotrophs.

A 45-kDa membrane polypeptide that is associated with activity of the particulate methane monooxygenase (pMMO) has been purified from three methanotrophic bacteria, and the N-terminal amino acid sequence was found to be identical in 17 of 20 positions for all three polypeptides and identical in 14 of 20 positions for the N terminus of AmoB, the 43-kDa subunit of ammonia monooxygenase. DNA from a variety of methanotrophs was screened with two probes, an oligonucleotide designed from the N-terminal sequence of the 45-kDa polypeptide from Methylococcus capsulatus Bath and an internal fragment of amoA, which encodes the 27-kDa subunit of ammonia monooxygenase. In most cases, two hybridizing fragments were identified with each probe. Three overlapping DNA fragments containing one of the copies of the gene encoding the 45-kDa pMMO polypeptide (pmoB) were cloned from Methylococcus capsulatus Bath. A 2.1-kb region was sequenced and found to contain both pmoB and a second gene, pmoA. The predicted amino acid sequences of these genes revealed high identity with those of the gene products of amoB and amoA, respectively. Further hybridization experiments with DNA from Methylococcus capsulatus Bath and Methylobacter albus BG8 confirmed the presence of two copies of pmoB in both strains. These results suggest that the 45- and 27-kDa pMMO-associated polypeptides of methanotrophs are subunits of the pMMO and are present in duplicate gene copies in methanotrophs.     

Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.     

Chromatographic separation of a small subunit (PsbW/PsaY) and its assignment to Photosystem I reaction center

By using a hydroxyapatite column, the five major Photosystem I (PSI) subunits (PsaA,-B,-C,-D,-E) solubilized by sodium dodecyl sulfate (SDS) were fractionated from a spinach PSI reaction center preparation. Another small (5-6 kDa) polypeptide was also separated, and purified to homogeneity. Mass spectroscopy yielded its molecular weight to be 5942 +/- 10. This polypeptide had an N-terminal sequence homologous to those of previously reported 5-kDa subunits from spinach and wheat and a 6.1-kDa subunit of Chlamydomonas, which had all been assigned to Photosystem II (PSII) and designated as PsbW. However, we found similar 5-kDa polypeptides with highly conserved N-terminal sequences ubiquitously in PSI particles from other plants including Daikon (Raphanus sativus, Japanese radish), Chingensai (Brassica parachinensis, Chinese cabbage), parsley and Shungiku (Chrysanthemum coronarium, Garland chrysanthemum) as well. Preparations of spinach PSI particles prepared by using a mild detergent (digitonin) had this 5-kDa subunit, while PSII particles did not. Moreover, a bare-bone PSI reaction center preparation consisting of PsaA/B alone had a more than stoichiometric amount of this 5-kDa polypeptide. A mechanically (without detergent) fractionated stroma thylakoid preparation from Phytolacca americana, which lacked other PSII subunits, also contained this 5-kDa subunit. Thus, we propose that this 5-kDa polypeptide, previously designated as a PSII subunit (PsbW), is an integral subunit of PSI as well.     

Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol--cytochrome-c reductase complex.

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein II and the 14-kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N-terminal region. Proteolytic cleavage of 7-11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton-motive activity of the complex.     

The asymmetric IscA homodimer with an exposed [2Fe-2S] cluster suggests the structural basis of the Fe-S cluster biosynthetic scaffold

It has been shown that the so-called scaffold proteins are vital in Fe-S cluster biosynthesis by providing an intermediate site for the assembly of Fe-S clusters. However, since no structural information on such scaffold proteins with bound Fe-S cluster intermediates is available, the structural basis of the core of Fe-S cluster biosynthesis remains poorly understood. Here we report the first Fe-S cluster-bound crystal structure of a scaffold protein, IscA, from Thermosynechococcus elongatus, which carries three strictly conserved cysteine residues. Surprisingly, one partially exposed [2Fe-2S] cluster is coordinated by two conformationally distinct IscA protomers, termed alpha and beta, with asymmetric cysteinyl ligation by Cys37, Cys101, Cys103 from alpha and Cys103 from beta. In the crystal, two alphabeta dimers form an unusual domain-swapped tetramer via central domains of beta protomers. Together with additional biochemical data supporting its physiologically relevant configuration, we propose that the unique asymmetric Fe-S cluster coordination and the resulting distinct conformational stabilities of the two IscA protomers are central to the function of IscA-type Fe-S cluster biosynthetic scaffold.     

Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.     

Plant mitochondrial F0F1 ATP synthase. Identification of the individual subunits and properties of the purified spinach leaf mitochondrial ATP synthase.

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1 delta subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N'-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 mumol.mg-1.min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 mumol.mg-1.min-1) and ITP (4.4 mumol.mg-1.min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.     

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.     

Solubilization and purification of alpha-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae

Yeast alpha-mannosidase, a marker enzyme of vacuolar membranes, was solubilized and purified from commercial bakers' yeast. The alpha-mannosidase was solubilized efficiently with 10 mM Na2CO3. A high pH (greater than 8.5) and a sufficient amount of a detergent such as 0.2% (w/v) Triton X-100 were required to keep the enzyme in a soluble state. This suggested that the enzyme is either a peripheral membrane protein or an ecto-type integral membrane protein. After 4,300-fold purification by conventional chromatography, the alpha-mannosidase gave a single band on nondenaturing polyacrylamide gel electrophoresis, but could be fractionated into active isoforms, which consisted of 107-, 73-, and 31-kDa polypeptides, with a Mono Q anion exchange fast protein liquid chromatography system. Apparent molecular weight of the native enzyme was determined as 560,000. It suggested that the composition of isoforms will be described as (107 kDa)n (73 kDa)6-n (31 kDa)6-n, where n is 0-6. The 107- and 73-kDa polypeptides were purified further under denaturing conditions. One-dimensional peptide map analysis and immunological analysis of these polypeptides indicated that they are closely related proteins. Immunoblotting of crude cell lysates revealed that the 107-kDa polypeptide appeared first, and then the 73-kDa polypeptide appeared along growth phase. It suggested that proteolytic conversion of the 107-kDa polypeptide occurs to form the 73- and 31-kDa polypeptides and leads to formation of isoforms of the enzyme.     

Cloning of cDNA encoding the membrane-bound form of bovine beta 1,4-galactosyltransferase

UDPgalactose: N-acetyl-D-glucosamine 4-beta-D-galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi-membrane-bound enzyme that participates in the biosynthesis of the oligosaccharide structures of glycoproteins and glycolipids. Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine-liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206-base-pair open reading frame that codes for 402 amino acids, including a presumptive N-terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non-overlapping amino acid residues which were positively identified by N-terminal sequencing of two polypeptides isolated from the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones. The finding of an N-terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C-terminal region of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51-kDa and 42-kDa soluble forms of the enzyme which encompass the C-terminal 324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.