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.     

Polypeptide chains of the large and small subunits of fraction I protein from tobacco

Crystalline Fraction I protein from tobacco has been dissociated and separated into three large subunit polypeptides and two small subunit polypeptides by isoelectric focusing in polyacrylamide gels containing 8m urea. The three large subunit polypeptides, resolved by isoelectric focusing, could not be differentiated by amino acid analysis or by fingerprinting of trypsin or chymotrypsin hydrolysates of the individual polypeptides. The two small subunit polypeptides, resolved by hydroxylapatite chromatography in 0.1% sodium dodecyl sulfate as well as by isoelectric focusing, were shown to be distinct polypeptides. The two polypeptides were shown to have different tyrosine:tryptophan ratios, shown by ultraviolet spectra in 0.1m NaOH, and different tyrosine contents shown by amino acid analysis, and they gave different peptide fingerprints after trypsin hydrolysis. The two small subunit polypeptides are concluded to be separate gene products but the three large subunit polypeptides are believed to be the result of modification of a single gene product.     

Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition

The seven major desmosomal polypeptides from isolated bovine muzzle desmosomes ranging from Mr 75 000 to 250 000 were separated by gel electrophoresis, isolated and characterized with respect to their amino acid composition and sugar content. The two largest polypeptides (bands 1 and 2), i.e. desmoplakins I and II, are similar in their amino acid composition, confirming our previous immunological and biochemical data, and display a relatively high glycine content. In contrast, the other two cytoplasmic components also believed to be associated with the desmosomal plaque, i.e. polypeptides of bands 5 (Mr 83 000) and 6 (Mr 75 000), differ significantly in their amino acid composition from the desmoplakins and from each other. All four candidate polypeptides for plaque association, i.e. bands 1, 2, 5, and 6, show no significant glycosylation. The glycoproteins 4a and 4b (Mr 115 000 and 130 000) are similar in their amino acid composition, peptide analysis and immunological reactivity. Both are relatively rich in mannose and galactose but also contain sialic acid. Our determinations also indicate that the two polypeptides differ significantly in their N-acetylglucosamine and mannose content. Most, if not all, of the sugar residues are associated with a water-soluble fragment of Mr 15 500 obtained after limited digestion with V8 protease. The glycopolypeptides obtained in band 3 (Mr 164 000-175 000) are distinct from the glycopolypeptides 4a and 4b in amino acid composition, sugar content, isoelectric pH values, certain antigenic determinants and in their pattern of cleavage products obtained by treatment with proteases or cyanogen bromide. The results identify polypeptides of bands 3, 4a and 4b as glycosylated with characteristic sugar compositions. It is suggested that the major glycoproteins (bands 3, 4a, 4b) of the desmosome are integral membrane components arranged in a special way conferring resistance to detergent treatment. The possible roles of these glycoproteins in cell recognition and in adhesive functions of the desmosome are discussed.     

The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E coli K12.

We have determined the nucleotide sequence of the rpoD gene which codes for the sigma subunit of RNA polymerase from E. coli K12. The gene, which we formerly cloned as a HindIII restriction fragment in the transducing phage, charon 25, was recloned into several plasmids. We have determined a 2600 base pair DNA sequence which includes the entire structural gene for sigma. The resulting amino acid sequence agrees with previous information obtained about sigma including the amino acid composition, partial sequence data for the N-terminus, the highly acidic nature of the polypeptide, and the cleavage pattern at cysteines. The molecular weight of 70,263 daltons calculated for the 613 amino acid polypeptides is significantly lower than had been determined previously by SDS polyacrylamide gel analysis.     

Brassica napus plastid and mitochondrial chaperonin-60 proteins contain multiple distinct polypeptides.

