Vladimír Kostka

Complete amino acid sequence of human serum albumin

The complete understanding of the binding and transport functions of human serum albumin, the main protein constituent of plasma, requires the knowledge of its chemical structure..The results of sequence studies on human serum albumin reported till the end of 1970 have been summarized in the Atlas of Protein Sequence and Structure [ 11; the largest continuous part of the sequence known by then was that of the 24-residue N-terminal region [2,3]. These studies had shown that the heterogeneity of albumin preparations [4,5] does not originate in its primary structure. In 1970 we began in this Laboratory systematic studies of human serum albumin aimed at the determination of the complete amino acid sequence of the protein. The primary structure of human serum albumin was concurrently studied also at the University of Buffalo [6,8], at Craighton University Medical School [9], and at the University of Texas [IO]. This paper reports on the complete amino acid sequence of human serum albumin, including the positions of all amides; the results presented are confronted with sequential data reported from other laboratories.

Complete primary structure of thermitase from Thermoactinomyces vulgaris and its structural features related to the subtilisin-type proteinases

Thermitase, a thermostable alkaline proteinase, consists of a single polypeptide chain, containing 279 amino acid residues (M r = 28 369). The enzyme shows remarkable structural features of proteinases of the subtilisin type as shown by pronounced sequential homologies. The amino acid replacements, insertions and deletions observed when the amino acid sequence of the enzyme is compared with the sequences of several subtilisins are discussed with respect to substrate specificity and expected tertiary structure. The existence of a cysteinecontaining subgroup of subtilisin‐like proteinases is postulated.

Primary structure of cathepsin D inhibitor from potatoes and its structure relationship to soybean trypsin inhibitor family.

A novel effective procedure for the purification of cathepsin D inhibitor from potatoes (PDI) was developed. The amino acid sequence of PDI was determined by analysis of the cyanogen bromide digest and of the limited tryptic and chymotryptic digests of the protein. The inhibitor is a single polypeptide chain protein consisting of 188 residues with a simple sugar moiety attached to Asn‐19. The tentative disulfide pairings are also suggested. The sequence data clearly indicate that PDI is homologous with the soybean trypsin inhibitor (STI) (Kunitz) family. The active center of PDI for trypsin inhibition was identified as Pro‐Val‐Arg‐Phe in analogy to STI.

Hydrolysis of synthetic chromogenic substrates by HIV-1 and HIV-2 proteinases.

Kinetic constants (Km,Kcat) are derived for the hydrolysis of a number of chromogenic peptide substrates by the aspartic proteinase from HIV-2. The effect of systematic replacement of the P2 residue on substrate hydrolysis by HIV-1 and HIV-2 proteinases is examined.

Sub-site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV-1.

A series of synthetic, chromogenic substrates for HIV‐1 proteinase with the general structure Ala‐Thr‐His‐Xaa‐Yaa‐Zaa∗Nph‐Val‐Arg‐Lys‐Ala was synthesised with a variety of residues introduced into the Xaa, Yaa and Zaa positions. Kinetics parameters for hydrolysis of each peptide by HIV‐1 proteinase at pH 4.7, 37°C and u = 1.0 M were measured spectrophotometrically and/or by reverse phase FPLC. A variety of residues was found to be acceptable in the P3, position whilst hydrophobic/aromatic residues were preferable in P1. The nature of the residue occupying the P2; position had a strong influence on k cat (with little effect on k m;β‐branched residues Val or Ile in this position resulted in considerably faster peptide hydrolysis than when e.g. the Leu‐containing analogue was present in P2.

Cleavage of oligopeptide side-chains in N-(2-hydroxypropyl)meth-acrylamide copolymers by mixtures of lysosomal enzymes☆

Abstract N-(2- Hydroxypropyl ) methacrylamide copolymers containing oligopeptide side-chains terminating in drug have been developed as lysosomotropic drug carriers. To obtain more information on the degradation process of such copolymers inside the lysosomes, two polymeric prodrug models have been synthesized: P-Ala-Gly-Phe-Gly-NAp and P-Ala-Gly-Phe-Leu-NAp ( P = polymeric backbone ; NAp = p - nitroanilide ). These substrates were incubated with lysosomal enzymes isolated from bovine spleen — cathepsin B and mixtures of cathepsin B with cathepsin C or H. The products of cleavage (peptide fragments) were quantitatively determined by gel permeation chromatography on a calibrated Sephadex G-15 column and the time course of cleavage followed for 48 hours.The inhibition of cathepsin C by one of the products of cleavage (Phe-Gly) was observed.

