Subunit structure of the reconstitutively active cytochrome b-c1 complex. Determination of amino acids and molar distribution of subunit fractions from gel electrophoresis.

A quantitative method has been developed to analyze the amino acid composition of protein subunits directly from the Coomassie Blue-stained band of polyacrylamide gel columns after electrophoresis. It is an improved method originally reported by Houston (Houston, L. L. (1971) Anal. Biochem. 44, 81--88). The results obtained can be thus used for the calculation of the molar ratios of subunit components of protein. The manipulation of the method and computation of the results are illustrated by a very complicated lipoprotein complex. The subunit molar ratios of the reconstitutively active cytochrome b-c1 complex were determined to be 2, 2, 2, 3, 2, 2, and 5 among the seven bands of the corresponding molecular weights of 53 000, 50 000, 37 000, 30 000, 28 000, 17 000, and 15 000, from gel electrophoretic columns. The amino acid composition of each subunit fraction determined directly from hydrolysis of gel was comparable with that obtained by actual isolation of each subunit.     

Physical properties and subunits of DNA dubia erythrocruorin.

The erythrocruorin of the freshwater leech Dina dubia possessed an S20,w of 61 S and exhibited a slightly sigmoid oxygenation curve with n congruent to 1.6 and P50 = 2.4 mm at pH 7.4. A minimum mol. wt of 23 000 +/- 2100 per heme group was determined from the iron and heme contents, 0.22 +/- 0.02 and 2.92 +/- 0.35 weight %. The subunit composition of this erythrocruorin was investigated using polyacrylamide gel electrophoresis and gel filtration in sodium dodecyl sulfate at neutral pH and gel filtration at pH 9. Sodium dodecyl sulfate electrophoresis of Dina erythrocruorin revealed the presence of five subunits (1-5) with mol. wts of about 13 000, 21 000, 23 000, 25 000 and 31 000, respectively. When the erythrocruorin was reduced with mercaptoethanol prior to dodecyl sulfate electrophoresis, three subunits (I-III) were observed, two possessing molecular weights in the range 12 000-14 000 (I and II) and one possessing a molecular weight of about 28 000. One of the subunits I, II, was provided by the dissociation of the 31 000 subunit. Subunit III (28 000) consisted of subunits 2, 3, and 4. It is likely that not all of the polypeptide chains constituting Dina erythrocruorin are associated each with a heme group.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

On the molecular weight of mitochondrially synthesized subunits of rat liver cytochrome oxidase.

The subunit composition of cytochrome c oxidase from rat liver mitochondria was studied by dodecylsulfate polyacrylamide gel electrophoresis. The apparent molecular weight of the seven subunits are in reasonable agreement with published data on cytochrome c oxidase subunits from other sources. Two additional subunits were found if the electrophoresis was performed with 8m urea, due to splitting of the smallest subunit. Performic acid oxidation of the isolated subunits I and II increased the apparent molecular weights from 38000 to 48000 and from 24500 to 29000, respectively, accompained by a normalization of the anomalous behaviour of subunit I in the Ferguson plot. It is suggested that performic acid, by splitting extremely inaccessible disulfide bridges, mediates full complexing of the subunits by dodecylsulfate, thus permitting the determination of the real molecular weights by dodecylsulfate polyacrylamide gel electrophoresis.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Amino acid incorporation in relation to molecular weight of proteins in young and adult brain

Rates of protein synthesis were studied in immature and adult rat brain tissue. After an amino acid incorporation period, in vivo or in incubated slices from brain, the soluble protein was fractionated according to molecular weight by column chromatography. In examining soluble whole proteins, no direct correlation between molecular weights and synthesis rates could be established; the highest synthesis rates were found in fractions around 70,000 MW and below 10,000. Incorporation into the subunits after fractionation by SDS gel electrophoresis was proportional to subunit molecular weight, with rates of incorporation into the largest subunits being the highest. The results suggest a relationship between turnover rate and structure of subunits of brain proteins.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin.

The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine.     

Subunit composition of mitochondrial F1-ATPase isolated from Saccharomyces carlsbergensis

1. The subunit stoicheiometry of mitochondrial F1-ATPase from yeast (Saccharomyces carlsbergensis), grown in the presence of [3H]leucine and uniformly labelled [14C]glucose, has been determined. 2. The stoicheiometry on the basis of radioactivity is : alpha: beta: gamma: epsilon = 3 : 3 : 1 : 1. The amount of the smallest subunit, epsilon, could not be measured by this method. 3. The molecular weights of the subunits, determined by urea-SDS gel electrophoresis, are 53 000, 50 000, 33 000, 12 500 and 6500, respectively. The calculated molecular weight of the ATPase is 360 000, assuming the presence of one epsilon subunit per F1. 4. The amino acid composition of the total ATPase and of the individual subunits has been determined. 5. The aurovertin-binding properties of F1 are discussed in relation to the subunit stoicheiometry.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

