Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificans

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. When the Paracoccus NADH dehydrogenase complex was irradiated by UV light in the presence of [adenylate-32P]NAD, radioactivity was incorporated exclusively into one of three polypeptides of Mr approximately 50,000. Similar results were obtained when [adenylate-32P]NADH was used. The labeling of the Mr 50,000 polypeptide was diminished when UV irradiation of the enzyme with [adenylate-32P]NAD was performed in the presence of NADH, but not in the presence of NADP(H). The labeled polypeptide was isolated by preparative sodium dodecyl sulfate gel electrophoresis and was shown to cross-react with antiserum to the NADH-binding subunit (Mr = 51,000) of bovine NADH-ubiquinone oxidoreductase. Its amino acid composition was also very similar to that of the bovine NADH-binding subunit. These chemical and immunological results indicate that the Mr 50,000 polypeptide is an NADH-binding subunit of the Paracoccus NADH dehydrogenase complex.     

Identification of the NADH-binding subunit of energy-transducing NADH-quinone oxidoreductase (NDH-1) of thermus thermophilus HB-8

The energy-transducing NADH--quinone oxidoreductase (NDH-1) isolated from Thermus thermophilus HB-8 is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN and at least three iron-sulfur clusters [Yagi, T., Hon-nami, K., and Ohnishi, T. (1988) Biochemistry 27, 2008-2013]. When NDH-1 of T. thermophilus HB-8 was irradiated by short UV light in the presence of [adenylate-32P]NADH or [adenylate-32P]NAD, radioactivity was incorporated into a single polypeptide of Mr 47,000. The labeling of the Mr 47,000 polypeptide was diminished when UV irradiation of the enzyme complex with [adenylate-32P]NAD was carried out in the presence of NADH or deamino-NADH which act as substrates for the NDH-1, but not in the presence of NADP(H) or AMP which act neither as substrates nor as competitive inhibitors. These results strongly suggest that the Mr 47,000 polypeptide is an NADH-binding subunit of the NDH-1 of T. thermophilus HB-8.     

In vivo and in vitro acylation of polypeptides in Vibrio harveyi: identification of proteins involved in aldehyde production for bioluminescence.

Incubation of soluble extracts from Vibrio harveyi with [3H]tetradecanoic acid (+ ATP) resulted in the acylation of several polypeptides, including proteins with molecular masses near 20 kilodaltons (kDa), and at least five polypeptides in the 30- to 60-kDa range. However, in growing cells pulse-labeled in vivo with [3H]tetradecanoic acid, only three of these polypeptides, with apparent molecular masses of 54, 42, and 32 kDa, were specifically labeled. When extracts were acylated with [3H] tetradecanoyl coenzyme A, on the other hand, only the 32-kDa polypeptide was labeled. When luciferase-containing dark mutants of V. harveyi were investigated, acylated 32-kDa polypeptide was not detected in a fatty acid-stimulated mutant, whereas the 42-kDa polypeptide appeared to be lacking in a mutant defective in aldehyde synthesis. Acylation of both of these polypeptides also increased specifically during induction of bioluminescence in V. harveyi. These results suggest that the role of the 32-kDa polypeptide is to supply free fatty acids, whereas the 42-kDa protein may be responsible for activation of fatty acids for their subsequent reduction to form the aldehyde substrates of the bioluminescent reaction.     

Low molecular weight GTP-binding proteins in cardiac muscle. Association with a 32-kDa component related to connexins.