Complete amino acid sequence of hog pepsin

The amino acid sequence of hog pepsin or of some parts of its polypeptide chain has been studied in a number of laboratories. The most recent summary of the results obtained is presented in the 1972 issue of the Atlas of Protein Sequence and Structure [ 11. Our aim has been to determine the complete covalent structure of this enzyme and thus to provide, together with the complete structures of bovine trypsinogen [2] and chymotrypsinogen [3] elucidated in this laboratory earlier, complete and independent sequence information on the fundamental proteolytic enzymes of the digestive tract. The determination of the complete structure of the whole molecule was based on the results of sequential studies on large fragments designated CB [4] obtained by cyanogen bromide cleavage of pepsin at its four methionine residues [5] (fig. 1). The studies on the individual cyanogen bromide fragments were begun after the determination of the disultide bonds of the 6 half-cystine residues of pepsin [6]. The complete amino acid sequence of fragment CBl [7], representing the 37-residue C-terminal part of the molecule, was determined. Later, the 55residue N-terminal amino acid sequence of pepsin [8,9] was

Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B

The action of bovine spleen cathepsin B as a dipeptidyl carboxypeptidase on newly synthesized substrates of the type peptidyl-X-p-nitrophenylalanyl (Phe(NO2))-Y (X,Y = amino acid residue) or 5-dimethylaminonaphthalene-1-sulfonyl (Dns)-peptidyl-X-Phe(NO2)-Y was investigated. The kinetic parameters of hydrolysis of the X-Phe(NO2) bond were determined by difference spectrophotometry (delta epsilon 310 = 1600 M-1 cm-1) or by spectrofluorometry by following the five- to eightfold increase of Dns-group fluorescence with excitation at 350 nm and emission at 535 nm. The substrates were moderately sensitive to cathepsin B; kcat varied from 0.7 to 4 s-1 at pH 5 and 25 degrees C; Km varied from 6 to 240 microM. The very acidic optima of pH 4-5 are characteristic for dipeptidyl carboxypeptidase activity of cathepsin B. Bovine spleen cathepsins S and H had little and no activity, respectively, when assayed with Pro-Glu-Ala-Phe(NO2)-Gly. These peptides should be a valuable tool for routine assays and for mechanistic studies on cathepsin B.

Comparison between prochymosin and pepsinogen from lamb and calf

1. Prochymosin (EC 3.4.23.4) and pepsinogen A (EC 3.4.23.1) from Mongolian lamb (Ovis platyurea) were purified to homogeneity by salt precipitation, gel filtration and ion-exchange chromatography. 2. Immunoelectrophoresis shows partial immunochemical identity between chymosins and pepsins from lamb and cattle, respectively. 2. Activity determinations, N-terminal amino acid sequences and amino acid compositions also show a close relationship between the proteinases from lamb and cattle. 4. Lamb prochymosin and pepsinogen are both glycosylated.

Purification of pepsins and cathepsin D by affinity chromatography on Sepharose 4B with an immobilized synthetic inhibitor.

Val-D-Leu-Pro-Phe-Phe-Val-D-Leu, a specific inhibitor of aspartate proteinases of the pepsin type, was synthesized. Its bonding to activated 6-aminohexanoic acid-Sepharose 4B afforded an affinity support suitable for the purification of human, porcine, and chicken pepsin, human gastricsin, and bovine cathepsin D. These enzymes bind to the support over the pH range 2-5 at 0-1.5 M concentration of NaCl. A buffer at pH greater than or equal to 6, low ionic strength, and containing 20% dioxane can serve as a general desorption agent. The proteinases were isolated from the crude extracts by a single-step procedure in a high degree of purity and in yields exceeding 70%; human pepsin, however, was not separated from human gastricsin. The support does not show any binding capacity for rat plasma renin at pH 7.4 and for some cysteine endopeptidases (cathepsin B, H, and L) at pH 3-5. The cathepsin D preparations isolated by affinity chromatography on the new support and on pepstatin-Sepharose were of the same degree of purity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequences, and specific activity.