Characterisation of a tryptic peptide from human placental ribonuclease inhibitor which inhibits ribonuclease activity.

Affinity-purified human placental ribonuclease inhibitor (PRI) was digested by trypsin. Subsequent fractionation of the hydrolysate by HPLC yielded 44 fractions, 3 of which retained the ability to inhibit ribonuclease. One of these, the most active, was a 15 amino acid peptide which had an amino acid composition corresponding to a tryptic fragment of PRI. This peptide was synthesised, and preliminary experiments were carried out on its interactions with ribonuclease. These experiments suggested that the behaviour of the peptide in terms of effect of pH, and effect of salt concentration were similar to the protein from which it was derived. These studies together with the strategic positioning of the peptide in the sequence of the ribonuclease inhibitor, suggest that this segment of PRI has an important role in the inhibitory activity of the intact protein.     

Complete amino acid analysis of proteins from a single hydrolysate.

An analytical procedure which affords the precise amino acid composition of a protein or a peptide from a single hydrolysate is described. This method utilizes 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole, rather then 6N HCl as a catalyst for hydrolysis. The hydrolysis is carried out in vacuo (20 mu) at 115 degrees for 22 to 72 hours. Half-cystine is determined as S-sulfocysteine by treating the hydrolysate with dithiothreitol followed by an excess of tetrathionate. The values of all amino acids, including tryptophan and half-cystine, were close to the expected theoretical values for the proteins examined. The method has the advantage that the neutralized hydrolysate can be applied directly to an ion exchange column. Further, the method is capable of distinguishing between free sulfhydryl groups as S-carbosymethylcysteine and disulfides as S-sulfocysteine. A limitation of the procedure is that tryptophan remains sensitive to the presence of carbohydrate in the sample.     

Pre-proparathyroid hormone: analysis of radioactive tryptic peptides and amino acid sequence.

The amino acid sequence of bovine pre-proparathyroid hormone has been partially determined by analysis of the polypeptide labeled selectively with radioactive amino acids. Analysis of tryptic peptides containing methionine or lysine indicated that parathyroid hormone, proparathyroid hormone, and pre-proparathyroid hormone had several common peptides. Two lysine-containing peptides present in proparathyroid hormone but not in parathyroid hormone were also present in pre-proparathyroid hormone. In addition, pre-proparathyroid hormone contained several additional lysine- and methionine-containing peptides not present in parathyroid hormone or proparathyroid hormone. Analysis by repetitive Edman degradation of the polypeptide labeled with lysine, methionine, and other amino acids indicated that pre-proparathyroid hormone contained 25 additional amino acids at the amino terminus of proparathyroid hormone; the identities of 17 of the 25 amino acids have been established. An unusual feature found was the presence of methionyl-methionyl at the amino terminus and the presence of 5 methionines within the first 14 amino acids.     

Complete amino acid sequence of human phosphoglycerate kinase. Isolation and amino acid sequence of tryptic peptides

As a part of the elucidation of the complete amino acid sequence of human phosphoglycerate kinase, 46 tryptic peptides, ranging in length from 1 to 26 residues, were isolated and characterized from the reduced and S-carboxymethylated enzyme. The isolated peptides were subjected to sequence analysis by the modified dansyl-Edman degradation procedure and automated Edman degradation technique. The results, together with the data on cyanogen bromide peptides and two additional tryptic peptides from cyanogen bromide peptides reported in the accompanying paper, established the complete amino acid sequence of human erythrocyte phosphoglycerate kinase.     

