Miroslav Baudyš

Structural relationship between the soluble and membrane-bound forms of human monocyte surface glycoprotein CD 14

The carboxy-terminal amino acid sequence of the soluble form of the 53,000 mol. wt monocyte surface antigen, CD14, was determined by carboxypeptidase Y digestion and compared with the complete amino acid sequence of this protein as predicted from the structure of cloned cDNA [Goyert et al. Science 239, 497-500 (1988)]. The soluble antigen isolated from urine appears to lack eight C-terminal amino acid residues predicted for the full-size translation product, but possesses a major part of the C-terminal hydrophobic domain originally suggested as the membrane-spanning segment. The CD14 antigen can be removed from the monocyte surface by phosphatidylinositol-specific phospholipase C treatment, indicating that this glycoprotein is anchored in the membrane by a phospholipid and is not a transmembrane protein. The soluble form occurring in serum and in supernatants of cultured monocytes thus probably arises by phospholipase-mediated cleaving off the cell surface antigen. A sensitive sandwich ELISA was developed using a monoclonal anti-CD14 antibody, MEM-18, and polyclonal rabbit anti-CD14 antiserum for quantitation of the soluble antigen concns in sera and cell culture supernatants. Using this assay, the antigen present in the supernatant of phospholipase treated peripheral blood mononuclear cells could be estimated. The assay was also used for estimation of the concns of the soluble form of the CD14 antigen in human sera.

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.

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.

Inhibition of aspartic proteinases by propart peptides of human procathepsin D and chicken pepsinogen

Two propart peptides of aspartic proteinases. the propart peptide of chicken pepsin and human cathepsin D, respectively, were investigated from the point of view of their inhibitory activity for a set of aspartic proteinases. These peptides display a very broad inhibitory spectrum. The strongest inhibition was observed for pepsin A‐like proteinases where propart peptides can be used as titrants of active enzymes.

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.

Identification of the active site cysteine and of the disulfide bonds in the N-terminal part of the molecule of bovine spleen cathepsin B.

Cathepsin B (CB), the most intensively studied lysozomal thiol endopeptidase, plays an important role in intracellular protein catabolism [1,2]. Pig liver CB exists in the single-chain (M) and two-chain form. The latter consists of a light (L-) and a heavy (H-) chain and most likely is the result of proteolytic cleavage in the N-terminal part of the molecule of the M-form [3,4]. The active cysteine is localized in the L-chain. Rat liver CB exists in the two-chain form only. The primary structure of its L-chain and of the N-terminal part of its H-chain have been determined [5]. The enzymatic characteristics of bovine spleen CB, its interaction with protein inhibitors, and its similarity with papain have been studied [6-9]. We have found that spleen CB exists in both forms. This paper describes the determination of the primary structure of the L-chain and the identification of active cysteine. The behavior of 3 other half-cystines present in the structure of the L-chain as well as the manner in which the Land H-chain are joined one to another have been elucidated. The similarity between CB and papain is discussed.

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.

Structure of Thermitase, A Thermostable Serine Proteinase from Thermoactinomyces Vulgaris , and its Relationship with Subtilisin-Type Proteinases

Thermitase (EC 3.4.21.14) is an extracellular thermostable serine proteinase isolated from Thermoactinomyces vulgaris culture filtrate [1–3]. The enzyme resembles in its characteristics the subtilisins. This is strongly indicated especially by the structure of the active site peptide [4]. The enzyme contains one methionine and one cysteine residue; these two residues are apparently functionally important [4]. Closely related seem to be the alkaline proteinases of Bacillus cereus and Bacillus thuringiensis [5] and proteinase K from the mold Tritirachium album [6]. Our interest in structural investigation of thermitase was stimulated by the problem of its apparently essential cysteinyl residue, by the lack of information on evolutionary relations between thermitase and other subtilisin-type enzymes and on the structural basis of the increased thermostability of thermitase. Another factor not to be neglected was the practical importance of the enzyme. Since the localization of the methionine residue was known from one of the early studies we decided to start our sequence work with the cyanogen bromide digest of the enzyme.

CHICKEN PEPSIN: STRUCTURE AND HOMOLOGY WITH OTHER ACID PROTEINASES

ABSTRACT The amino acid sequence of chicken pepsin (CP) has been studied to provide a basis for the elucidation of (a) the catalytic mechanism of CP at the molecular level, and (b) the evolutionary relationship of CP with other carboxyl proteinases. For more efficient. preparation of the enzyme CP was isolated directly from acid extracts of chicken proventriculi mucosa by covalent and affinity chromatography; these procedures were based on the coupling of CP either via its SH-group to mercurial Sepharose or to Sepharose with immobilized A. lumbricoides pepsin inhibitor as affinant. The latter support was used to advantage also for affinity chromatography purification of other carboxyl proteinases. As starting material for sequence studies the zymogen (CPG) was isolated by anion exchange chromatography of alkaline extracts of the mucosa. The process of CPG activation to CP starts (at the amino acid sequence level) by scission of the bond between Phe (26) and Leu (27). The 26-residue N-terminal activation peptide released inhibits to different degrees CP, calf chymosin, and hog pepsin. Sequence studies carried out by automatic degradation of CPG and CP derivatives and by the classical approach based on overlapping peptides permitted a partial structure of CPG to be derived. The latter accounts for 322 residues arranged in 19 segments of interchangeable order. The alignment of this structure with those of hog pepsinogen and calf prochymosin shows sequential homologies in the terminal parts of the molecule, around half-cystines and the reactive aspartic acid residues.