Low molecular weight GTP-binding proteins and their cellular interactions were examined in cardiac muscle. Heart homogenate was separated into various subcellular fractions by differential and sucrose density gradient centrifugation. Various fractions were separated by sodium dodecyl sulfate-gel electrophoresis, blotted to nitrocellulose, and GTP-binding proteins detected by incubating with [alpha-32]GTP. Three polypeptides of M(r) 23,000, 26,000, and 29,000 were specifically labeled with [alpha-32P]GTP in all the fractions examined and enriched in sarcolemmal membranes. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 26- and 29-kDa polypeptides. A polypeptide of M(r) 40,000 was weakly labeled with [alpha-32P]GTP in the sarcolemmal membrane and tentatively identified as Gi alpha by immunostaining with anti-Gi alpha antibodies. Cytosolic GTP-binding proteins were labeled with [alpha-32P]GTP and their potential sites of interaction investigated using the blot overlay approach. A polypeptide of 32 kDa present in sarcolemmal membranes, intercalated discs, and enriched in heart gap junctions was identified as a major site of interaction. The low molecular weight GTP-binding proteins associated with the 32-kDa polypeptide through a complex involving cytosolic components of M(r) 56,000, 36,000, 26,000, 23,000, and 12,000. A monoclonal antibody against connexin 32 from liver strongly recognized the 32-kDa polypeptide in heart gap junctions, whereas polyclonal antibodies only weakly reacted with this polypeptide. The low molecular weight GTP-binding proteins associated with a 32-kDa polypeptide in liver membranes that was also immunologically related to connexin 32. These results indicate the presence of a subset of low molecular weight GTP-binding proteins in a membrane-associated and a cytoplasmic pool in cardiac muscle. Their association with a 32-kDa component that is related to the connexins suggests that these polypeptides may be uniquely situated to modulate communication at the cell membrane.     

Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum

Ferredoxin-NAD(P)(+) reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP(+) and NAD(+). When concentrations of NADP(+) exceeded 10 microM, NADP(+) photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD(+). It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.     

Identification of 3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate- and N,N'-dicyclohexylcarbodiimide-binding subunits of a higher plant H+-translocating tonoplast ATPase

The polypeptide composition of the NO-3-sensitive H+-ATPase of vacuolar membrane (tonoplast) vesicles isolated from red beet (Beta vulgaris L.) storage root was investigated by affinity labeling with [alpha-32P]3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate [( alpha-32P]BzATP) and [14C]N,N'-dicyclohexylcarbodiimide [( 14C]DCCD). The photoactive affinity analog of ATP, BzATP, is a potent inhibitor of the tonoplast ATPase (apparent KI = 11 microM) and the photolysis of [alpha-32P]BzATP in the presence of native tonoplast yields one major 32P-labeled polypeptide of 57 kDa. Photoincorporation into the 57-kDa polypeptide shows saturation with respect to [alpha-32P]BzATP concentration and is blocked by ATP. [14C]DCCD, a hydrophobic carboxyl reagent and potent irreversible inhibitor of the tonoplast ATPase (k50 = 20 microM) labels a 16-kDa polypeptide in native tonoplast. The tonoplast ATPase is purified approximately 12-fold by Triton X-100 solubilization and Sepharose 4B chromatography. Partial purification results in the enrichment of two prominent polypeptides of 67 and 57 kDa. Solubilization, chromatography, and sodium dodecylsulfate-polyacrylamide gel electrophoresis of tonoplast labeled with [alpha-32P]BzATP or [14C]DCCD results in co-purification of the 57- and 16-kDa labeled polypeptides with ATPase activity. It is concluded that the tonoplast H+-ATPase is a multimer containing structurally distinct BzATP- and DCCD-binding subunits of 57 and 16 kDa, respectively. The data also suggest the association of a 67-kDA polypeptide with the ATPase.     

The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: gene cloning and deduced primary structure

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. The NADH-binding subunit (Mr = 50,000) of this enzyme complex was identified by direct photoaffinity labeling with [32P]NADH [Yagi, T., & Dinh, T.M. (1990) Biochemistry 29, 5515-5520]. Primers were synthesized on the basis of the N-terminal amino acid sequence of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction. This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P. denitrificans. The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the alpha subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16. When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex I were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster.     

Localization of the tight ADP-binding site on the membrane-bound chloroplast coupling factor one

The photoaffinity analog 2-azido-ADP (2-azidoadenosine 5'-diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2-azido-ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2-azido-ADP competitively inhibited ADP tight binding (Ki = 1.4 microM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 microM) was similar to that observed for ADP (2 microM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re-energization; (d) the tight binding of 2-azido-[beta-32P]ADP was completely prevented by ADP; (e) the analog inhibited the light-triggered ATPase activity at micromolar concentrations. Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[beta-32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the beta subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the beta subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.     

Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc

Photoaffinity labeling of purified cellulose synthase with [beta-32P]5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of [beta-32P]5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.     

