Automated amino acid sequence of small peptides utilizing Polybrene

Klapper, David G.; Wilde, Charles E.; Capra, J. Donald

doi:10.1016/0003-2697(78)90282-8

Cited by 322 publications

(102 citation statements)

References 5 publications

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“…The liquid-phase sequencing was performed on a Beckman model 890C sequencer, with the 0.25 M quadrol program in the presence of polybrene [40]. The amino acid phenylthiohydantoins were identified by reversed-phase, high-performance liquid chromatography [41].…”

Section: Preparation Of L7/l12 Proteinsmentioning

confidence: 99%

Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy

1986

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The amino acid sequences of mutationally altered ribosomal protein L7/L12 from four different rplL mutants of Escherichia coli were determined and correlated with some features of the mutant ribosomes. Two of the rplL mutations are deletions around position 40, which give rise to a shortened hinge region between the two domains of L7/L12. The other two mutants harbor point mutations at position 74 (Gly+Asp) or at position 82 (Glu+Lys), which are in or close to an evolutionarily conserved sequence in the C-terminal domain. The two latter mutations are associated with decreased rates of growth and translational elongation. All four mutants show increased nonsense codon read-through in vivo. Ribosomes from one of the deletion mutants show clearly increased missense error rates in vitro.Some mutationally altered ribosomal proteins affect translational accuracy both in vivo and in vitro. The best characterized examples of far are mutants with changed S12 (rpsL), which show a more accurate phenotype, and with altered S4 (rpsD), which are less accurate during translation [l -81. These mutants [9, 101 are primarily defective in translational proof-reading step(s) [ll -151. Recent observations show that S5 (rpsE) mutations, which are associated with a ribosomal ambiguity (Ram) phenotype in vivo and in vitro, produce ribosomes which have a decreased proof-reading intensity [16,17,441. Furthermore, mutations in rpsQ (S17) and in rplF (L6) might give a more accurate translation but such mutants are not so well characterized as those affecting S4 and S12 [18, 191. Recently ribosomal protein L7/L12 was shown to be involved in the maintenance of translational accuracy. It was found that some mutations in the gene coding for this protein (rplL) increase the translational error rates both in vivo and in vitro. Furthermore, the increased error level, characteristic of the mutant ribosomes containing altered forms of L7/L12, can be correlated with a decreased proof-reading efficiency. The increased translational misreading in vivo is reversed by the introduction of an rpsL allele, which gives a Str-R phenotype. Therefore it appears that some mutants with altered L7/L12 in many respects are similar to S4 and S5 mutants [7, 9, 20, 441 in that they both increase error frequencies and suppress streptomycin-dependent phenotypes.In the present investigation we have purified the L7/L12 protein [21] from four different rplL mutants and localized the corresponding alterations by determining their amino acid sequences. In addition, we describe the effects of these mutational alterations on translation rates both in vivo MATERIALS AND METHODS Construction of strainsThe E. coli K12 strains used in this study are listed in Table 1. The rplL159, rplL265 and rplL564 alleles were introduced by PI -mediated cotransduction with argE+ into UY128 and UD132 followed by screening for rifampicin sensitivity (Rif-S) as described elsewhere [20]. Strain VT564 was used as the donor of rplL564.Construction of the rplL518 derivatives UY258 and UY278 was...

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Section: Preparation Of L7/l12 Proteinsmentioning

confidence: 99%

Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy

1986

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“…Automated sequencing was performed with a Beckman Sequencer 890C and PTH-amino acids were identified as recently described (8,18,20,41,43). PTH-2-pe-Cys was identified in the water phase.…”

Section: Amino Acid Sequence Analysismentioning

confidence: 99%

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Svensson

Larsen

Svendsen

et al. 1983

Carlsberg Res. Commun.

189

158

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The primary structure ofglucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of GI were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517-527 (1983)), identification of 574 of the 614 amino acid residues in Gl. Sequencing of glucoamylase Gl cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000.The majority of the carbohydrate of Gl is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various at-amylases.Abbreviations: BNPS-skatole = 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; ca = citraconyl; Con A = concanavalin A; DFP = diisopropylfluorophosphate; DPCC = diphenylcarbamyl chloride; EDTA = ethylenediaminetetraacetic acid, disodium salt; Hepes = N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; Nemac = N-ethylmorpholine acetate; HPLC = high pressure liquid chromatography; PTH = phenylthiohydantoin; SDS-PAGE = polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; 2-pe = 2-pyridylethyl; G I designates the larger and G2 the smaller of the forms ofglucoamylase from A. niger (44).

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“…Amino acid sequences were determined on isolated chains and CNBr fragments with a Beckman 890 automatic sequencer, using a 0.33 M Quadrol program, in the presence of Polybrene (20).…”

Section: Methodsmentioning

confidence: 99%

Organization of the diversity--joining region in rabbit immunoglobulin heavy chains as revealed by cleavage of a specific methionine residue in a100 allotype.

Tonnelle¹,

Cazenave²,

Brézin³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Three anti-micrococcus antibodies of restricted heterogeneity have been isolated from the antisera ofhomozygous alOO/alOO rabbits. Heavy chains contained an unusual methionine residue at position 87 that may correlate with the alOO specificity. From this position, the sequence of a stretch of 35-50 amino acid residues was determined, permitting the definition ofvariable (V), diversity (D), and joining (J) segments in rabbit Ig heavy chains, with their most probable boundaries. Rabbit D regions so defined appear to be highly variable, both in sequence and in length, which varies, in the heavy chains analyzed, between 6 and 11 residues. The J regions are highly homologous to the mouse J2 segment.Allotypic specificities of the a series in the rabbit, described by Oudin (1-3) over 20 years ago, have provided a valuable genetic marker of rabbit immunoglobulins, because they were shown to be expressed on the variable regions ofthe heavy chains (VH) (4). Correlations ofal, a2, a3, and a-allotypes with the presence of certain amino acids located in the VH framework have long been reported (5-9). Other allotypic specificities of the a series were more recently described in wild rabbits Oryctolagus cuniculus (10-13), including a100, alOl, and a102 specificities; these are crossreactive with certain domestic allotypes of the same series. We have looked for amino acid residues correlating with the a100 specificity. MATERIALS AND METHODSAnimals. Homozygous al00/al00 rabbits were obtained by first mating wild rabbits [trapped in the Rambouillet Forest, France, as described by Cazenave et al. (10)], heterozygous at the a locus (WR90) was a2/al00, WR78 was a3/al00) with domestic animals homozygous at the a locus (E674 was a3/a3 and E673 was al/al). Successive breedings were performed between offspring and domestic animals as indicated in Fig. 1.Immunization Procedure. Each of the three alOO/alOO homozygous rabbits F276, F479, and F490 received three weekly intravenous injections of 5 mg of Micrococcus lysodeikticus (Worthington) in saline for 20 weeks, in order to favor production of antibodies of restricted heterogeneity in large amounts (14).Preparation of Immunoglobulins and Antibodies. IgG-containing fractions were isolated from F276 antiserum by precipitation with (NH4)2SO4 (40% saturation, final concentration). After extensive dialysis against buffered saline, were purified by immunoabsorption on a column of M. lysodeikticus polysaccharide linked to activated Sepharose (15). In the case of F479 and 490 antisera, total IgG was directly isolated by ion-exchange . E674

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Automated amino acid sequence of small peptides utilizing Polybrene

Cited by 322 publications

References 5 publications

Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy

Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Organization of the diversity--joining region in rabbit immunoglobulin heavy chains as revealed by cleavage of a specific methionine residue in a100 allotype.

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