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P Elödi

Publications and source records attributed to P Elödi.

At least 19 recordsLinked to original sources
PubMed1993

Substrate specificity of 'elastomucoproteinase': an enzyme which can degrade cartilage aggrecan.

The substrate specificity of elastomucoproteinase (EMP), an enzyme which was first isolated from crude pancreatic elastase and described as a proteoglycan-degrading enzyme, determined on tripeptide-p-nitroanilide substrates indicates the existence of a 'new' chymotrypsin-like enzyme. EMP, however, did not cleave any glycosaminoglycans, i.e., its 'mucolytic' effect has been excluded. Activity of EMP on synthetic or protein substrates (e.g., collagen type-II and aggrecan of cartilage) was completely inhibited by serine proteinase inhibitors, which was also found when using cartilage proteoglycan monomers. EMP cleaves the core protein of proteoglycan monomer (aggrecan) into small peptides, some containing glycosaminoglycan chains resulting in an unusual elution profile on Sepharose CL-6B chromatography when compared to the effects of pancreatic and granulocyte elastases, chymotrypsin, cathepsin G and stromelysin. EMP-like activity also was detected in neutrophil granules of bovine leukocytes and polyclonal antibodies were raised against purified bovine EMP to detect the enzyme in both crude elastase preparations and the granule fraction of bovine leukocytes.

Adult↗
PubMed1992

Synthesis and functional evaluation of a peptide derivative of 1-beta-D-arabinofuranosylcytosine.

We have synthesized a peptidyl prodrug derivative of 1-beta-D-arabinofuranosylcytosine (1) designed to be a selective substrate of plasmin. D-Val-Leu-Lys-ara-C (2) was obtained by coupling the protected peptide Cbz-D-Val-Leu-(N6-Cbz)Lys-OH and ara-C (1) by a water-soluble carbodiimide (EDCI), followed by the removal of the Cbz groups by using catalytic hydrogenolysis over Pd/C. The kinetic constant of hydrolysis of 2 in the presence of plasmin demonstrated effective release of 1. The amino group of 1, which is sensitive to the removal by cytidine deaminase, is protected in 2 by the formation of the amide bond resulting in a prolonged half-life of 2 in biological milieu. The antiproliferative efficiency of 2 against L1210 leukemic cells was significantly higher than that of 1. The activity of 2 was abolished in the presence of serine proteinase inhibitor, (4-amidinopheny)methanesulfonyl fluoride. These data indicate that 2 is a prodrug form of 1 in systems generating plasmin.

Animals↗
PubMed1990

The enzyme capable to cleave trypsin active site titrant as substrate is a carboxylesterase with electrophoretic mobility similar to albumin.

An enzyme isolated from Ehrlich ascites plasma and capable of cleaving trypsin active site titrant 4-nitrophenyl-p-guanidinobenzoate (Steven, F.S. and Al-Achmad, R.K. (1983) has been further investigated. The substrate hydrolysis follows Michaelis-Menten kinetics. The molecular mass of the enzyme is 50-70 kDa by gel filtration and SDS polyacrylamide gel electrophoresis. It has the mobility of albumin and coelutes with a carboxylesterase activity on a cation exchange column. (Cbz-Arg-NH)2-Rhodamine, the specific noncompetitive inhibitor of guanidinobenzoatase, also inhibits the carboxylesterase activity. Therefore, the guanidinobenzoatase activity of Ehrlich ascites plasma is a carboxylesterase (EC 3.1.1.1.) which likely originates from blood.

Amino Acid Sequence↗
PubMed1989

Effect of polyamines on the carbamoyl-phosphate synthase activity of CAD protein.

The effects of polyamines were studied on carbamoyl-phosphate synthase II (EC 6.3.5.5.) which is the first and rate limiting enzyme in mammalian pyrimidine synthesis. Polyamines in physiological concentrations (0.1-1 mM) strongly inhibited the carbamoyl-phosphate synthesis. Of the polyamines tested spermine was the most effective followed by spermidine and putrescine. Spermine increased the KM for ATP and the requirement for Mg++ of carbamoyl-phosphate synthase reaction. UTP, an inhibitor, had similar, while phosphoribosyl-pyrophosphate, an activator of the enzyme had an opposite effect. Increasing concentrations of phosphoribosyl-pyrophosphate completely reversed the inhibition caused by spermine, while did not influence the degree of inhibition caused by UTP. A possible physiological role of polyamines in synchronizing the substrate and activator functions of phosphoribosyl-pyrophosphate in pyrimidine synthesis is suggested.

Adenosine Triphosphate↗
PubMed1989

Plasminogen activator and plasmin-like activities in experimental rat tumours.

