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K Rose

Publications and source records attributed to K Rose.

At least 145 records · Page 8Linked to original sources
PubMed1985

Sequence determination of the Sendai virus fusion protein gene.

From a genomic DNA library of Sendai virus, we have identified and sequenced clones corresponding to the F glycoprotein gene. The limits of the F gene region were defined by mapping the 5' and 3' ends of the mRNA with S1 nuclease. The Sendai virus F gene is 1821 nucleotides long. The predicted primary translation product of the single long open reading frame would code for a protein of 565 amino acids, containing a putative signal peptide, three carbohydrate addition sites, a hydrophobic region corresponding to the known cleavage/activation site of FO, and a long, very hydrophobic region near the C-terminus which probably represents the transmembrane region of the protein. The signal peptide cleavage site of the mature protein was determined by mass spectrometry. Interestingly, the amino acid sequence surrounding the cleavage/activation site of the Sendai virus F protein shows significant homology to the same region of the influenza B and C virus HA proteins, suggesting that these genes may have evolved from a common ancestor. The ability of the Sendai virus F protein to fuse membranes relative to its primary structure is discussed.

Animals↗
PubMed1984

A case of spurious product formation during attempted resynthesis of proteins by reverse proteolysis. Some batches of 'pure' glycerol contain cross-linking agents.

In cases where enzyme-catalysed synthesis of a peptide bond is being used to re-form a protein from two large peptide fragments, the organic co-solvent chosen has so far been glycerol, for most solvents in use in small-molecule systems are potent protein denaturants. We have found, however, that impurities contaminating certain batches of glycerol are effective in cross-linking the complexes formed by these peptide fragments, thus mimicking the enzyme-catalysed process. In one such case, the reported re-formation of cytochrome c from a two-fragment complex system, cytochrome c-T, the extent and rate of conjugate formation duplicates that reported for enzymic resynthesis. We observed no difference between mixtures containing or lacking enzyme. We warn of the danger of confusion possible to those engaged in studies of resynthesis, and suggest a simple control of purchased glycerol to avoid it. We recommend similar caution to those (X-ray crystallographers and others) who seek to stabilize protein solutions by adding large quantities of glycerol.

Amino Acids↗
PubMed1984

A mass-spectrometric investigation of the mechanism of the semisynthetic transformation of pig insulin into an ester of insulin of human sequence.

In the trypsin-mediated semisynthetic transformation of pig insulin into an ester of insulin of human sequence, the B30 alanine residue of the pig hormone is replaced by an ester of threonine. The mechanism of this reaction was investigated by carrying out the transformation in a medium containing water enriched with 18O. Subsequent analysis by combined g.l.c.-mass spectrometry demonstrated that the oxygen isotope, is incorporated into the B29 carbonyl group of the insulin ester product. This result, together with those of appropriate controls, supports the conclusion that the transformation occurs, in the system studied, by a mechanism involving hydrolysis followed by coupling, and not by direct transpeptidation as has been previously found the case for another such system [Markussen & Schaumberg (1983) in Peptides 1982 (Bláha & Malon, eds.), pp. 387-394, W. de Gruyter, Berlin and New York].

Amino Acid Sequence↗
PubMed1984

An improved procedure, involving mass spectrometry, for N-terminal amino acid sequence determination of proteins which are N alpha-blocked.

A modification to a previously described procedure [Gray & del Valle (1970) Biochemistry 9, 2134-2137; Rose, Simona & Offord (1983) Biochem. J. 215, 261-272] for mass-spectral identification of the N-terminal regions of proteins is shown to be useful in cases where the N-terminus is blocked. Three proteins were studied: vesicular-stomatitis-virus N protein, Sendai-virus NP protein, and a rabbit immunoglobulin lambda-light chain. These proteins, found to be blocked at the N-terminus with either the acetyl group or a pyroglutamic acid residue, had all failed to yield to attempted Edman degradation, in one case even after attempted enzymic removal of the pyroglutamic acid residue. The N-terminal regions of all three proteins were sequenced by using the new procedure.

