Correlation of 30S ribosomal proteins of Escherichia coli isolated in different laboratories

Summary Ribosomal proteins isolated from 30S subunits ofE. coli in four laboratories have been correlated by using two-dimensional gel electrophoresis, immunological techniques, amino acid compositions and molecular weights. The results are given in the Table. A common nomenclature for naming 30 S ribosomal proteins and their genetic loci is proposed.     

Isolation of 3-Hydroxy-β-ionol from Burley Tobacco

Foxtail millet (Setaria italica) proteins were fractionated into five fractions, i. e., water-, salt-, ethanol-, sodium dodecyl sulfate (SDS)- and 2-mercaptoethanol (ME)-soluble fractions, by successive extraction with various solvents from millet flour. The proportion in each fraction was 7.2, 5.6, 40, 25 and 20% respectively, of total flour nitrogen. The proteins of the ethanol- and SDS-soluble proteins were similar in amino acid composition and molecular weight distribution. More than 15 different molecular weight classes of proteins ranging from 11,000 to 150,000 were distinguished by SDS-polyacrylamide gel electrophoresis without prior reduction of their disulfide bonds. These major protein bands in the gel were estimated to be homo-olygomers (monomer, dimer, trimer, etc.) of subunit A or subunit B. The molecular weights of subunits A and B were 12,000 and 17,000, respectively. Subunits A and B were also different in amino acid composition: subunit A had higher content of methionine.     

Structure of cytochromec oxidase from baker's yeast—a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochromec binding site

Summary Cytochromec oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K).In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtartion. Their amino acid composition as well as their amino- and carboxy-terminal amino acid residues have been determined. Sequence determinations of sub-units IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids.The binding site of yeast cytochrome oxidase for cytochromec was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochromec from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochromec and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochromec and from subunit III of cytochrome oxidase.Recipient of a fellowship from the Swiss National Science Foundation. Present address: Department of Biology, University of California at San Diego, La Jolla, Calif. 92037 (USA).     

Purification of Escherichia coli 50 S ribosomal proteins by high performance liquid chromatography

The 50 S subunit proteins from the Escherichia coli ribosome were purified by size-exclusion, ion-exchange or reversed phase high performance liquid chromatography (HPLC) avoiding any precipitation or desalting procedures during isolation. Best resolution of this complex protein mixture was achieved by reversed phase chromatography on supports with short alkyl chains and C18 hydrocarbon-bonded phases; 23 out of the 32 proteins from the 50 S subunit were purified as shown by two-dimensional gel electrophoresis, amino acid analysis and direct micro-sequencing. Protein recoveries varied between 25 and 84% as determined by amino acid analysis. Ribosomal proteins of other organisms can be separated under similar conditions.     

Purification and properties of a ribosomal protein methylase from Eschericha coli Q13.

Ribosomal protein methylase has been purified from Escherichia coli strain Q13 using methyl-deficient 50S subunits as substrates. The purified enzyme (or enzyme complex) which is devoid of rRNA methylating activity is quite stable and has a pH optimum around 8.0. The Km for S-adenosyl-L-methionine is 3.2 muM. The molecular weight of the enzyme is 3.1 X 10(4); minor methylating activity was also detected for protein peaks with molecular weights of 1.7 X 10(4) and 5.6 X 10(4). Protein L11 is the major protein methylated by the purified enzyme. Product analysis revealed the presence of N epislon-trimethyllysine, a methylated neutral amino acid(s) previously observed in protein L11 and N epislon-monomethyllysine. Free ribosomal proteins were much better substrates for the methylation, indicating that methylation of 50S ribosomal proteins can occur before the complete assembly of the 50S ribosomal subunit.     

Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes

Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis. The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000. The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000. The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively. Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively. On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions.     

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.     

四季豆种子中两种蛋白质的分离纯化及其性质初步研究

四季豆（ＰｈａｓｅｏｌｕｓｖｕｌｇａｒｉｓＬ．）属于豆科蝶形花亚科野百合属,在我国分布极广,普植于温带地区．虽然四季豆是一种极普通的营养作物,但贮藏过久或煮沸不透常发生中毒．四季豆种子中成分复杂,除了蛋白组分外,还含有多种小分子组分［１］．一般认为,四季豆种子的毒性是这些组分复合作用的结果一直引人关注的则是其中的有毒蛋白．四季豆蛋白中很多是凝集素［２～４］,四季豆凝集素是一种典型的植物血细胞凝集素（Ｐｈｙｔｏｈｅｍａｇｇｌｕｔｉｎｉｎ,ＰＨＡ）,是一种寡糖结合专一性的糖蛋白,具有凝聚血细胞,刺激…     

Purification and structural properties ofRhodospirillum rubrum ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase fromRhodospirillum rubrum has been purified to homogeneity or near homogeneity using affinity chromatography techniques. The subunit molecular weight of the enzyme is 50,000. Thus, the enzyme is similar in subunit molecular weight to that found for other bacterial ADPglucose pyrophosphorylases. The amino acid composition is similar to that found for theRhodospirillum tenue enzyme. However, the N-terminal amino acid sequence of theR. rubrum enzyme shows no apparent homology with theR. tenue enzyme N-terminal amino acid sequence.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Ribosomal proteins

Summary The proteins of E. coli ribosomes were separated by a specially developed type of preparative polyacrylamide gel electrophoresis and proteins corresponding to 16 of the separated bands have so far been isolated. The amino acid compositions and the tryptic peptide maps of these proteins show certain degree of similarity as well as distinct differences. Determination of molecular weights revealed a wide range: The lowest molecular weight was 9,000 and the highest 41,000 for the proteins so far studied.The similarities between various ribosomal proteins in their amino acid compositions and their peptide maps on one hand and the wide range in their molecular weights on the other hand can be explained by a hypothesis involving gene duplications.     