Plasminogen activator activity and plasmin-like amidolytic activity were investigated in two experimental rat tumours, using human plasminogen and chromogenic peptide substrate, S-2251. The invasive hepatocarcinoma and non-invasive nephroma were induced with the same chemical carcinogen, dimethylnitrosamine, in F-344 rats and they were continuously transplanted under the renal capsule. While there was no difference in plasmin-like activities of the tumours, the plasminogen activator activity was very low in the nephroma, but high in the hepatocarcinoma. Since the activator activity was completely inhibited by amiloride, it was considered to be of urokinase-type. These results were in accordance with the assumed role of urokinase in the invasion. However, of the respective control organ, kidney was rich in both activities but rat liver contained only very low activities. Therefore the comparison of the plasminogen activator activity of a tumour to the control organ probably does not provide information concerning the malignant transformation as it is suggested in the literature.

Animals↗
PubMed1988

Properties of chymotrypsin bound covalently to dextran.

The kinetic properties dextran-chymotrypsin conjugate were studied by means of low molecular weight substrates. It was found that KM, kcat and kcat/KM of dextran chymotrypsin for the hydrolysis of benzoyl-L-tyrosine-ethyl-ester did not differ substantially from those of the free enzyme. However, the data found for kcat of dextran-chymotrypsin and free chymotrypsin assayed for the hydrolysis of three tripeptidyl-p-nitroanilide D-Arg-Val-Trp-pNA, D-Arg-Val-Tyr-pNA, Z-Phe-Pro-Phe-pNA, were definitely different. The inhibition of the modified chymotrypsin with soybean trypsin inhibitor was found to be less pronounced than that with the free enzyme. The effect of potassium and magnesium salts on the inactivation of both enzymes was also studied. The effect of dextran matrix on the catalytic properties and the conformational stability of modified chymotrypsin is discussed.

Animals↗
PubMed1986

Specific inhibition of human granulocyte elastase with peptide aldehydes.

The kinetic features of human granulocyte elastase, chymotrypsin, porcine pancreatic elastase and elastomucoproteinase were compared. Amino acyl ester substrates were assayed and Km and kcat values were defined. Aldehyde analogues of the p-nitroanilide substrates designed for granulocyte elastase as optimal for Km appeared to be potent inhibitors. Suc-D-Phe-Pro-valinal (Ki = 40 microM) was found to inhibit granulocyte elastase competitively and specifically when measured with synthetic substrates, and the Ki was 3 microM with the natural protein substrate, elastin.

Aldehydes↗
PubMed1986

Active centre studies on bovine pancreatic chymotrypsin with tripeptidyl-p-nitroanilide substrates.

The kinetic behaviour of bovine pancreatic chymotrypsin was studied with 22 N-protected and 17 N-unprotected tripeptidyl-p-nitroanilide substrates. The contribution of the individual side chains to the kinetic parameters were calculated by regression analysis. At subsite P1 (notation of Schechter and Berger, 1967, Biochem. Biophys. Res. Commun. 27, 157) Tyr seems to be better than Phe and Trp, concerning kcat values. At P2 subsite the best KM values were obtained with Gly and Ser, whereas the hydrophobicity of P2 subsite appears to be necessary for efficient catalytic activity. At P3 mainly polar amino acids, both with D and L configuration, were tested. They improve the solubility of substrates in aqueous medium, as well as the kinetic parameters. Suc(OMe) and Suc protecting groups at P4 increase significantly the catalytic activity compared to the aromatic ones. The obtained data were compared to the known substrate binding site of bovine pancreatic chymotrypsin.

Anilides↗
PubMed1986

Mapping of the substrate binding site of human leukocyte chymotrypsin (cathepsin G) using tripeptidyl-p-nitroanilide substrates.

The kinetic constants, KM, kcat and kcat/KM of human leukocyte chymotrypsin (Cathepsin G, E.C.3.4.21.20.) were determined with 33 N-protected and 16 N-unprotected tripeptidyl-p-nitroanilide substrates. The individual contributions of the amino acid side chains of the substrates at P1-P4 subsites to the kinetic parameters were calculated by regression analysis. As far as KM is concerned, the highest contributions yielded the structure of an "optimum" substrate PhCO-Ala-Val-Tyr-pNA. The contribution values permitted us to characterize the S1-S4 binding segment in the enzyme's binding site, interacting with the P1-P4 moieties of the substrate. The enzyme prefers uniformly hydrophobic substituents at the S1-S4 sites. The S1 primary specificity subsite of the enzyme can bind Leu and 2-aminohexanoic acid as well, besides the aromatic amino acids Phe, Tyr, Trp. Data were compared with those obtained when studying pancreatic chymotrypsin with the same substrates.

