Further characterization of the photoreaction center from Ectothiorhodospira sp. detection of the H subunit by monoclonal antibodies

Abstract The photoreaction center isolated from Ectothiorhodospira sp. is known to contain polypeptides L,M and C (a four heme c cytochrome) in a 1:1:1 molar ratio. As a test for the presence of a H subunit, Western blots of the chromatophore and photoreaction center polypeptides were reacted with a monoclonal antibody directed against the H subunit of Rhodospirillum rubrum . The chromatophores but not the isolated photoreaction center cross-reacted with this antibody. It is concluded that the H polypeptide is lost during the purification of the photoreaction center which, in its native state, therefore contains four polypeptides including a genuine H subunit.     

由铬革屑制备可溶性蛋白及氨基酸的研究

成品铬革屑中蛋白质含量在 70 %以上 ,处理成小颗粒后与水、Ca(OH) 2 按 1∶4 0∶0 .4的比例混合 ,90℃反应 5h ,可溶性蛋白转化率近 6 0 % ,且在升温后加入Ca(OH) 2 有利于退鞣。以 6mol/L盐酸水解皮革蛋白粉制备复合氨基酸 ,水解 14h ,水解液以HD -I树脂脱色 ,氨基酸损失较少 ,且动态脱色效率明显高于静态脱色 ,采用 717树脂脱酸可获得pH4 .5 - 5的复合氨基酸溶液 ,总得率为 6 0 .1%。     

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.     

Photoreaction center of photosynthetic bacteria. 2. Size and quaternary structure of the photoreaction centers from Rhodospirillum rubrum strain G9 and from Rhodopseudomonas sphaeroides strain 2.4.1.

The photoreaction center from Rhodospirillum rubrum strain G9 binds about 6 times as much sodium dodecyl sulfate as certain proteins commonly used as molecular weight markers for sodium dodecyl sulfate--polyacrylamide gel electrophoresis. This presumably explains the apparent discrepancy between the molecular weight of the photoreaction center determined by electrophoresis (76 000) and its minimal molecular weight (87 000). The molecular weight of the photoreaction center solubilized with Triton X-100 was determined by three different methods: conventional sedimentation equilibrium, a combination of sedimentation velocity and gel filtration measurements, and sedimentation equilibrium in H2O and in D2O. Each technique required a determination of the amount of bound detergent. All three methods gave molecular weight values close to 60 000. A similar molecular weight was found for the photoactive beta gamma dimer obtained from the photoreaction center of Rhodopseudomonas sphaeroides strain 2.4.1 which, as a whole, had a molecular weight of 87 000. These results indicate that the photoreaction center from Rp. sphaeroides is an oligomer of the type alpha 1 beta 1 gamma 1. In contrast, the photoreaction center from Rs. rubrum appears to be dissociated, in solution, into a photoactive beta gamma dimer and a free polypeptide alpha.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.     

The photoreaction center of Rhodospirillum rubrum mutant strain F24.1

Rhodospirillum rubrum strain F24.1 is a spontaneous revertant of nonphototrophic mutant F24 derived from wild-type strain S1. Strain F24 shows no detectable photochemical activity and contains, at most, traces of the photoreaction center polypeptides. Strain F24.1 has a phototrophic growth rate close to that of the wild-type strain (Picorel, R., del Valle-Tascón, S. and Ramírez, J.M. (1977) Arch. Biophys. Biochem. 181, 665–670) but shows little photochemical activity. Light-induced absorbance changes in the near-infrared, photoinduced EPR signals and ferricyanide-elicited absorbance changes indicate that strain F24.1 has a photoreaction center content of 7–8% as compared to strain S1. Polyacrylamide gel electrophoresis of isolated F24.1 chromatophores shows the photoreaction center polypeptides to be present in amounts compatible with this value. Photoreaction center was prepared from strain F24.1 and showed no detectable difference with that of strain S1. It is concluded that strain F24.1 photosynthesis is due entirely to its residual 7–8% of typical photoreaction center.     

Selective activation of cyclic AMP dependent protein kinase by calcitonin in a calcitonin secreting lung cancer cell line

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of [14C]Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme. After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs. at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared. Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of [14C]Nbf-N-Lys per mol. A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues. The amino acid sequence immediately around the lysine residue labeled with [14C]Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G... .     

Characterization of methylated neutral amino acids from Escherichia coli ribosomes.