Location of amino acid substitutions in human hemoglobin. Mass spectrometric rapid analysis of tryptic peptides

Point mutations alpha 58 His----Tyr (Hb M Boston), beta 6 Glu--lys (Hb C) and beta 26 Glu----Lys (Hb E) have been identified in abnormal hemoglobins by means of tryptic hydrolysis of their alpha- and beta-chains followed by mass-spectrometry coupled with direct extraction of ions from solution. The abnormal hemoglobin Hb M Boston alpha 58 (E7) His----Tyr has been for the first time found in the blood of a patient from the USSR. This express-method is generally applicable for the identification of point mutation in proteins. The amount of protein necessary for the analysis is 100-1000 pmole. The stability, proteolytic degradation of the identified abnormal Hb's and Hb Bart's were investigated. The molecular pathogenesis of the hemoglobinopathies are discussed from the point of view of the observed properties.     

Comparison of tryptic peptide maps of eight isoperoxidases from tobacco tissue cultures

Eight isoperoxidases from tobacco suspension culture WR-132 (termed C_n, C₃, C₄, A_c, and A_f) and tobacco callus culture W-38 (termed A₁, A₂, and A₃) have been subjected to trypsin digestion followed by peptide mapping. The peptide maps of isoperoxidases A_f and A₃ are identical. All other isoperoxidases do not appear to be dramatically dissimilar in certain portions of their sequence, since many matching peptides have been found when various isoperoxidases are cross-compared. However, only two, and possibly three, highly homologous peptides are present in all of the isoperoxidases.     

Dihydroorotase from Escherichia coli. Cloning the pyrC gene and production of tryptic peptide maps

We have inserted a 1.7-kilobase pair Escherichia coli DNA fragment containing the 1-kilobase pair pyrC gene into the high copy number plasmid pKC16. Dihydroorotase expressed by the pyrC plasmid in E. coli constituted 6.3% of the soluble protein in frozen cell paste. Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E. coli strain lacking the pyrC plasmid: tryptic peptide maps from the two dihydroorotase preparations, produced using reverse-phase high performance liquid chromatography, were indistinguishable. We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.     

Primary structure of intracellular serine proteinase from Bacillus amyloliquefaciens. I. Isolation of the enzyme and amino acid sequence of peptides of tryptic hydrolysate

Method of isolation of intracellular serine protease was modified. Gramicidin S-sepharose CL-4B with a higher content of the ligand, synthesized through a modified procedure, was used as an affinity sorbent which simplified the purification and led to the pure enzyme with high specific activity and 90% yield. Trypsin hydrolyzate of the protease was separated by ion-exchange chromatography on a sulphocationite resin followed by paper chromatography and paper electrophoresis to yield twenty-five individual peptides. Their complete or partial sequences, corresponding in total to 146 amino acid residues, were determined by the manual Edman procedure.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. The tryptic peptides.

The NADP-specific glutamate dehydrogenase of Neurospora crassa was digested with trypsin, and peptides accounting for 441 out of the 452 residues of the polypeptide chain were isolated and substantially sequenced. Additional experimental detail has been deposited as Supplementary Publication SUP 50052 (11 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem J. (1975) 145, 5.     

The amino acid sequence of hamster pancreatic ribonuclease

The amino acid sequence of golden hamster pancreatic ribonuclease was determined by analysis of tryptic, chymotryptic, thermolytic, and CNBr peptides and by automatic sequence analysis of the intact protein. Like all RNases with an Asn-Met-Thr sequence at positions 34-36, hamster RNase is glycosylated at position 34 with a complex-type carbohydrated chain. Val-17, Ala-18, His-55, His-76 and Ala-90 have never been observed in other pancreatic RNases. Ala-90 replaces Ser-90, which had been invariant in all mammalian RNases studied so far. The amino acid sequence of hamster RNase differs at 15 positions from that of another Cricetidae rodent, the muskrat. The similarity between both ribonucleases was used to confirm a few less certain parts of the muskrat RNase sequence. The replacement rate of the RNases of the Cricetidae appeared to be higher than the average rate in the mammals, but much lower than the rate in another myomorph family, the Muridae (mouse and rat). Possibly, in many respects, the Cricetidae underwent less evolutionary change in recent times than the evolutionarily highly successful Muridae.