The subunit structure of the cytochrome c oxidase complex.

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.     

Characterization of a seventh different subunit of beef heart cytochrome c oxidase. Similarities between the beef heart enzyme and that from other species.

Beef heart cytochrome c oxidase has been resolved into seven subunits by electrophoresis in highly cross-linked gels containing urea and sodium dodecyl sulfate. The molecular weights of the polypeptides are estimated to be I, 35 400; II, 24 100; III, 21 000; IV, 16 800; V, 12 400; VI, 8200; and VII, 4400. It has been shown that subunits II and III can coelectrophorese on standard sodium dodecyl sulfate-polyacrylamide gels and appear as a single component with an apparent molecular weight of 22 500. This accounts for previous reports that the beef heart enzyme contains only six subunits. Amino acid analysis of the isolated subunits I, II, and III revealed that they have polarities of 35.5, 44.7, and 39.9%, respectively. All three subunits have an extremely high leucine content and a low percentage of basic amino acids relative to subunits IV-VII. The size, number, and properties of subunits in the beef heart cytochrome c oxidase complex suggest that it has essentially the same subunit structure as the complexes isolated from Saccharomyces cerevisiae and Neurospora crassa.     

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.     

Cytochrome c oxidase from bakers' yeast. III. Physical characterization of isolated subunits and chemical evidence for two different classes of polypeptides.

Earlier studies have shown that cytochrome c oxidase from bakers' yeast is an oligomeric enzyme which contains three polypeptides (I to III) synthesized on mitochondrial ribosomes and four polypeptides (IV to VII) synthesized on cytoplasmic ribosomes. These polypeptide subunits have now been isolated by a simple protocol which utilizes differences in polypeptide charge, solubility, and size. Their molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, gel filtration in the presence of guanidine hydrochloride, and amino acid analysis were: I, 40,000; II, 33,000; III, 22,000; IV, 14,500; V, 12,700; VI, 12,700; and VII, 4,600. All seven polypeptide subunits exhibited acidic isoelectric points; cytoplasmically made subunits were more acidic than mitochondrially made ones. The amino acid composition of two mitochondrially made subunits and two cytoplasmically made subunits was determined. The two mitochondrial translation products, I and II, contained only 34.7% and 42.1% polar amino acids, respectively, whereas the two cytoplasmic translation products, IV and VI, contained 48.3% and 49.3%, respectively. This agreed with the observation that Subunits I and II are very insoluble, requiring detergents for solubility, whereas Subunits IV and VI are water-soluble in the absence of any added detergent. These results indicate that the cytochrome c oxidase subunits synthesized on mitochondrial and cytoplasmic ribosomes are fundamentally different in size, isoelectric properties, and hydrophobicity. They also suggest the possibility that at least some of the mitochondrially made subunits are buried in the lipid phase of the mitochondrial inner membrane.     

Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure

A sodium dodecyl sulfate-gel electrophoretic procedure which allows the separation of isolated cytochrome c oxidase from different mammalian sources into 13 different polypeptides is described. Application of the silver-staining procedure results in the same protein pattern as obtained by Coomassie blue staining. From the correlation of the gel bands with 12 isolated polypeptides from which the complete amino acid sequence is known, it is concluded that mammalian cytochrome c oxidase consists of 13 different polypeptides which can all be separated by the described procedure.     

Studies on cytochrome c oxidase, VIII. The amino acid sequence of polypeptide VII.

The sequence determination of polypeptide VII from beef heart cytochrome c oxidase is described. The amino acid sequence is deduced from overlapping tryptic peptides and peptides obtained after cleavage with Staphylococcus aureus protease. The protein consists of 85 amino acids corresponding to a Mr of 10026, in agreement with a value of 9500 obtained by sodium dodecyl sulfate gel electrophoresis. The amino acid sequence around the only methionine present is very similar to sequences around the invariant heme binding methionine of the cytochrome c family. This similarity suggests that the protein is one of the heme bindings subunits of the oxidase.     

Identification of subunits of bovine heart cytochrome oxidase.