Regulation of C4 photosynthesis: inactivation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and activation by phosphorolysis

These studies provide information about the mechanism of the light/dark-mediated regulation of pyruvate, Pi dikinase (EC 2.7.9.1) in leaves. It is shown that inactivation is due to a phosphorylation of the enzyme from the beta-phosphate of ADP, and that activation occurs by phosphorolysis to remove the enzyme phosphate group. During ADP plus ATP-dependent inactivation of pyruvate, Pi dikinase in chloroplast extracts, 32P was incorporated into the enzyme from [beta-32P]ADP. Approximately 1 mol of phosphate was incorporated per mol of monomeric enzyme subunit inactivated. There was very little incorporation of label from ADP or ATP labeled variously in other positions with 32P or from the nucleotides labeled with 3H in the purine ring. Purified pyruvate, Pi dikinase was also labeled from [beta-32P]ADP during inactivation. In this system, phosphorylation of the enzyme required the addition of the "regulatory protein" shown previously to be essential for catalyzing inactivation and activation. During orthophosphate-dependent reactivation of pyruvate, Pi dikinase, it was shown that the enzyme loses 32P label and that pyrophosphate is produced. The significance of these findings in relation to regulation of the enzyme in vivo is discussed.     

Identification of low molecular weight GTP-binding proteins and their sites of interaction in subcellular fractions from skeletal muscle

The presence of low molecular weight GTP-binding proteins was investigated in subcellular fractions from skeletal muscle. Skeletal muscle homogenate, transverse tubules, triads, sarcoplasmic reticulum membranes, and cytosol fractions were separated in sodium dodecyl sulfate-gel electrophoresis and blotted onto nitrocellulose. The presence of GTP-binding proteins was explored by incubation of these blots with [alpha-32P] GTP. GTP labeled two polypeptides of Mr = 23,000 and 29,000 in all the fractions examined. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 29-kDa polypeptide, although both were enriched in transverse tubule fractions. A GTP-binding polypeptide of 40 kDa was also enriched in transverse tubule preparations and identified as Gi alpha by immunostaining with anti-Gi alpha. Using a blot overlay approach and [alpha-32P]GTP-labeled cytosolic components, several polypeptides were identified that interact with the 23- and 29-kDa GTP-binding proteins. Among these components were polypeptides of Mr = 60,000, 47,000, 44,000, 42,000, and 38,000, which were mainly of cytosolic origin but also associated with triads and transverse tubule membranes. The 47-, 44-, 42-, and 38-kDa polypeptides were found to be structurally related to the glycolytic enzymes enolase, 3-phosphoglyceric phosphokinase, aldolase, and glycoeraldehyde-3-phosphate dehydrogenase, respectively. The purified glycolytic enzymes specifically bound the 23- and 29-kDa GTP-binding proteins under both denaturing and nondenaturing conditions. The association of the GTP-binding proteins with these polypeptides was resistant to detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), Triton X-100, and Tween. A 23-kDa GTP-binding protein purified from chromaffin cells bound to a 157-kDa polypeptide in triads and chromaffin cell membranes. The 157-kDa polypeptide was a minor component in these membranes and not related to the subunits of the dihydropyridine receptor. In view of the proposed function of low molecular weight GTP-binding proteins in processes such as membrane communication and secretion coupling, the association of these proteins with transverse tubules and triads in skeletal muscle is discussed in terms of a role in signal transmission.     

Association of Ferredoxin-NADP Oxidoreductase with the Chloroplastic Pyridine Nucleotide Dehydrogenase Complex in Barley Leaves

Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2′-monophosphoadenosine-5′-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2′-monophosphoadenosine-5′-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex.     

Subunit composition of vacuolar membrane H(+)-ATPase from mung bean

The vacuolar H(+)-ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidycholine and purified by QAE-Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X-100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purified ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43-kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X-100. The 16-kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino-terminal amino acid sequence of the isolated 68-kDa polypeptide generally agreed with that deduced from the cDNA for the carrot 69-kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112]. Thus, mung bean vacuolar H(+)-ATPase seems to consist of nine distinct subunits.     