不吸水链霉菌葡萄糖异构酶的物化表征

葡萄糖异构酶是一种催化葡萄糖异构为果糖的酶。本文用紫外吸收光谱、红外光谱、氨基酸组分分析、聚丙烯酰胺凝胶梯度电泳、SDS-聚丙烯酰胺凝胶电泳、超薄 层聚丙烯酰胺凝胶等电聚焦电泳技术研究了不吸水链霉菌嗜热亚种M1033菌株产生的葡萄糖异构酶的一些物化性质。结果表明由本实验室制备的均一葡萄糖异构酶的A₂₈₀与A₂₆₀的比值是1.76。它是由一个亚单位组成的酶分子。最小分子量是49000。pI值是5.2。氨基酸组分与其它来源的葡萄糖异构酶的氨基酸组分相比较有一些差异,其中Glu,Gly,ALa和Leu的含量都此其它异构酶的高,而Met,Trp,Asp,Thr则比其它葡萄糖异构酶的低。     

Studies on proteins of animal ribosomes

Summary Total ribosomal protein from rat liver ribosomes can be separated into about 20 chief electrophoretic fractions by preparative polyacrylamide gel electrophoresis. Ten electrophoretically homogeneous fractions have been isolated from the total mixture of ribosomal protein, respectively from proteins, prefractionated by CM-cellulose chromatography. Amino acid composition and molecular weights of some fractions have been determined. The amino acid composition of these fractions and of the total protein mixture are basically similar but there are also significant differences with regard to some amino acids. The molecular weights of the proteins studied are in the range between 7,000 and 29,000.     

Capsid Polypeptides of Mouse Elberfeld Virus I. Amino Acid Compositions and Molar Ratios in the Virion

The four major polypeptide chains (alpha, beta, gamma, delta) constituting the capsid protein of mouse Elberfeld (ME) virus were isolated by preparative electrophoresis on polyacrylamide gels, and the amino acid composition of each chain was determined. In addition, the molecular weights of the smallest chains of ME virus, mengovirus, and poliovirus, which had previously been determined by gel electrophoretic methods, were redetermined by gel filtration chromatography in 6 m guanidine hydrochloride. Each was found to have a molecular weight about 7,300. Using the reevaluated molecular weights and the known amino acid compositions of the chains, the molar ratio of each chain in the ME virion was determined by quantitative analysis of the distribution of radioactivity in the electrophoretically separated chains of virus which had been specifically radiolabeled with leucine or with methionine. Equimolar proportions of all four chains were found in the virion.     

Molecular weight hetergeneity of proteins of the ribosomes and ribosomal subunits from dry pea seeds]

The molecular weight distribution of the total protein of ribosomes and ribosomal subunits isolated from dry pea seeds was studied by electrophoresis in polyacrylamide gel, containing sodium dodecyl sulfate. It was demonstrated that overall protein of 80 S ribosomes is separated into a number of fractions with molecular weights of 10000-64000. Treatment of ribosomes with 0.5 per cent tritone, 0.5 per cent and 1 per cent deoxycholate does not change the general pattern of the molecular weight distribution of ribosomal proteins. The large subunit reveals 19 protein zones (14 major and 5 minor zones), their molecular weights are varying from 10000 to 54000. The majority of proteins of the large subunit have molecular weights of 14000--32000. The molecular weights of 17 protein zones of the small subunit (7 major and 10 minor zones) vary from 10000 to 64000. The majority of proteins of both large and small subunits have molecular weights of 14000--32000. Electrophoretic separation of proteins in the split gel confirmed the fact that the proteins of large subunit differ in molecular weights from those of the small subunit. Thus, ribosomal proteins of pea seeds are shown to produce a typical (for 80S ribosomes) pattern of molecular weight distribution under polyacrylamide gel electrophoresis in the presence of sodium dodecul sulphate.     

Purification and properties of the latent F1-APTase of Micrococcus lysodeikticus.

The latent coupling factor (F1)-ATPase of Micrococcus lysodeikticus has been purified to homogeneity as determined by a number of criteria including, nondenaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release of F1 from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) can be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a molecular weight of 400 000. The ATPase contained five different subunits alpha, beta, gamma, delta, and espsilon and their molecular weights determined by SDS-polyacrylamide gel electrophoresis were 60 000, 54 000, 37 000, 27 000 and 9000, respectively. The subunit composition was determined with 14C-labelled, F1-ATPase prepared from cells grown on medium containing [U-14C]-labelled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3 : 3 : 1 : 1 : 3.     