Amino Acid Sequence↗
PubMed1984

Preparation and analysis of the nucleocapsid proteins of vesicular stomatitis virus and sendai virus, and analysis of the sendai virus leader-NP gene region.

A procedure is presented for isolating the nucleocapsid proteins, N and NP from vesicular stomatitis virus and Sendai virus respectively, in soluble form. These proteins were suitable for the determination of their blocked amino-terminal peptide sequences by gas-liquid chromatography/mass spectrometry at the low nanomole level. The N protein prepared by this procedure was previously shown to retain some of its expected biological activity. The sequence of 626 nucleotides from the 3' end of the Sendai virus genome, which includes the first one-third of the NP gene, was determined. Using this information, primer extension studies on intracellular Sendai virus mRNAs allowed the determination of the structure of the leader-NP intervening sequence and the 5' end of the NP mRNA. Comparison of the amino termini of the nucleocapsid proteins with their respective mRNA sequences revealed that these proteins are similarly processed in vivo.

Amino Acid Sequence↗
PubMed1984

Analysis of the Sendai virus M gene and protein.

The nucleotide sequence of the Sendai virus M (matrix or membrane) gene region was determined from cloned genomic DNA, and the limits of the M mRNA were determined by S1 nuclease mapping. The M mRNA is 1,173 nucleotides long and contains a single long open reading frame coding for a protein of 348 amino acids. The amino acid sequences of the N- and C-terminal peptides of the M protein were obtained by mass spectrometric analysis and correspond to those predicted from the open reading frame, with the N terminus modified in vivo by cleavage of the initiating methionine and acetylation of the following amino acid. The amphiphilic nature of the M protein structure is discussed.

Amino Acid Sequence↗
PubMed1983

The semisynthesis of analogues of cytochrome c. Modifications of arginine residues 38 and 91.

The arginine residues at positions 38 and 91 of horse cytochrome c are absolutely conserved throughout eukaryotic evolution. For studies of the functional roles of these residues, we have prepared, by semisynthetic techniques, analogues of cytochrome c in which one or the other of the arginine residues has been modified. The products of modification by adduct formation with pentane-2,4-dione were purified and extensively characterized. In biological tests, the arginine-91-modified cytochrome c showed little difference in behaviour from native horse cytochrome c. Modification of arginine-38, however, led to extensive changes in biological and chemical properties. We also prepared and tested adducts with cyclohexane-1,2-dione and camphorquinone-10-sulphonic acid. The same effects on biological properties were noted irrespective of the nature of the modifying group. We suggest reasons for the differences in sensitivity of the two sites.

Amino Acids↗
PubMed1983

Amino acid sequence determination by g.l.c.--mass spectrometry of permethylated peptides. Optimization of the formation of chemical derivatives at the 2-10 nmol level.

A new technique is described that permits the permethylation of acylated peptides at the 2-10 nmol level. The presence of up to 400 micrograms of sodium dodecyl sulphate per sample does not affect the reaction yields. The technique, which is a miniaturization of the widely used methyl iodide/dimethylsulphinyl carbanion procedure, employs a layer of hexane to exclude moisture and oxygen from the reaction mixture. Analysis of the peptide derivatives by combined g.l.c.--mass spectrometry permits amino acid sequence information to be obtained. In addition to studies of digests of a model substrate (glucagon), the new permethylation technique has been used to identify a peptide of interest from a digest of a cytochrome and to define the N-termini of two proteins at the 5 nmol level.

Acetylation↗
PubMed1983

A new mass-spectrometric C-terminal sequencing technique finds a similarity between gamma-interferon and alpha 2-interferon and identifies a proteolytically clipped gamma-interferon that retains full antiviral activity.