Characterization of multiple active forms of the NADPH dehydrogenase component of the oxidase complex from rabbit peritoneal neutrophils by photolabeling with an arylazido derivative of NADP+

A NADPH cytochrome c oxidoreductase purified from membranes of rabbit peritoneal neutrophil was shown to behave as the NADPH dehydrogenase component of the O2- generating oxidase complex. A photoactivable derivative of NADP+, azido nitrophenyl-gamma-aminobutyryl NADP+ (NAP4-NADP+), was synthesized in its labeled [3H] form and used to photolabel the NADPH cytochrome c reductase at different stages of the purification procedure. Control assays performed in dim light indicated that the reduced form of NADP4-NADP+ generated by reduction with glucose-6-phosphate and glucose-6-phosphate dehydrogenase was oxidized at virtually the same rate as NADPH. Upon photoirradiation of the purified reductase in the presence of [3H]NAP4-NADP+ and subsequent separation of the photolabeled species by sodium dodecyl sulfate polyacrylamide gel electrophoresis, radioactivity was found to be present predominantly in a protein band with a molecular mass of 77-kDa and accessorily in bands of 67-kDa and 57-kDa. Evidence is provided that the 67-kDa and 57-kDa proteins arose from the 77-kDa protein by proteolysis. Despite removal of part of the sequence, the proteolyzed proteins were still active in catalyzing electron transport from NADPH to cytochrome c and in binding the photoactivable derivative of NADP+.     

A novel adenylylation process in liver plasma membrane-bound proteins

Rat liver plasma membrane contains five distinct polypeptides of apparent molecular mass of 130, 120, 110, 100, and 86 kDa which are labeled upon incubation with [alpha-32P]ATP as well as with [gamma-32P]ATP. Covalently bound adenosine 5'-monophosphate to some of the polypeptides was identified using nonhydrolyzable analogues of ATP. Chase experiments of alpha-32P-nucleotide-labeled polypeptides with different nonradiolabeled phosphocompounds and sensitivity to different inhibitors demonstrate that the 86-kDa polypeptide is a phosphoesterase, forming a catalytic intermediate. On the other hand, the comparative slow rate of turnover of the polypeptides of higher molecular mass (130, 120, 110, and 100 kDa) suggests that the bound AMP could play a regulatory rather than a catalytic role. Using the nonhydrolyzable ATP analogue [alpha, beta-methylene]ATP and dilution experiments with Triton X-100-solubilized membranes, it has been possible to identify the 130-kDa adenylylated polypeptide as a possible target of an adenylylating system. These polypeptides, except the 86-kDa phosphoesterase, are affected in their electrophoretic mobility in the absence of beta-mercaptoethanol. An intercatenary disulfide bond(s) appear(s) to link the polypeptide(s) of 120 kDa and/or 110 kDa in a dimeric structure of apparent molecular mass of 240 kDa. All five polypeptides labeled with [alpha-32P]ATP are glycoproteins bound to the cell plasma membrane.     

The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.

Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.     

Subunit composition of photosystem I and identification of center X as a [4Fe-4S] iron-sulfur cluster

A photosystem I (PS-I) preparation from barley (Hordeum vulgare L.) containing the reaction center protein P700-chlorophyll a-protein 1 (CP1) and smaller polypeptides with apparent molecular masses of 18, 16, 14, 9.5, 9, 4, and 1.5 kDa has been analyzed with respect to subunit stoichiometry. CP1 contains two homologous subunits with approximate masses of 82 kDa. CP1 and the smaller polypeptides were isolated, and the amino acid composition of each component and of the PS-I preparation was determined. Based on the amino acid composition data and the determined ability of each isolated polypeptide to bind Coomassie Brilliant Blue, the PS-I complex is shown to contain 1 mol of each of the homologous 82-kDa polypeptides as well as 1 mol of the 18-, 16-, 9.5-, and 9-kDa polypeptides for each mol of P700. The total polypeptide mass of the PS-I complex is 209 kDa excluding tryptophan and approximately 220 kDa including tryptophan. The two 82-kDa subunits present/P700 provide cysteine residues for binding only one Fe-S center. In conjunction with the earlier reported binding of four iron and four acid-labile sulfides to CP1/P700 (H?j, P. B., Svendsen, I., Scheller, H. V., and M?ller, B. L. (1987) J. Biol. Chem. 262, 12676-12684), this demonstrates the center X is a [4Fe-4S] cluster and eliminates the possibility of center X being composed of two [2Fe-2S] clusters.     