Developmental profile, isolation, and biochemical characterization of a novel lipoglycoheme-carrier protein from the American dog tick, Dermacentor variabilis (Acari: Ixodidae) and observations on a similar protein in the soft tick, Ornithodoros parkeri (Acari: Argasidae)

A novel lipoglycoheme-carrier protein (CP) in the American dog tick, Dermacentor variabilis (Say) has been purified and characterized. CP was purified by native-PAGE from partially fed virgin females. CP has a density of 1.25 g/ml with a molecular weight of 200 K by native-PAGE and 340 K by gel filtration chromatography. CP is comprised of two majour subunits, 98 K and 92 K in molecular weight by SDS-PAGE. Separate amino acid composition of the two subunits indicated high contents of As(x), Gl(x) and leucine. However, the N-terminal amino acid sequence of the two subunits was only 13% identical. The lower molecular weight subunit showed 61% identity to artemocyanin (biliprotein) in fairy shrimps, 46% identity to minor vitellogenin in chickens and 13% identity to vitellin of the black-legged tick. No similarity match was found for the other subunit. CP is a lipoglycoheme-protein as indicated by selective staining of native-PAGE gel for lipids, carbohydrates and heme. Lipid analysis by thin layer chromatography revealed the presence of cholesterol, phospholipids, monoacylglycerides, triacylglycerides and free fatty acids. Heme associated with purified CP demonstrated a lambda(max) of 397.5 nm while the lambda(max) of crude hemolymph plasma was 402.5 nm. The presence of CP in whole body homogenates of eggs, unfed and fed larvae and fed nymphs as well as in the plasma of unfed and fed adults including vitellogenic females was demonstrated by native-PAGE. Although a protein of analogous size was not found in the soft tick, Ornithodoros parkeri Cooley, a high molecular weight protein (500 K) is the predominant plasma protein in both unfed and fed male and female adults of that species as determined by native-PAGE. Also, CP appears to function as a biliprotein which sequesters heme.     

Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.     

Heterogeneity of delta-crystallins of the embryonic mallard lens. Correlation between subunit compositions and isoelectric points.

delta-Crystallins from the lenses of embryonic mallards (Anas platyrhynchos) were analyzed with respect to native and subunit molecular weight, subunit composition, and isoelectric point. NaDodSO4-urea-polyacrylamide gel electrophoresis showed that unfractionated mallard delta-crystallins are composed of approximately equal amounts of subunits with molecular weights near 47 000 and 48 000. Agarose gel chromatography showed that the embryonic mallard delta-crystallins have native molecular weights slightly less than 200 000. Thus, embryonic mallard delta-crystallins appear to be tetramers. Five major and nine minor delta-crystallins were resolved by isoelectric focusing. The five predominant delta-crystallins each cross-reacted with antichick delta-crystallin antiserum, and each had a different proportion of the larger and smaller subunits, indicating a direct relationship between the isoelectric point and the subunit composition. The presence of numerous, minor species of native delta-crystallins with different isoelectric points suggested that the subunits possess charge heteogeneity as well as size heterogeneity.     

Zein of maize grain. Preparation and characterization]

A laboratory procedure for isolation and purification of zein from grains of 4 varieties of Maize was described. The preparations were characterized by their physicochemical properties. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS), native zein (from INRA 260 hybrid) was resolved into 2 major classes with average molecular weights of 45,000 and 22,000. After reduction with mercaptoethanol zein contained only two subunits of 22,000 and 24,000 daltons. Upon starch gel electrophoresis in 6 M urea at pH 3.5, native zein exhibited five major or medium intensity bands and several minor ones. The latter, under reducing conditions, disappeared to reinforce the major bands or to yield some new minor bands. Amino acid analysis revealed a very low content of lysine. The NH2-terminal amino acids were determined to be threonine and phenylalanine with a preponderance of the former. Zeins isolated from the varieties studied appeared tohave the same NH2-terminal residues and similar amino acid compositions with an arginine/histidine ratio ranging from 1.1 to 1.2. They differed in relative importance of components, detected by electrophoresis in the presence of SDS or urea. Changes in zein characteristics with the grain genotype allow one to conclude that the components of molecular weights of 22,000 and 24,000 consist of several subunits differing in charge and amino acid content.     

Isolation of a heme binding subunit from bovine heart cytochrome C oxidase.

A subunit which retains heme has been isolated and purified up to a homogenous form on polyacrylamide gel electrophoretic column in the presence of sodium dodecyl sulfate and β-mercaptoethanol from cytochrome oxidase. The separation of the subunit does not rely on any detergent except cholate used in the preparation of cytochrome oxidase. The purification involves a reaction with pyridine, pH precipitation, and DEAE-cellulose column chromatography. The purified subunit has a molecular weight of 11,600 daltons and contains more than 40 nmol Fe per mg protein; the lower iron content than the calculated value is apparently due to the loss of heme $\text{a}$ in the course of the purification. The subunit is freely soluble in aqueous solution at neutral pH to give a dark green color. Spectral properties and amino acid composition of this subunit have been studied.