Plastid chaperonin-60 protein was purified to apparent homogeneity from Brassica napus using a novel protocol. The purified protein, which migrated as a single species by nondenaturing polyacrylamide gel electrophoresis, contained four polypeptides: three variants of p60cpn60 alpha and p60cpn60 beta. Partial amino acid sequence determination demonstrated that each variant of p60cpn60 alpha is a distinct translation product. During this study, additional chaperonin-60 proteins were purified. These proteins, which were free from contaminating plastid chaperonin-60, were separated into at least two high molecular weight species that were resolved only by nondenaturing polyacrylamide gel electrophoresis. These proteins contained three 60-kD polypeptides. Two of these polypeptides were recognized by existing antisera, whereas the third was not. Partial amino acid sequence data revealed that each of these, including the immunologically distinct polypeptide, is a chaperonin-60 subunit of putative mitochondrial origin. The behavior of chaperonin-60 proteins during blue A Dyematrex chromatography suggests that this matrix may be generally useful for the identification of chaperonin-60 proteins.     

Isolation and partial characterization of an arginine-rich apolipoprotein from human plasma very-low-density lipoproteins: apolipoprotein E.

A water-insoluble apoprotein was isolated from apo-VLDL by column chromatography on Sephadex G-200 in sodium dodecylsulfate followed by preparative polyacrylamide gel electrophoresis in a discontinous sodium dodecylsulfate system, or by preparative electrophoresis alone. The protein was similar in amino acid composition to the "arginine-rich protein" reported by Shore and Shore. It represented about 10% of the total protein mass of VLDL. The apoprotein showed one single band with an apparent Mr of 39000 in sodium dodecylsulfate gel electrophoresis, and was homogeneous in gel electrophoresis at pH 8.9 In 8M urea. Immunochemical studies also showed homogeneity of this protein, and antisera prepared against it did not react with any other of the well known apolipoproteins, but did react with VLDL and apo-VLDL preparations. Analytical isoelectric focusing in 8M urea resulted in a heterogeneous banding pattern showing three major polypeptides with pI values of 5.5, 5.6 and 5.75. Thus this apolipoprotein clearly differs from the apo-B and apo-C polypeptides of VLDL as well as from apoproteins A and D in its molecular weight, amino acid composition, focusing behavior and immunochemical properties.     

Chemical and spectroscopic evidence on the homology of three antitumor proteins: α-sarcin,mitogillin, and restrictocin

α-Sarcin, restrictocin, and mitogillin, three antitumor proteins, have been compared in terms of chemical composition as well as secondary and tertiary structure. The amino acid composition of the three polypeptides showed that restrictocin and mitogillin are essentially identical, α-Sarcin is also very similar to the other two proteins, although it lacks one methionine in its amino acid composition. Peptide maps of restrictocin and mitogillin coincide, except that one additional peptide is present in the mitogillin fingerprint. Although the α-sarcin map is different from the other two fingerprints, seven tryptic peptides with identical chromatographic and electrophoretic properties as well as amino acid composition were identified. The secondary and tertiary structures of mitogillin and restrictocin are identical from circular dichroism and difference spectroscopy studies. α-Sarcin has slightly different spectroscopic properties than the other two proteins. From these studies, the three proteins could be considered homologous polypeptides.     

The polypeptide composition of ubiquinone-cytochrome c reductase (complex III) from beef heart mitochondria.

The subunit structure of ubiquinone-cytochrome c reductase (complex III) has been examined and eight different polypeptides have been identified. Apparent molecular weights for each have been obtained by one or more methods including polyacrylamide gel electrophoresis in sodium doecyl sulfate and in sodium dodecyl sulfate-8 M urea and by gel filtration in sodium dodecyl sulfate and in 6 M guanidine hydrochloride. Values obtained are as follows: I, 47 500; II, 45 500; III, 29 500; IV, 27 800; V, 24 800; VI, 13 900; VII, 10 700; VIII, 4 800-9 00. Individual polypeptides have been purified and the amino acid composition of several of these have been determined. At least one polypeptide, the apoprotein of cytochrome b, is hydrophobic in character and this is a mitochondrially synthesized component (B. Lorenz, W. Kleinow, and H. Weiss (1974), Hoppe-Seyler's Z. Physiol. Chem. 355, 300). Other polypeptides including the hemoprotein of cytochrome c1 are more hydrophilic in amino acid composition.     