Dissecting the Diphenylene Iodonium-Sensitive NAD(P)H:Quinone Oxidoreductase of Zucchini Plasma Membrane

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) in zucchini (Cucurbita pepo L.) hypocotyls. In the presence of NADPH, lipophilic ubiquinone homologs with up to three isoprenoid units were reduced by intact PM vesicles with a Km of 2 to 7 [mu]M. Affinities for both NADPH and NADH were similar (Km of 62 and 51 [mu]M, respectively). Two NAD(P)H:quinone oxidoreductase forms were identified. The first, labeled as peak I in gel-filtration experiments, behaves as an intrinsic membrane complex of about 300 kD, it slightly prefers NADH over NADPH, it is markedly sensitive to the inhibitor diphenylene iodonium, and it is active with lipophilic quinones. The second form (peak II) is an NADPH-preferring oxidoreductase of about 90 kD, weakly bound to the PM. Peak II is diphenylene iodonium-insensitive and resembles, in many properties, the soluble NAD(P)H:quinone oxidoreductase that is also present in the same tissue. Following purification of peak I, however, the latter gave rise to a quinone oxidoreductase of the soluble type (peak II), based on substrate and inhibitor specificities and chromatographic and electrophoretic evidence. It is proposed that a redox protein of the same class as the soluble NAD(P)H:quinone oxidoreductase (F. Sparla, G. Tedeschi, and P. Trost [1996] Plant Physiol. 112:249-258) is a component of the diphenylene iodonium-sensitive PM complex capable of reducing lipophilic quinones.     

Identification of the UDP-glucose-binding polypeptide of callose synthase from Beta vulgaris L. by photoaffinity labeling with 5-azido-UDP-glucose

The photoaffinity probe 5-azidouridine 5'-[beta-32P]diphosphate glucose (5N3[32P]UDP-Glc) was used to identify a 57-kDa polypeptide as a strong candidate for the UDP-Glc-binding polypeptide of UDP-glucose: (1,3)-beta-glucan (callose) synthase from red beet (Beta vulgaris L.) storage tissue. Unlabeled 5N3UDP-Glc was a competitive inhibitor of callose synthase with a Ki of 310 microM. Callose synthase was purified from plasma membranes by a two-step solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, followed by product entrapment, and photoincorporation of radioactivity from 5N3[32P]UDP-Glc was used to identify UDP-Glc-binding polypeptides that copurified with callose synthase activity. Photoinsertion into the 57-kDa band was closely correlated with all catalytic properties examined. Photolabeling of the 57-kDa polypeptide was enriched upon purification of callose synthase by product entrapment, was abolished with increasing levels of unlabeled UDP-Glc, was dependent upon the presence of divalent cations, and the pH dependence of photolabeling correlated with the pH activity profile of callose synthase. In addition, photolabeling of the 57-kDa band did not occur after phospholipase treatment, which destroys enzyme activity. The extent of labeling of this polypeptide thus correlates closely with the activity of callose synthase under a wide variety of conditions. These results imply that the polypeptide at 57 kDa represents the substrate-binding and cation-regulated component of the callose synthase complex of higher plants.     

Purification and characterization of a bioluminescence-related fatty acyl esterase from Vibrio harveyi

Vibrio harveyi extracts contain three polypeptides (32, 42, and 57 kDa) which are involved in long-chain aldehyde biosynthesis and can be labeled with [3H] tetradecanoic acid (+ATP) and/or [3H]tetradecanoyl-CoA. These proteins have been separated from other labeled bands by ammonium sulfate fractionation, and the 32-kDa polypeptide has been further purified to homogeneity by ion-exchange, gel filtration, and hydroxylapatite chromatography. In aqueous buffers at pH 7, the 32-kDa protein catalyzes the hydrolysis of tetradecanoyl-CoA at a low rate (0.01 mumol/min/mg) to form free fatty acids. The thioesterase rate is slightly increased by phosphate, which also protects the enzyme against inhibition by the sulfhydryl reagent N-ethylmaleimide. Acyl-CoA cleavage is dramatically stimulated (up to 100-fold) by certain organic solvents, in particular glycerol and ethylene glycol, with the fatty acyl group being transferred to the alcohol acceptors. These enzymatic properties may be related to the role of the 32-kDa esterase in generating fatty acids for subsequent use in the V. harveyi bioluminescent system.