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V Kostka

Publications and source records attributed to V Kostka.

At least 55 records · Page 3Linked to original sources
PubMed1983

Covalent structure of chicken pepsinogen.

Chicken pepsinogen is a glycoprotein consisting of a single polypeptide chain and containing the following 367 amino acid residues: Asp23, Asn16, Thr26, Ser41, Glu14, Gln11, Pro18, Gly31, Ala17, Cys7, Val25, Met9, Ile23, Leu28, Tyr22, Phe20, His8, Lys17, Arg7, Trp4. The Mr-value of the protein is 42 074. This value includes the carbohydrate moiety of the protein, i.e. Man3, (GlcNAc)7, (-SO3H)5. The primary fragmentation of the molecule was effected by limited trypsinolysis at arginine residues after preceding modification of the lysines with citraconic anhydride. All eight peptides expected in theory were obtained and their size, amino acid composition, and N-terminal amino acid sequence were characterized. To elucidate the amino acid sequence of these large fragments the latter were subjected to secondary cleavage by CNBr, trypsin (after removal of the protecting groups from the lysines), the proteinase from Staphylococcus aureus V8 strain, alpha-chymotrypsin, hydroxylamine, or dilute acid; the resulting peptides were isolated by gel permeation and ion-exchange chromatography and by the fingerprint techniques. Overlaps at sites of the arginine residues were obtained in an earlier study [Baudys, M. & Kostka, V. (1982) Collect. Czech. Chem. Commun. 47, 2814-2832]. Chicken pepsinogen shows the highest degree of homology with the primary structures of pepsinogens A. The internal homologies are apparent in the neighborhood of the two active aspartic acid residues. We have assigned tentatively chicken pepsinogen to the group of pepsinogens A (EC 3.4.23.1); this assignment is a result both of our sequence studies and of an investigation of the kinetic characteristics of the enzyme.

Amino Acid Sequence↗
PubMed1983

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: a novel assay.

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (delta epsilon = 860 M-1 cm-1) and by ninhydrin colorimetry (substrate I, epsilon 570 = 2.31 X 10(4) M-1 cm-1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5-6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.

Animals↗
PubMed1983

Amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins.

The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val-Val-Gly-Gly-Trp-Asp-Phe-Val-Asp-Asn-Asp-Ser-Thr- Pro-Gln-Asn-Gly-Asn-Gly-64His-Gly-Thr-His-68Cys-Ala- Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn-Asn-Ser-Thr-Gly-Ile- Ala-Gly-Thr-Ala-Pro-Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-14CH2-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.

Amino Acid Sequence↗
PubMed1982

Amino acid sequence of the small cyanogen bromide peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins.

The following amino acid sequence of the small cyanogen bromide peptide (mol. wt. 5399) of thermitase from Thermoactinomyces vulgaris has been determined: Ala-Thr-Pro-His-Val-Ala-Gly-Val-Ala-Gly-Leu-Leu-Ala-Ser-Gln-Gly-Arg-Ser-Ala-Ser -Asn-Ile-Arg-Ala-Ala-Ile-Glu-Asn-Thr-Ala-Asp-Lys-Ile-Ser-Gly-Thr-Gly-Thr-Tyr-Trp-Ala-Lys-Gly-Arg-Val-Asn-Ala-Tyr-Lys-Ala-Val-Gln-Tyr. The results obtained support the classification of the enzyme as a serine proteinase of the subtilisin type as proposed in a previous paper (1). This partial sequence extending from the serine residue involved in the active site to the C-terminal amino acid of the enzyme shows a 40% homology with the corresponding part of the subtilisin BPN' or subtilisin Carlsberg molecule but a 56% homology as regards conservative amino acid replacements. The secondary structure of this polypeptide fragment, predicted from the data obtained by the method of Chou & Fasman (2) agrees fairly well, within the limit or error of the method, with the structure of the corresponding part of the subtilisin BPN' molecule. Therefore, as expected, no dramatic changes in the spatial structure appear to account for the higher thermostability of thermitase, at least in this area of the polypeptide chain.

Amino Acid Sequence↗
PubMed1977

The first step in the activation of chicken pepsinogen is similar to that of prochymosin.

Chicken pepsinogen was incubated at pH2.5 with pepstatin. The zymogen activated itself by a sequential mechanism and an intact peptide derived from residues 1-26 in the protein was released in the first step. This peptide was found to inhibit the milk-clotting activities of pig and chicken pepsins and calf chymosin but to different extents.

Amino Acid Sequence↗