The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.

A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.     

Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms

We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A. R., Kahan, L., and Cooperman, B. S. (1982) Anal. Biochem. 123, 342-348). In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes. Using an octadecasilyl silica column and a trifluoroacetic acid/acetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins. All of the known ribosomal proteins have been identified on the chromatograms with the exception of L31 and its variant, L31'. Three protein peaks, not corresponding to known ribosomal proteins, have been observed in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear. The reverse phase high performance liquid chromatography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under standard gradient conditions and containing several proteins is reapplied to the column and eluted with a shallower gradient. Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatography.     

The molecular weight of, and evidence for two types of subunits in, the molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

The weight-average molecular weight of the Mo-Fe protein isolated from Azotobacter vinelandii has been determined by sedimentation-equilibrium techniques. In buffer, the value is 245000+/-5000; in 8M-urea, the value is 61000+/-1000. The protein was separated into two components by chromatography on CM-cellulose in 7M-urea, pH 4.5. These components have similar molecular weights but were shown to differ in charge, amino acid content and arginine-containing peptides. It is proposed that the tetramer has the subunit composition (nalpha2nbeta2).     

A study of mitochondrial ribosomes from the higher plant Solanum tuberosum L.

Ribosomal subunits are isolated from potato tuber mitochondria devoid of contaminating organelles. The sedimentation constants of the two mitochondrial ribosomal subunits are 33S and 50S respectively. The apparent sizes of the high molecular weight RNAs are 19S and 25S.The proteins of these ribosomes have been analyzed by two-dimensional electrophoresis in SDS polyacrylamide gels to determine their number and molecular weights. The small subunit contains 35 protein species ranging from 8 to 60 kDa. The 50S large subunit contains 33 protein species ranging from 12 to 46 kDa. These preliminary results are the first analysis made on mitochondrial ribosomes from a higher plant.     

Major hemolymph proteins from larvae of the black swallowtail butterfly,Papilio polyxenes

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II. Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.     

Soluble lactose-binding vertebrate lectins: a growing family

Extracts of rat intestine contain nine soluble lactose-binding lectins with subunit molecular weights ranging from 14,500 to 19,000 that were purified by affinity chromatography and ion-exchange chromatography. Two of them are either identical with or closely related to other known rat lectins. A third appears to be the isolated carbohydrate-binding C-terminal domain of a known lectin but lacks the N-terminal domain presumed to mediate a different function. The others have not been described previously. Among them, the major rat intestinal lectin, RI-H, and a related protein, RI-G, have N-terminal amino acid sequences with similarities to sequences found in other known rat lectins. Therefore, these results introduce new members of a growing family of these structurally homologous soluble lactose-binding proteins.     

Rapid separation of Escherichia coli 30S ribosomal proteins by fast protein liquid chromatography (FPLC)

The proteins of the 30S ribosomal subunit from Escherichia coli have been separated by reverse-phase high-performance liquid chromatography on a short alkyl chain (C1/C8)-coated phase. The reverse-phase column was connected to a fast protein liquid chromatography (FPLC) system. The 21 proteins of the 30S ribosomal subunit were resolved into 16 peaks. Eleven proteins were isolated in purified form in a single chromatographic run as shown by polyacrylamide gel electrophoresis and amino acid analysis. Interestingly, the retention times of some proteins differed from the retention times observed on other reversed-phase support materials. The results show the speed and resolution of reverse-phase FPLC for both analytical and semi-preparative separations of 30S ribosomal proteins.     

Studies of basic proteins of 60S- and 40S-subunits of pea seed ribosomes by two-dimensional polyacrylamide gel electrophoresis]

Basic proteins of 60S- and 40S-subunits of pea seed ribosomes were studied by two-dimensional electrophoresis in polyacrylamide gel (PAAG) with subsequent electrophoresis of separated proteins in the gels containing sodium dodecyl sulfate. The proteins under study were found to be electrophoretically heterogenous and showed considerable variations in the staining by amido black and a specific distribution between the two subunits. 47 protein components were detected in the protein preparations of the 60S subunit: 18--as intensively stained, 12--as moderately stained and 17--as weakly stained spots. Presumably, the 60S subunit does not contain proteins whose molecular weights are over 60.000 or below 14.000. Two proteins have mol. weight over 50.000; other proteins have mol. weights varying between 15.000 and 30.000. 32 proteins components were revealed in the protein preparations of the 40S subunit: 15--as intensively coloured, 8--as moderately coloured and 9--as weakly coloured spots. The 40S subunit does not contain proteins whose molecular weights are over 33.000 and below 10.000. Three proteins have mol. weights over 30.000, the other proteins have mol. weights within the interval of 15.000--30.000. The amount of basic proteins in the 80S plant ribosomes is, in all probability, higher as compared to that in animal ribosomes, and this is due to the 60S subunit.     

江苏菜豆同工凝集素的分离纯化及性质研究

江苏菜豆经酸水(PH2.0)抽提,硫酸铵分级沉淀,分离植物血球凝集素(PHA-P),分子量为128000的糖蛋白,活性回收率在80％以上,PHA-P经SP-sephadexc-50离子交换层析,分成L_4,L_3E_1,L_2E_2,L_1E_3,和E_4同工凝集素。 L_4和E_4等电点为5.4和6.5。亚基分子量分别是31000和33000,并有类似的氨基酸组成。PAGE分析为单一蛋白带。红细胞凝集活性随电泳迁移速度的加快而增强,促淋性细胞分裂活性则减弱。E_4血凝活性受CalNAc,EDTA抑制和Zn~(++)的促进。