Purified lipid-depleted cytochrome oxidase, at purity of 12--14 nmol heme a per mg protein, has been shown to contain seven non-identical subunits in the ratio of unity. Their molucular weights on polyacrylamide gel are, in thousands, 40, 21, 14.8, 13.5, 11.6, 9.5, and 7.6 from gel electrophoresis after dissociation in sodium dodecyl sulfate and beta-mercaptoethanol. The molar ratio is determined by the amino acid composition of each subunit obtained from direct hydrolysis of the stained polyacrylamide gel slices. The amino acid composition of the isolated subunits I and II determined by regular hydrolysis method is found practically the same as that from direct hydrolysis of gel slices. The heme-associated polypeptides are identified with subunits of molecular weights of 40.10(3) and 11.6.10(3). One of the two coppers associated with the polypeptide of molecular weight of 21 000. The second copper may be associated with heme in the subunit of 40.10(3). Evidence of the existence of interpolypeptide disulfide linkages is presented.     

Resolution and reconstitution of succinate-cytochrome c reductase: preparations and properties of high purity succinate dehydrogenase and ubiquinol-cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c1 III complex). An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinae dehydrogenase fraction and the cytochrome b-c1 complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c1 complex was separated into chtochrome b-c1 III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate. The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 anbd 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low Km ferricyanide reductase activity of 85 mumol succinate oxidized per min per mg protein at 38 degrees C. Chemical composition analysis of cytochrome b-c1 III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation. When these three components were mixed in a proper ratio, a thenoyltrifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH2: cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands. 2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c1, band 5 and Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c1 and identified with the so-called oxidation factor, and band 7 a polypeptide peptide associated with cytochrome b. 3. The validity of molecular weight estimate for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively. 4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide band.     

Preparation and chemical characterisation of calf Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein preparations were obtained from calf urine by 1.0 M NaCl precipitation followed by 4 M urea/Sepharose 4B chromatography. By using 0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis a molecular weight of 86 500 +/- 4500 (n = 12) was calculated for the glycoprotein. Amino acid and carbohydrate analyses were performed, the carbohydrate composition being (in residues per 100 amino acid residues in the glycoprotein): fucose, 0.90; galactose, 4.82; mannose, 4.63;N-acetylglucosamine, 7.36; N-acetylgalactosamine, 1.38; sialic acid, 2.93. Under conditions of mild acid hydrolysis (0.05 M H2SO4, 80 degrees C, 1 h) the calf Tamm-Horsfall glycoprotein preparations were degraded partially into two lower molecular weight fragments (approximate Mr 66 000 and 51 000), as shown by polyacrylamide gel electrophoresis, both fragments being periodic acid-Schiff reagent positive.     

Zein of maize grain: I — isolation by gel filtration and characterization of monomeric and dimeric species

Unreduced zein chromatographed on Sephadex G 200 in 8 M urea, on G 100 in 1.5 or 2.5% sodium dodecyl sulfate (SDS) and on hydroxypropylated G 100 in 70% ethanol was resolved into two minor fractions A and B and two major ones D and M irrespective of the medium. The quantitative importance of the fraction M was dependent on the isolation conditions of zein. It decreased from 53% of the proteins contained in ethanolic extract and chromatographed as they were extracted, to 40% of the purified zein. The molecular weight values obtained from SDS-polyacrylamide gel electrophoresis and amino acid compositional data indicated that fractions D and M, as isolated from purified zein in the presence of ethanol, represented respectively dimeric and monomeric forms of a mixture of M_r 22 000 and 24 000 polypeptides with threonine or phenylalanine as NH₂-terminal residue.Electrophoretic analysis of selectively carbamylated fraction M on starch gel at pH 3.5 revealed that zein subunits comprised several polypeptides differing in the number and the nature of basic amino acids. At least one of these polypeptides contained one lysyl residue.     

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix

A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be -190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7, G. metallireducens cytochrome c did not show Fe(III)-reducing activity.     

Isolation and characterization of light harvesting bacteriochlorophyll · protein complexes from Rhodopseudomonas capsulata

The isolation of two native light harvesting bacteriochlorophyl · protein complexes from Rhodopseudomonas capsulata is described. The light harvesting bacteriochlorophyll I (B 875) has been isolated from the blue-green mutant Ala⁺ lacking both carotenoids and light harvesting bacteriochlorophyll II. Light harvesting bacteriochlorophyll I is associated with a protein (light harvesting band 2) of 12 000 molecular weight.Light harvesting bacteriochlorophyll II complex has been isolated from the mutant Y5 lacking a reaction center and light harvesting bacteriochlorophyll I. Light harvesting bacteriochlorphyll II (B 800 + 850) together with carotenoids is associated with two polypeptides (light harvesting bands 3 and 4) having molecular weights of about 8000 and 10 000 (sodium dodecyl sulfate polyacrylamide gel electrophoresis). A third protein (light harvesting band 1) is in the purified light harvesting II fraction (mol. wt. approx. 14 000), but not associated with bacteriochlorophyll or carotenoids. The amino acid composition of the 3 antenna pigment II proteins is given. The polarity of these proteins was found to be 48%. From the amino acid composition the following molecular weights were calculated band 1: 17 350, band 3: 13 350 and band 4: 10 500.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V--VII were rather resistant to degradation. From the relative inaccessibility of subunits V--VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Modifications of Δ5-3-ketosteroid isomerase induced by ultraviolet irradiation in the presence of the solid-phase photoaffinity reagent Δ6-testosterone agarose