Biochemical characterization of rice glutelin

The two major subunits of rice glutelin, the acidic (α) and basic (β) polypeptides were purified by chromatofocusing and cation exchange chromatography, respectively. The molecular weight range of the α polypeptides was 28.5 to 30.8 kilodaltons and the molecular weight range of the β polypeptides was 20.6 to 21.6 kilodaltons. Electrofocusing in polyacrylamide gels showed that the isoelectric points of the α and β polypeptides were 6.5 to 7.5 and 9.4 to 10.3, respectively. At least 12 polypeptides of the α-group and nine polypeptides of the β-group could be separated by electrofocusing. The amino acid compositions of whole glutelin, and the purified α and β subunits were analyzed. The α subunit contained more glutamic acid/glutamine, serine, and glycine, and less alanine, lysine, aspartic acid/asparagine, and isoleucine than the β subunit. A comparison of the amino acid composition of rice glutelin subunits with those of the 11S proteins from eight other plant species indicated that there is more similarity between the β subunits than the α subunits of several diverse plant species.     

Protein Synthesis in Newcastle Disease Virus-Infected Chicken Embryo Cells

A double-isotopic label difference analysis of polyacrylamide gels has been used to distinguish between cellular and viral protein accumulation in infected cells and to quantify the kinetics of accumulation of viral polypeptides. This technique, coupled with the determination of total radioactive amino acid incorporation in infected cultures, has revealed the following kinetic patterns. Viral polypeptides are first detected in infected cultures 2.0 to 2.5 h postinfection. The rate of accumulation of radioactive amino acids in viral polypeptides increases to a maximum (30 to 35% of the rate of accumulation in uninfected control cultures), whereas the rate of accumulation of radioactive amino acids in host-cell protein decreases to a minimum (20% of the rate of accumulation in uninfected control cultures) by 5 to 6 h postinfection. All of the viral polypeptides detected late in infection are also present at the earlier times, and the major virion structural polypeptides are present in approximately the same (N/G-2, 53K) or slightly increasing (L, G-1, M) relative amounts. One peak area containing a nonstructural glycopeptide with an apparent molecular weight of 66,000 shows significant alterations in rates of accumulation during infection. Inhibition in the rate of radioactive amino acid incorporation into both trichloroacetic acid-soluble and acid-precipitable material during infection has been demonstrated. However, these two inhibition phenomena can be uncoupled temporally by incubating infected cultures at 36 C instead of the usual 40 C, suggesting that they may not be directly related.     

HLA-D region antigen-associated invariant polypeptides as revealed by two-dimensional gel analysis. Glycosylation and structural inter-relationships

Two-dimensional polyacrylamide gel analyses of immunoprecipitates of HLA-D region antigens prepared from [35S]methionine-labeled B lymphoblastoid cells revealed a number of invariant polypeptides (Ii and theta) that co-precipitate with the alpha and beta polypeptides of the class II (Ia) antigens. The invariant polypeptides comprised at least three Ii spots of Mr = 31,000 (Ii1-Ii3) and a series of six theta spots of Mr = 34,000 (theta 1-theta 6). The structural inter-relationships of these polypeptides have been investigated. Tryptic peptide fingerprints showed that Ii and theta have closely related amino acid sequences. In contrast, the fingerprints of the HLA-DR alpha and beta polypeptides clearly differed from those of theta and Ii as well as from each other. Analyses of immunoprecipitates prepared from cells cultured in the presence of tunicamycin revealed the presence of two N-linked oligosaccharides on each invariant polypeptide and suggested that the more acidic theta polypeptides (theta 1 and theta 2) differed from the other invariant polypeptides by the presence of sialic acid on one or both N-linked oligosaccharides. Removal of sialic acid by neuraminidase simplified the pattern of theta spots into three distinct Ii-related polypeptides. Endo-beta-N-acetylglycosaminidase H digestion indicated that the individual theta polypeptides represent stages in carbohydrate processing whereby Ii with two N-linked immature oligosaccharides are converted initially to theta 6-theta 3 with one immature and one complex, but nonsialylated, oligosaccharide and finally to theta 2-theta 1 with two complex oligosaccharides. Digestion of the theta polypeptides with N-acetylgalactosamine oligosaccharidase indicated that the theta spots are also derived by O-glycosylation from the Ii polypeptides. This assignment is supported by results obtained using monensin to block glycosylation within the Golgi. At least three spots persisted after complete removal of the N- and O-linked oligosaccharides, suggesting the presence of a family of invariant polypeptides differing in amino acid sequence.     

