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Biomedical subjects

B Wittmann-Liebold

Publications and source records attributed to B Wittmann-Liebold.

At least 109 records · Page 6Linked to original sources
PubMed1983

The core protein of alphaviruses. 1. Purification of peptides and complete amino-acid sequence of Semliki Forest virus core protein.

The primary structure of the core protein of Semliki Forest virus has been established by protein chemical characterization of 102 peptides, generated by digestion with trypsin, pepsin, thermolysin, and by partial acid cleavage of the protein. Besides a difference in one position, the sequence as established by these experiments is in agreement with the sequence predicted from the nucleotide sequence of the mRNA [Garoff et al. (1980) Proc. Natl Acad. Sci. USA, 77, 6376-6380]. The core protein has a blocked N terminus, consists of 267 amino acid residues, and has the following amino acid composition: Asp12, Asn9, Thr16, Ser10, Glu11, Gln15, Pro23, Gly20, Ala23, Val19, Met8, Ile11, Leu9, Tyr7, Phe6, His7, Lys37, Arg15, Trp5, Cys4, and an Mr of 29919. It contains 22.1% basic amino acids, mainly lysines, compared with a total of 8.6% acidic residues. The resulting surplus of positive charge is located in the N-terminal half of the protein (predominantly arginines at positions 12-21 and lysines at positions 66-114). Other amino acids are also unevenly distributed; proline and glutamine are accumulated in the N-terminal half of the sequence whereas histidine, glycine and the acidic residues are mainly present in the C-terminal part. This distribution suggests that the virus core protein consists of two or more structural domains.

Amino Acid Sequence↗
PubMed1983

The core protein of alphaviruses. 2. Purification of peptides and complete amino-acid sequence of Sindbis virus core protein.

The primary structure of the core protein of Sindbis virus has been established by protein chemical characterization of peptides derived by enzymatic digestion with trypsin, pepsin and thermolysin and by chemical cleavage with cyanogen bromide. The peptide chain consists of 264 amino acids and has the composition Asp8, Asn8, Thr17, Ser12, Glu12, Gln14, Pro28, Gly24, Ala22, Val16, Met10, Ile8, Leu14, Tyr4, Phe9, His6, Lys25, Arg23 and Trp4 and an Mr of 29 382. Comparison of this structure with the primary structure of the SF virus core protein revealed several important common characteristics of alphavirus core proteins. 1. The N-terminal halves (1-110) of the proteins are rich in basic amino acids and proline. 2. The C-terminal part (approximately equal to 110-264/267) is highly conserved: 70% of the amino acid residues are in identical positions. 3. The conserved part contains a possible catalytic centre for the presumed protease activity of the core protein. The similarities between the primary structures of both core proteins are reflected in their predicted secondary structures.

Amino Acid Sequence↗
PubMed1983

Reversed-phase liquid chromatography of peptides for direct micro-sequencing.

Tryptic and cyanogen bromide peptides derived from yeast aspartyl-tRNA synthetase and from Escherichia coli ribosomal proteins were separated by reversed-phase liquid chromatography, employing volatile buffers of low ionic strength. The conditions used allow the performance of micro-sequencing without desalting or extensive lyophilization, and can therefore be applied to peptide mixtures containing hydrophobic fragments which tend to precipitate. To prevent losses of peptides, direct ultra-violet detection of the peptides was preferred, to detection by post-column derivatization with an additional stream splitting device. Preparative separations were performed with 5-10 nmol of peptide mixture; analytical runs were made with 5-10 micrograms of protein hydrolysate.

Amino Acid Sequence↗
PubMed1983

Direct micro-sequence analysis of peptides from Escherichia coli ribosomal proteins S11, L9 and L29 after separation by reversed phase chromatography.

Tryptic peptides of the ribosomal proteins S11, L9 and L29 were separated by reversed phase chromatography under conditions which enabled direct micro-sequencing with the 4-(dimethylamino)azobenzene-4'-isothiocyanate/phenylisothiocyanate double coupling method [Chang, Brauer, Wittmann-Liebold (1978) FEBS Lett. 93, 205-214]. The peptides were separated on a RP-18 column employing volatile buffers at pH 2.0, 4.1 and 7.8. Depending on the different chromatographic behaviour of the peptide mixture, the elution gradient was optimised for each hydrolysate using 20 micrograms of the hydrolysed protein. Preparative separations were made with 150-250 micrograms. At least 80% of the peptides could be isolated by these techniques and used for direct micro-sequencing without further purification or desalting. The results show that the high-performance liquid chromatographic method employed allows easy isolation and sequencing with minute amounts of peptides.