The nature of the products formed during the photoinactivation of Δ⁵-3-ketosteroid isomerase in the presence of the solid-phase photoaffinity reagent Δ⁶-testosterone succinyl agarose has been investigated after ultraviolet irradiation. The polypeptide products eluted from the agarose phase by sodium cholate, sodium dodecyl sulfate, and pH 10.5 triethylamine buffer have been characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, pH 4–6 gel isoelectric focusing, and amino acid analysis. The amino acid compositions of the cholate eluted and SDS eluted products are found to be similar to that of native isomerase, whereas the covalently bound polypeptide eluted by pH 10.5 triethylamine possesses a distinetly different composition. Digestion of the covalently bonded isomerase polypeptide with trypsin yields an agarose-bound peptide fraction that has been characterized by its amino acid composition. This composition is different from that of the undigested covalently bound polypeptide and suggests that the site of covalent attachment lies somewhere between residues 28 and 45 of the isomerase polypeptide.     

Extraction of fixed, stained protein bands from gels for micro amino acids analysis using o-phthaldialdehyde.

A method is presented for extraction of fixed, stained protein bands from polyacrylamide gels suitable for automated fluorescence analysis of amino acids using o-phthaldialdehyde. Bands, containing microgram quantities of protein and stained with Coomassie blue, are extracted from homogenized gel slices with sodium dodecyl sulfate. The Coomassie blue and sodium dodecyl sulfate do not interfere with the amino acid determination, and contamination by ammonia from the gels is low. The method has been applied to the analysis of human carbonic anhydrase C, and the amino acid composition is found to be similar to that obtained by other methods requiring larger amounts of protein.     

Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III

By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron-sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Isolation of coconut storage proteins by polyacrylamide-gel electrophoresis

By combining sodium dodecyl sulfate (SDS)-gel electrophoresis with a new chilling technique for visualization of protein-SDS complexes in polyacrylamide gels, a process has been developed which will permit the isolation of milligram quantities of pure polypeptides. Using this technique, we have isolated two molecular weight classes of polypeptides from coconut storage globulins and determined the amino acid composition of each. When the two amino acid compositions were summed on a molar basis, the result agreed reasonably well with the amino acid composition of the starting material with the exception of cystine. Apparently, some contaminant from the polyacrylamide caused its destruction to be accelerated during hydrolysis.     

Large scale isolation and properties of subunits from bovine heart cytochrome oxidase

The subunits of the cytochrome oxidase from bovine heart were isolated in large quantities suitable for amino acid sequence studies. The preparation of subunits III, IV, V, VI, and VII for sequence determination can be achieved without employing sodium dodecyl sulfate. The method presented essentially involves pyridine extraction, pH fractionation, ammonium sulfate fractionation, and various types of column chromatography. However, subunits I and II can be prepared only in the presence of sodium dodecyl sulfate by molecular sieve chromatography; subunit III can also be isolated in this manner. The separation of subunits is found to be hindered by phospholipids associated with the enzyme and therefore the phospholipid-depleted preparation is used as the starting material. The molecular weights of subunits I, II, III, IV, V, VI, and VII are 40,000, 21,000, 14,800, 13,500, 11,600, 9,500, and 7,600, respectively. These values are based on the results of the conventional Weber and Osborn method of gel electrophoresis in the presence of sodium dodecyl sulfate. The amino termini of subunits I and II have been determined as N-formylmethionine, and those of subunits III, IV, V, VI, and VII are alanine, alanine, serine, alanine, and an N-acetyl-blocked residue, respectively. The carboxyl termini for subunits I to VII are lysine, leucine, lysine, histidine, valine, isoleucine, and valine, respectively. The complete amino acid sequence of some subunits has been published and that of other subunits will be reported elsewhere in collaboration with the Amino Acid Sequence Group of Cytochrome Oxidase at the University of Hawaii.