A novel mass-spectrometric technique is described that permits the identification of the C-terminal peptide of a protein. The technique involves the incorporation of 18O into all alpha-carboxy groups liberated during enzyme-catalysed partial hydrolysis of the protein, followed by mass spectrometry to identify as the C-terminal peptide the only peptide that did not incorporate any 18O. The technique has been used to identify the true C-terminal tryptic peptide of a bacterially produced gamma-interferon and to distinguish it from a peptide produced by anomalous tryptic cleavage. It was found that a closely similar sequence segment of bacterially produced alpha 2-interferon undergoes an analogous cleavage. The technique was also used to identify the C-terminus of a clipped gamma-interferon that retains full antiviral activity.

Amino Acid Sequence↗
PubMed1983

Evidence for the vitamin K-dependent gamma-carboxylation of the first glutamic acid residue in peptide substrates containing a diglutamyl sequence.

The peptide substrate commonly used in vitamin K-dependent carboxylation, Phe-Leu-Glu-Glu-Val, has been shown, by the use of high-voltage paper electrophoresis, to be degraded from the N-terminus by a microsomal leucine amino-peptidase. The replacement of phenylalanine with a N-t-butoxycarbonyl group resulted in a tetrapeptide substrate with a blocked N-terminus resistant to enzymic degradation. Vitamin K-dependent carboxylation of this non-degradable substrate gave a unique carboxylated product, which was separated from microsomal protein and unchanged substrate by using DEAE-Sephadex A25 as a final step. The carboxylated product was subsequently decarboxylated in 2HCl and analysed by using g.l.c. coupled to a mass spectrometer. This showed that only the first glutamic acid residue in the peptide substrate was carboxylated.

Animals↗
PubMed1983

Rapid preparation of human insulin and insulin analogues in high yield by enzyme-assisted semi-synthesis.

Reaction conditions are described that permit the enzyme-assisted semi-synthetic replacement of residue B30 of pig insulin (or of analogue) to proceed in very high yield in 2 h or less. Immobilized trypsin may be used as catalyst, and excess amino acid ester may be recycled after a simple extraction. Alanine-B30 may be replaced by a variety of nucleophiles, including threonine O-t-butyl ether t-butyl ester, in which case the yield of crude product is about 99%. De-protection of the B30 threonyl ester analogue of insulin thus formed then affords human insulin in an overall yield of about 92%, based on pig starting material. The product has full biological potency, as determined by depression of blood glucose concentration in rats, and showed the expected behaviour on radioimmunoassay.

Animals↗
PubMed1983

Development of a gas chromatography mass spectrometry assay for the two eosinophil chemotactic factors of anaphylaxis.

Gas chromatography mass spectrometry has been used for over a decade for the determination of the amino acid sequences of fragment peptides derived from larger parent molecules. The majority of these fragments have from four to seven residues and several different methods of derivatization have been devised. Few reports have been published in which similar techniques have been used for the quantification of such peptides, but there is a growing list of small peptides which have been shown to have biological activity in their own right. This report is concerned with the development of a gas chromatographic mass spectrometric assay for the two eosinophil chemotactic peptides, Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu, which appear to have a role to play in the course of the inflammatory process in skin disorders.

Anaphylaxis↗
PubMed1981

The role of the arginine-B22 residue in insulin action.

We describe the modification of the side chain of the arginine-B22 residue of insulin by the N8N9-(1, 2-dihydroxycyclohex-1,2-ylene) group and by the adipoyl group. These are the first insulin derivatives described that contain a modified arginine residue in an otherwise unaltered molecule. When tested for their ability to lower blood sugar concentration, both modified insulins showed a specific activity indistinguishable from that of insulin. In view of the fact that the substituent groups involved are very bulky and in one case of opposite charge to that of the side chain, the retention of biological activity casts doubt on the idea, previously generally accepted, that the arginine-B22 residue is essential to the activity of the hormone.

Animals↗
PubMed1981

Bacteriophage T5 growth in Escherichia coli containing PstI fragments of the colicin Ib plasmid.