Anilides↗
PubMed1985

Investigation on the substrate specificity of human plasmin using tripeptidyl-p-nitroanilide substrates.

The hydrolysis of 35 tripeptidyl-p-nitroanilides was studied with human plasmin and the kinetic parameters were determined. The individual contribution of the various side chains to the kinetic parameters was calculated by regression analysis. Considering Km, substrates having Z-D-Ile-Phe-Lys as well as H-D-Ile-Phe-Lys sequences were found to be the best, while Bz-Ile-Leu-Lys and pGlu-Leu-Lys sequences are the best for kcat. The Km values of substrates protected at N-terminus are lower, their kcat values are higher than those of the unprotected ones with the same sequence.

Amino Acid Sequence↗
PubMed1985

Is elastomucoproteinase a double-headed enzyme?

During isolation pancreatic elastase is accompanied by a minor component, elastomucoproteinase (EMPase), which can be separated in pure state by anion-exchange chromatography. EMPase exhibits both proteolytic and mucolytic activities. Applying powdered aortic preparations as a substrate, the enzyme can split off carbohydrate moieties, but it can also cleave peptide bonds as a proteinase. Assaying with tripeptide-p-nitroanilide substrates, the enzyme can decompose only those substrates which contain Phe or Tyr at the C-terminal. It appeared that the binding of Bz-Ala-Gly-Phe-pNA was the best, while the hydrolysis of Bz-Ala-Pro-Tyr-pNA was the fastest. Proteolytic activity of EMPase can be completely inhibited with phenylmethanesulphonyl fluoride, whereas its mucolytic activity only by 20%. The two activities may be located at two separate sites. The enzyme seemed to be composed of a single polypeptide chain by SDS-gel electrophoresis in the presence of urea and beta-mercapto-ethanol.

Amidohydrolases↗
PubMed1983

The interaction of heparin with human plasmin.

1. The interaction of heparin with human plasmin was investigated measuring plasmin activity and enzyme inactivation in the presence of heparin. Hydrolysis of synthetic substrates (H-D-Val-Leu-Lys-pNA, H-D-Val-Phe-Lys-pNA and H-D-Pro-Phe-Lys-pNA) by plasmin was enhanced by heparin through an increase in kcat values. 2. This effect was the consequence of a change of Vmax since Km values were not altered in the presence of heparin. The polysaccharide also enhanced the rate of enzyme inactivation using TLCK as an active site blocking reagent. 3. Furthermore, heparin increased the heat sensitivity of plasmin, when synthetic substrate H-D-Val-Leu-Lys-pNA was used but it did not affect enzyme activity towards N-benzoyl-L-arginine-ethylester substrate. 4. The data show that microenvironmental conformation around the active center of plasmin is influenced by heparin.

Binding Sites↗
PubMed1983

Surface-governed molecular regulation of blood coagulation.

Among extracellular biological processes the spatial control of blood clotting is a unique phenomenon. Localization in space has very important consequences in both normal and pathological conditions. Under physiological circumstances a clot is formed only in the vicinity of injury, albeit the prerequisites of coagulation are almost completely given in the whole circulation. The local character of blood clotting is secured by the following major conditions: The regulatory signal initiating coagulation-the damaged vascular wall-is itself a surface on which the majority of clotting reactions take place. The first enzyme, factor XII, of the intrinsic coagulation pathway is activated on the collagen fibers exposed in the damaged vascular wall, although the significance of this reaction in respect of the clotting process is ambiguous. On the membrane of platelets adhered to the damaged blood vessel is activated factor XI, too, which is a well-established participant of the intrinsic clotting process. The further consecutive reactions of coagulation are confined to the surface produced by injury, because the enzymes involved contain gamma-carboxyl-glutamyl side chains which are anchored through calcium bridges to the phospholipids of the platelet membrane. The last enzyme of the sequence is thrombin, which is released from the surface. The reactions taking place on the surface form an enzyme cascade, which amplifies the relatively weak triggering signal by several orders of magnitudes. Amplification is ensured not only by the enzyme-substrate relationship of the consecutive reaction partners, but also by spatial confinement, which endows the process with higher efficacy than could be expected on a statistical basis from reactions in solution. It contributes to the efficiency of enzyme cascade that the non-enzymatic regulatory proteins increase the activity of factors IXa and Xa, and thereby the overall process. While the partner of factor IXa, factor VIII, is captured from plasma, factor V, the partner of factor Xa, is derived from the platelets adhered to the damaged surface and orients the binding of factor Xa. The surface localization ensures the protection of the members of clotting system: In the activator complexes found on the surface, the spatial arrangement of clotting factors prevents the inactivation of factors by physiological inhibitors or by proteolytic enzymes and specific antibodies that appear in the circulation in pathological conditions. Platelet factor 4, derived from platelets, binds heparin and thereby markedly decreases the reactivity of antithrombin III, the physiological inhibitor of clotting factors. The above two circumstances are

Blood Coagulation↗
PubMed1982

Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines.