Nematode chromosomal proteins--III. Some structural properties of the histones of Caenorhabditis elegans

The histones of Caenorhabditis elegans (Nematoda) have been identified by correlating criteria of electrophoresis and amino acid composition with the five main histones from calf thymus. C. elegans H1(1) consists of at least two subtypes with approximate molecular weights of 20,000 and 18,500 daltons as resolved by SDS polyacrylamide gel electrophoresis. They are some 10% smaller than the two subtypes of calf histone H1. The differences are also corrobated by the amino acid composition of the nematode and calf H1 complements. Nematode H2A resembles calf H2A in chromatographic and electrophoretic properties and in the amino acid composition, although it lacks histidine, which seems to be replaced by lysine. Like calf H2A, it is dimorphic as shown by Triton/acid/urea polyacrylamide gel electrophoresis. The H2B complement from C. elegans consists of two proteins with a molecular weight of approximately 12,500. They can be separated by ion-exchange chromatography, but they are very analogous to each other and to calf H2B in amino acid composition. Each form is also resolved into two more subtypes by Triton/acid/urea polyacrylamide gel electrophoresis. Nematode H3 resembles calf thymus H3 in its electrophoretic behaviour; three subfractions can be distinguished in Triton/acid/urea gels. C. elegans H4 is very similar to calf H4 in its chromatographic, electrophoretic and solubility properties, but differs significantly in composition. The meaning of this difference is discussed with regard to the generally observed stringent conservation of H4 sequences between distantly related species.     

Structural Proteins of Rabies Virus

Purified rabies virions, unlabeled or labeled with radioactive amino acids or d-glucosamine, were dissociated into their polypeptides by treatment with sodium dodecyl sulfate in a reducing environment and fractionated by electroiphoresis in sodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights of individual polypeptides were estimated by comparison of their rate of migration with that of protein markers of known molecular weight. Purified viral nucleocapsid and a mixture of envelope components, isolated from virions disrupted by sodium deoxycholate, were analyzed by the same procedure. The number of molecules per virion of each polypeptide was estimated from the proportions of the separated components, the known molecular weight of the viral ribonucleic acid, and the chemical composition of the nucleocapsid. The protein moiety of the nucleocapsid particle was estimated to consist of 1,713 molecules of a major polypeptide (molecular weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecular weight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein component (molecular weight, 80,000 daltons), the viral envelope contains 789 and 1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000 and 25,000 daltons).     