The methylated neutral amino acids from both 30S and 50S ribosomal subunits of an Escherichia coli K strain were characterized. The 50S ribosomal subunit contains three methylated neutral amino acids: N-monomethylalanine, N-monomethylmethionine, and an as yet unidentified methylated amino acid found in protein L11. Both N-monomethylalanine and N-monomethylmethionine were found in protein L33. The amount of N-monomethylmethionine in this protein, however, is variable but not more than 0.25 molecules per protein. Thus protein L33 from this E. coli K strain has heterogeneity in its N-terminal amino acid and can start with either N-monomethylalanine or N-monomethylmethionine. The N-monomethylmethionine residue was not derived from the reduction of N-formylmethionine in the protein. The 30S ribosomal subunit contains only one methylated neutral amino acid: N-monomethylalanine.     

Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride

1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3×10⁶) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15×10⁶). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2×10⁵ for mucoprotein A and 2.8×10⁴ for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.     

Purification and properties of the fatty acids synthetase complex from Neurospora crassa, and the nature of the fas-mutation.

A procedure is described for the purification of the fatty acid synthetase complex (FAS) from Neurospora crassa. The enzyme complex has a molecular weight of 2.3 times 10(6), contains 6 mol of 4'-phosphopantetheine per mol, and on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives a single band, or a closely spaced doublet, which comigrates with standard myosin (molecular weight, 2 times 10(5)). Since the slightly retarded component in the doublet accounts for all protein-bound 4'-phosphopantetheine, the complex appears to be made up of 11 to 12 equally sized subunits, 6 of which carry the acyl carrier protein function. In this unusual arrangement, notably the lack of the low-molecular-weight acyl carrier protein component seen in other FAS systems, as well as in its enzymatic properties, the Neurospora FAS complex is quite similar to the yeast enzyme. The FAS complex of a saturated fatty acid-requiring mutant, previously disignated cel-, contains less than 2% of the 4'-phosphopantetheine prosthetic groups found in the wild-type complex. The leaky phenotype of this mutant, here designated fas-, is accounted for by a residual fatty acid synthesizing activity in its FAS complex, which is several-fold higher than expected from its residual content of 4'-phosphopanthetheine.     

Photosynthetic unit size and electron-transport chain in a photoreaction center-depleted mutant of Rhodospirillum rubrum

The aim of this work was to explain the relatively fast growth of a mutant of Rhodospirillum rubrum (F24.1) which contains 7–8% of an apparently normal photoreaction center. We explored the double hypothesis that the size of its photosynthetic unit is larger than that of the wild type and that its electron-transport chain is organized in a network rather than in isolated loops. The first feature would allow faster growth under less than saturating light intensities and the second would allow faster maximal electron fluxes than would be predicted from the photoreaction center content. With respect to the first possibility, measurements of absorbance changes at 793 nm induced by short flashes of increasing intensity indicate that the photosynthetic unit of strain F24.1 is 5.6-fold larger than that of strain S1. The second possibility was verified by measuring relative electron fluxes at the photoreaction center in the two strains. This was established in the steady state from the amount of primary donor oxidized by a continuous light beam of increasing intensity. This electron flux was found to be about 70% as high in strain F24.1 as in strain S1. A more detailed study of the electron-transport chain indicated that cytochrome c₂ is by far the main secondary electron donor in strain F24.1. No evidence could be obtained for the existence of another secondary donor in that strain. The mole ratio of cytochrome c₂ to photoreaction center is about 6 in strain F24.1 as conpared to about 0.5 in strain S1. In strain 24.1, the pool of secondary donor appears to be collectively involved in the reduction of the oxidized primary donor. The replacement time at the photoreaction center of a first equivalent of oxidized cytochrome c₂ by a second equivalent of reduced cytochrome c₂ is less than or equal to 0.2 ms. The effect of the photoreaction center content on the size of the photosynthetic unit is discussed in terms of the different models proposed for the organisation of the photosynthetic unit. We propose that the electron-transport chain is organized in a network, perhaps by virtue of the lateral mobility of some of the electron carriers such as ubiquinone and cytochrome c₂.     

The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure.

Herpes simplex virus ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, which are required together for activity. Active R2 protein contains a tyrosyl free radical and a binuclear iron center. A truncated form of the R2 subunit, lacking 7 amino acid residues in the carboxyl terminus, was constructed, overexpressed in Escherichia coli and purified to homogeneity. In the presence of ferrous iron and oxygen, the truncated protein readily generated similar amounts of tyrosyl free radical as the intact protein. However, the radical showed differences in EPR characteristics in the truncated protein compared with the normal one, indicating an altered structural arrangement of the radical relative to the iron center. The truncated R2* protein was completely devoid of binding affinity to the R1 protein, demonstrating that the subunit interaction is totally dependent on the 7 outermost carboxyl-terminal amino acids of protein R2.     

Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs. I. Isolation and properties of the low-molecular-weight apoproteins.

In a continuing study of protein-lipid interactions in egg yolk, the total apoprotein mixture (i.e. the 'apovitellenins') from the high-lipid, low-density lipoprotein (density 0.97 g/ml) of the yolk from hen's eggs has been isolated in a soluble form. By gel-filtration chromatography in 6M urea the mixture has been separated into several fractions from which three new low-molecular-weight proteins (I, Ia, and II), making up about 30% of the total, have been isolated. The most plentiful of these (I) consists of stable aggregates with several identical subunits each of molecular weight about 10 000. This protein is analogous to the principal protein from the corresponding lipoprotein of emu's egg yolk, i.e. emu's apovitellenin I. Hen's apovitellenin I has a slightly different amino acid composition from that of the emu; notably it contains a sulphydryl group. The hen's protein also forms more stable aggregates that are dissociated by detergent and by guanidine hydrochloride but are stable in urea. The molecular weight of Ia is similar to that of I and the amino acid composition is the same, with the exception that Ia has a higher proportion of amide groups. It aggregates less readily than I under the same conditions. The third new protein (II, 'hens's apovitellenin II') has a molecular weight of about 20 000. It has no tyrosine or methionine residues, but contains glucosamine and has several disulphide groups. It has been isolated in very small amount only.     

The subunit structure of prealbumin

1. Prealbumin, a thyroxine-binding protein, is known to bind also to retinol-binding protein and is suspected on X-ray-crystallographic evidence of having a quaternary structure. 2. Experiments described in this paper suggest that the molecule is a tetramer of mol.wt. about 56000, which is disaggregated with some difficulty into subunits of mol.wt. about 14000. 3. The number and staining properties of the tryptic peptides indicate that the subunits are identical or closely similar. This conclusion is reinforced by comparing the sum of the amino acid compositions of the tryptic peptides with the amino acid composition of the whole protein. All minor peptides that were isolated could be shown to be derived from major peptides. 4. No evidence has been found, either from electrophoretic experiments or from amino acid sequence determination, for any dissimilarity between the subunits.     

Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties.

Kidney alkaline phosphatase was purified to homogeneity. It is a glycoprotein of about 172,000 molecular weight. Analyses of the subunit structure by sedimentation equilibrium in 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate indicate that the alkaline phosphatase is a dimer comprising two very similar or identical subunits of about 87,000 molecular weight. The native enzyme contains 4.5 +/- 0.2 g atoms of zinc per mol of protein. Reconstitution experiments from the apophosphatase show that binding of 4 Zn2+ per mol of dimer is essential for full activity. The kinetic data of Zn2+ binding to the apoprotein require at least a two-step mechanism, in which one of the steps corresponds to a conformational change within the enzyme. This paper also presents data concerning amino acid composition, sugar content, enzyme stability, absorbance index, and sedimentation velocity.     

Purification and properties of glutamate synthase from Nocardia mediterranei.

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.     

Navy (haricot)-bean (Phaseolus vulgaris) lectin. Isolation and characterization of two components from a toxic agglutinating extract

Two protein components were isolated in a highly purified state from a toxic fraction from the navy (haricot) bean (Phaseolus vulgaris). One, a lectin, strongly agglutinated horse erythrocytes and leucocytes, agglutination being readily observable with both types of cell at a lectin concentration of 4mug/ml. The other component (component 1), although possessing some similarity in composition, was thought to be non-agglutinating or, at most, only very weakly so. Component 1 had a mol.wt. of about 143000 and a subunit mol.wt. of about 37000, suggesting a tetrameric structure probably with identical subunits. Alanine was the only N-terminal amino acid identified and the molecule was notable in being devoid of tryptophan and cysteine, low in methionine and high in leucine, glutamic acid and aspartic acid. The lectin was somewhat smaller (mol.wt. about 114000) and apparently also composed of four identical subunits of mol.wt. about 30000. Dansylation showed that arginine occupied the N-terminus of the polypeptide chain. Aspartic acid, serine, threonine and leucine were the predominant amino acids of the lectin, and the sulphur-containing amino acids were entirely absent. Both constituents were glycoproteins and the compositions of the carbohydrate portions (4.9% for component 1 and 8.1% for the lectin) were generally similar, consisting of mannose and glucosamine with smaller amounts of glucose and traces of xylose and arabinose.     

Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas.

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.     

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 micromol. min(-1). mg(-1). The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP(+) oxidoreductase activity and catalyzed the reduction of NADP(+) with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.