Amino Acid Sequence↗
PubMed1983

Purification of Escherichia coli 30S ribosomal proteins by high performance liquid chromatography.

High performance liquid chromatography was applied to the separation of proteins derived from the Escherichia coli 30S ribosomal subunit. Several methods of separating this protein mixture has been tested: size-exclusion chromatography on hydrophilic phases; ion exchange and reversed phase chromatography (on C2 to C18 hydrocarbon-bonded supports). Various elution systems were examined in order to obtain pure proteins suitable for micro-sequence analysis. The resolution and yields of the proteins varied considerably, depending on the type of support and gradient system used. The best results were achieved with uniformly globular-shaped supports of large pore size, and by combining high performance size exclusion with rechromatography on reversed phase columns. Purification conditions for the individual proteins are listed. The methods employed avoid any precipitation step and allow easy identification of the proteins by one or two-dimensional gel electrophoresis, amino-acid analysis or direct manual or automatic micro-sequencing. Since the isolation time is much reduced compared with conventional purification procedures, the proteins obtained by the techniques described here are well suited for topographical and immunological studies or reconstitution assays. Ribosomal proteins of other organisms can be separated under similar conditions.

Amino Acid Sequence↗
PubMed1982

The primary structure of the acidic phosphoprotein P2 from rat liver 60 S ribosomal subunits. Comparison with ribosomal 'A' proteins from other species.

The primary structure of rat liver ribosomal protein P2 was deduced from the sequence of the peptides. Ten peptides were obtained by cleavage of P2 with trypsin. The peptides, which accounted for the 111 residues of P2, were isolated by high voltage electrophoresis and chromatography on cellulose thin layer sheets, and the partial or complete sequence was determined by micromanual or solid-phase procedures using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate and phenylisothiocyanate. In a similar manner, the sequence of 14 peptic peptides was determined. The sequence of the NH2-terminal 30 residues of P2 was obtained by automatic Edman degradation in a sequenator. The ordering of the tryptic peptides was aided by determination of the partial or complete sequence of fragments generated with chymotrypsin, or Armillaria mellea protease, or by secondary cleavage of peptic peptides with trypsin. The carboxyl-terminal sequence was obtained from a cyanogen bromide fragment and from hydrolysis with carboxypeptidase. The sequence of protein P3 was also determined. P3 differs from P2 only in that it lacks the carboxyl-terminal 8 residues, and hence, it is likely to be a proteolytic product of P2. Rat liver ribosomal protein P2 is homologous with yeast YP A1, with Artemia salina eL12, and with Halobacterium cutirubrum L20. It is likely that rat liver P2 is also homologous with the prokaryotic ribosomal "A" proteins, Escherichia coli L7/L12, Micrococcus lysodeikticus MA1, and Bacillus subtilis L9, but that during evolution, a transposition of a portion of the molecule occurred.

Amino Acid Sequence↗
PubMed1982

Primary structure of Escherichia coli ribosomal protein S1 and of its gene rpsA.

The primary structure of proteins S1, the largest protein component of the Escherichia coli ribosome, has been elucidated by determining the amino acid sequence of the protein (from E. coli MRE600) and the nucleotide sequence of the S1 gene (rpsA, of a K-12 strain). The two methods gave results in perfect agreement except of two positions where possible strain specific differences were found. Protein S1 (MRE600) is composed of 557 amino acid residues (no modified amino acids were detected) and has Mr 61,159. The DNA sequence for protein S1 (K-12) suggests 556 amino acid residues. A computer survey of the sequence revealed three regions in S1 with a high degree of internal homology. The ribosome binding domain of S1 (NH2 terminus) does not show any preponderance of basic amino acids. The two cysteine and the majority of tryptophan residues of S1 as well as two od the three homologous regions were located in its middle region which contains the nucleic acid binding domain. The pattern of degenerate codon usage in the S1 gene is nonrandom and similar to that reported for other ribosomal protein genes.

Amino Acid Sequence↗
PubMed1982

Purification and characterization of two forms of rat plasma proangiotensin.