Temperature-sensitive mutants of foot-and-mouth disease virus with altered structural polypeptides. I. Identification by electrofocusing

The structural polypeptides of foot-and-mouth disease virus were analyzed by electrofocusing in a polyacrylamide gel containing 9 M urea. Three versions of the technique were used to accomodate the widely differing isoelectric points of the four polypeptides. VP2 was identified by comparing mature virus with procapsids. The selective actions of proteases on virions of two serotypes and on their 12S particles were examined. From this emerged a simple test for distinguishing the similarly sized polypeptides: VP1, VP2, and VP3. The effects of carbamylation and succinylation on the charge of the polypeptides were investigated. From the properties of polypeptides modified either chemically or by mutation, it was concluded that all amino acid substitutions that might be expected to cause a charge change would be detected except for neutral-to-histidine substitutions in the most basic polypeptide, VP1. In a sample of 73 temperature-sensitive mutants, 11 classes of variant polypeptides were distinguished on the basis of charge. Their molecular weights were unchanged. Alterations were found in all structural polypeptides except VP4. Mutations affecting VP2 caused similar shifts in the precursor, VP0.     

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.     

Purification and reconstitution of the proton-pumping ATPase of fungal and plant plasma membranes

The 11S storage protein (glycinin) of soybean [Glycine max (L.) Merr., cv. Raiden] was studied by polyacrylamide gel electrophoresis and amino acid sequence analysis. It contained the following subunits composed of acidic (A) and basic (B) polypeptides: A1aB2, A1bB1b, A2B1a, and A3B4. However, it lacked polypeptides A4, A5, and B3 which are present in many other cultivars. A new acidic polypeptide called A6 was present in a low amount and was characterized by amino acid sequence analysis. It was homologous to A4, although of a smaller apparent molecular weight. Since Raiden has an average protein content of about 40% and its glycinin fraction can be purified as a 350,000 D complex which is typical of other cultivars, the results imply polymorphism with respect to glycinin subunit composition. Because there is a wide variation in the methionine content of the various subunits, these findings suggest the possibility of genetically manipulating the nutritional quality of soybean seed protein by altering glycinin subunit composition.     

Ejectisins: tough and tiny polypeptides are a major component of cryptophycean ejectisomes

Fragments of discharged ejectisomes were isolated from two Cryptomonas and a Chroomonas species by detergent treatment followed by Percoll density gradient centrifugation. The fragments withstand high concentrated detergent solutions, reducing agents and freeze-thawing. Disintegration was achieved in 6 M guanidine hydrochloride. Reassembly into long, filamentous, ejectisome-like structures occurred after dialysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the polypeptide patterns of isolated ejectisome fragments and of reconstituted ejectisome-like structures were dominated by polypeptides with relative molecular weights of approximately 6 kDa. The polypeptides were not glycosylated and did not cross-react with antisera directed against recombinant Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis. A polyclonal antiserum directed against reconstituted, ejectisome-like filaments cross-reacted with the 6-kDa polypeptides and immunolabeled extruded ejectisome filaments. Twenty amino acid residues, obtained by N-terminal amino acid sequence analysis, matched to polypeptide sequences deduced from cDNA sequences of the cryptophyte Guillardia theta. The term “ejectisins” is introduced for the 6-kDa polypeptides which represent a major component of cryptophycean ejectisomes.     

Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle

Digitonin and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine-Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine-binding activity of 2500-2800 pmol/mg protein (60-70% purity) from digitonin-solubilized membranes by a combination of wheat-germ-agglutinin--Sepharose, anion-exchange and dihydropyridine-Sepharose chromatography steps. The individual subunits were isolated in dodecyl-sulfate-denatured form from the preparation of the receptor, enriched by a two-step large-scale procedure applied to Chapso-solubilized membranes. The 160-kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160-kDa subunit is cross-linked by intramolecular S-S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N-glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.     

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 A1a+ 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.     

Multiple forms of ADPglucose pyrophosphorylase of rice endosperm

ADPglucose pyrophosphorylase from developing rice ( Oryza sativa ) endosperm was purified. The final preparation yielded 6 major protein spots as separated by two-dimensional polyacrylamide electrophoresis. All 6 polypeptides had similar molecular weights of ca 50 kDa and cross-reacted with polyclonal antibodies raised against two main protein bands among them. The results suggest that the rice endosperm ADPglucose pyrophorsphorylase is tetrameric and composed of multiple subunits with similar amino acid structure.