PstI restriction fragments of the colicin Ib (ColIb) plasmid have been cloned into the Apr gene of the pBR322 vector. Colicin-producing clones (Col+) all contained two common PstI (L and U) fragments, 2000 and 800 bp long, respectively. All of these colicin producers were found to permit the normal growth of bacteriophage T5; the presence of the whole ColIb plasmid causes an abortive T5 or BF23 phage infection. None of the other clones selected for their inability to propagate T5 produced colicin. The clones (Abi+) that permitted only an abortive infection by T5 contained a single restriction fragment, O (1600 bp), which was not found in any of the colicin producers. Likewise, the specific fragments (L and U) found in the Col+ clones were not found in the Abi+ clones. These data are very hard to reconcile with the hypothesis of a colicin-induced cell deterioration after T5 phage infection.

Bacteriocin Plasmids↗
PubMed1981

Colicin activity and abortive infection of T5 bacteriophage in Escherichia coli (ColIb).

We performed three types of experiments to test the hypothesis that abortive infection of T5 bacteriophage in Escherichia coli (ColIb+) is due to internally released colicin. (i) We measured the sensitivity of cells to colicin under a variety of conditions and then looked at the plating efficiency of T5 in ColIb+ cells under these same conditions. Cells grown at 42 degrees C or with hexanol had a reduced sensitivity to externally added colicin and an increased efficiency for T5 when the ColIb plasmid was present in the infected cells. Phage growth was far from normal, however. (ii) We measured the colicin sensitivity of a mutant bacterium that grew T5 normally even in the presence of the ColIb plasmid and measured the plating efficiency of T5 on another mutant that was colicin tolerant. Here again, the correlation between colicin activity and inhibition of phage replication was not complete. (iii) We looked for colicin-negative plasmid mutants and tested the ability of cells containing these plasmids to support the growth of T5. These experiments used Tn5, a kanamycin resistance transposon, as the mutagen. All possible combinations of colicin production and phage inhibition were found, including mutants that produced no colicin but still inhibited phage production.

Bacteriocin Plasmids↗
PubMed1980

A mass-spectrometric method for the estimation of the ratio of gamma-carboxyglutamic acid to glutamic acid at specific sites in proteins. Application to the N-terminal region of bovine prothrombin.

When a polypeptide containing gamma-carboxyglutamic acid is decarboxylated in 2H2O, residue of (gamma gamma-2H2)glutamic acid are formed. Subsequent proteolytic digestion produces peptides which contain at each site 2H2-substituted and unsubstituted glutamic acid in the same ratio as existed for gramma-carboxy-substitution. The peptides may be identified and this ratio determined by combined gas chromatography--mass spectrometry. We also discuss decarboxylation in 3H2O followed by amino-acid analysis and Edman degradation.

1-Carboxyglutamic Acid↗
PubMed1978

Neural and endocrine development after chronic tryptophan deficiency in rats: I. Brain monoamine and pituitary responses.

Caloric restriction and tryptophan deficient diets have been shown to delay aging in the immature laboratory rat. Studies of monoamine levels in the brain of developing female rats fed on these diets show changes in serotonin but not norepinephrine or dopamine levels: in tryptophan-restricted rats serotonin levels were reduced in all brain areas studied, whereas in caloric-restricted animals serotonin levels were increased in the cerebral hemispheres only. Another group of animals, in which growth and maturation was delayed by feeding d,1-parachlorophenylalanine (PCPA) showed decreases in serotonin, norepinephrine and dopamine concentrations in all brain regions investigated. All treatments employed to arrest growth and maturation resulted in pituitary alterations manifested by gross, histological and ultrastructural changes. It is postulated that there maturation- and age-retarding treatments delay the development of the central nervous system resulting in postponed maturation of the neuroendocrine axis, with consequent hypoactivity of certain pituitary functions and a resultant delay in the onset of maturation and senescence.

Animals↗