Two forms of rat plasma proangiotensin were purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose at pH 6.5, DEAE-Sepharose at pH 8.9, Sephadex G-150, hydroxyapatite and hexyl-agarose. Both forms were finally separated by affinity chromatography on concanavalin-A--Sepharose. Presence or absence of carbohydrate side chains seems to be the only difference between these forms of proangiotensin. Both proteins consist of single polypeptide chains having apparent molecular weights of 52000 and 55000 and isoelectric points around 4.7 and 4.4, respectively. No significant difference between the proteins could be observed with respect to the amino-terminal amino acid sequence which was found to be the same (H2N-Asp-Arg-Val) as for angiotensin I and II. Furthermore, extensive digestion with renin, releasing the decapeptide angiotensin I, did not significantly reduce the molecular weights of both polypeptides. It can therefore be concluded that the angiotensin I peptide is located at the amino terminus of the prohormone. Kinetic constants measured for the release of angiotensin I by renin were found to be Km = 5.0 microM proangiotensin and V = 270 nmol of angiotensin I h-1 unit renin-1 for the concanavalin-A-binding form and Km = 5.6 microM proangiotensin and V = 250 nmol angiotensin I h-1 unit renin-1 for the prohormone which did not bind to concanavalin-A--Sepharose. The form of proangiotensin not bound to concanavalin-A--Sepharose was found to be more thermally labile (tm of 59.0 degrees C) than the form binding to concanavalin A (tm of 61.5 degrees C, where tm = temperature at which 50% reactivity is lost).

Amino Acid Sequence↗
PubMed1982

Primary structure of Escherichia coli ribosomal protein S1 and features of its functional domains.

The complete covalent structure of ribosomal protein S1 of Escherichia coli has been determined and predictions made of its secondary structure. Protein S1 (E. coli MRE 600) is a single-chain, acidic protein with 557 amino acid residues of the composition Asp43, Asn23, Thr25, Ser25, Glu60, Gln14, Pro10, Gly48, Ala48, Val67, Met6, Ile30, Leu45, Tyr6, Phe17, His8, Lys43, Arg30, Trp7, Cys2 and an Mr of 61159. The two -SH groups of S1 are located in the central region of the chain at positions 292 and 349, the latter being the reactive group whose modification results in the reported loss of the nucleic-acid-unfolding ability of S1. The central region also contains the majority of the tryptophan, histidine and methionine residues of S1 and is predicted to have a secondary structure dominated by beta-sheets and turns. A direct proof for the location of the nucleic-acid-binding domain of S1 in the central region has recently been obtained [Subramanian et al. (1981) Eur. J. Biochem. 119, 245-249]. The N-terminal region of S1, which contains the ribosome-binding domain has a relatively high predicted alpha-helix content and no preponderance of basic amino acids. The facile trypsin-sensitive site in S1 is located at Arg-171, approximately at the border between the N-terminal and central regions. The acidic and basic amino acids of S1 (32.8% of all residues) are distributed throughout the chain, often in small clusters of between two and six residues. The amino acid sequence of S1 contains three 24-residue stretches with strong internal homology. Two of the stretches are located in the central, RNA-binding region, suggesting a possible role in the RNA-binding and helix-destabilizing functions of S1. A fragment of Mr 10(4) from the central region of S1 gives an anomalously high apparent Mr by dodecylsulfate gel electrophoresis, indicating a stable structural element therein and accounting for the apparent high Mr of S1 as determined by gel electrophoresis.

Amino Acid Sequence↗
PubMed1981

The number of copies of ribosome-bound proteins L7 and L12 required for protein synthesis activity.

Poly(U)-dependent poly(Phe) synthesis and elongation factor G (EF-G)-dependent GTPase activity were used to study the partial reconstitution of L7/L12-deficient ribosomes with proteins L7/L12 and fluorescent conjugates. Seventy-five per cent of these activities are restored when unmodified L7/L12 dimer is added to L7/L12-deficient cores at a ratio of 1:1. Various covalent fluorescent conjugates of L7/L12 bind to these cores about as well as unmodified protein. A fluorescein-5-isothiocyanate derivative of L12 shows almost no functional activity when bound. However, mixed reconstitutes of this conjugate and unmodified L12 have 75% functional activity when half the protein is unmodified. These results can be explained by a model in which there are two independent binding sites on the ribosome for two dimers of L7/L12. The binding of dimers to ribosomes is totally random and complete; the particle is 100% active so long as it has one active dimer bound to either one of the two sites. However, more complex models cannot be ruled out. An 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonic acid (IAEDANS) derivative of L7 is labeled semispecifically at the COOH terminus. This conjugate shows partial functional activity. When assay results are analyzed using the above model, it appears that the specific COOH-terminal modification has no effect on activity. However, all but a small fraction of the nonspecific IAEDANS modifications lead to inactivation.

Binding Sites↗