All the five enzymes of urea synthesis and the formation of urea in vitro can already be demonstrated in human liver as early as the 9th week of fetal development. At this stage the activity of carbamoyl phosphate synthetase is the highest, whereas that of ornithine carbamoyltransferase is the lowest as compared to those in the adult. The kinetic parameters of the urea cycle enzymes are the same in fetal liver as in adult liver, except that the Km values of ornithine carbamoyltransferase for L-ornithine are 3.5 mM and 0.42 mM in the fetus and in adult liver, respectively. Urea formation in vivo seems to begin in the second half of fetal life, and a gradual increase can be detected in the activity of the enzymes of urea synthesis. The activity of ornithine decarboxylase, the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase, however, changes in the opposite direction. The concentration of carbamoyl phosphate and aspartate remains constant, but that of ornithine gradually decreases during ontogenesis. The ornithine, carbamoylphosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.

Argininosuccinate Lyase↗
PubMed1981

Purification and characterization of the chymotrypsin-like enzyme of the bovine granulocyte.

A chymotrypsin-like enzyme (EC 3.4.21.20) was isolated from bovine granulocytes, and purified 14-fold by affinity chromatography on 4-phenylbutylamine Affi-gel. The molecular weights of the isoenzymes were estimated as 16 000, 19 300 and 24 000 by SDS-polyacrylamide gel electrophoresis. A Michaelis constant of 2 mM and a catalytic constant of 34.6 s-1 were determined with Bz-Tyr-OEt. The esterolytic activity of the enzyme could be inhibited both by PMSF and by TPCK. It was also inhibited by chymostatin (Ki = 0.11 microgram/ml) and by the cytosol inhibitor of the bovine granulocyte (K'i = 1 microM). The chymotrypsin-like enzyme of the bovine granulocyte shares a number of the kinetic properties common to the chymotrypsin-like enzyme of the human granulocyte. The two granulocytes showed nearly identical chymotrypsin-like enzyme activities per cell.

Animals↗
PubMed1981

Study of the specificity of thrombin with tripeptidyl-p-nitroanilide substrates.

The kinetic behaviour of human thrombin has been studied with 26 tripeptidyl-p-nitroanilide substrates protected at the N terminus and with 9 unprotected ones. By the regression analysis of experimentally determined 1/Km, kcat and kcat/Km values the individual contribution of each side chain of the various substrates to the kinetic parameters was calculated. The contributions to the kinetic parameters of the best substrates provide information about the structure of the binding site. The interaction of subsites S1 and P1, which determines primary specificity, proved to be marginal on the basis of contribution values, though it depends upon this contact whether the substrate is hydrolyzed at all. At subsite S2 proline appeared to be favourable. Subsite S3 plays an important role in efficiency. The best parameters were obtained here with the D configurations of bulky amino acid residues. The aromatic protecting groups applied did not improve the properties of substrates. BZDPhe-Pro-Arg-Nan was predicted by calculation to be better than the protected substrates assayed. The compound was synthesized and tested. Its experimentally determined 1/Km, 55.1 mM-1, was in good agreement with 50.9 mM-1 found by calculation.

Anilides↗
PubMed1981

Investigation of the substrate-binding site of trypsin by the aid of tripeptidyl-p-nitroanilide substrates.

The kinetic parameters of the tryptic hydrolysis of tripeptidyl-p-nitroanilide substrates were determined and the data were studied by regression analysis. The sequence of substrates optimal from the viewpoint of kinetic constants 1/Km, kcat and kcat/Km was established and the influence of amino acid side chains on the binding and reactivity of substrates was calculated. At subsite P3 [notation of Schechter and Berger (1967) Biochem. Biophys, Res. Commun. 27, 157] polar side chains (Asn, D-Arg) are favourable as regards 1/Km, whereas hydrophobic side chains are preferred definitely from the viewpoint of catalytic efficiency, just as at subsite P2. In the side chain contributions, calculated for the kinetic parameters, the P3-S3 interaction predominates, in spite of the fact that the properties of the residue at subsite P1 decide whether hydrolysis occurs at all. The ZAsn-Ile-Arg-Nan sequence was predicted as a better substrate than those tested experimentally. The compound was synthesized, and the calculated value of its 1/Km (116.4 mM-1) was in a good agreement with the measured value (100.2 mM-1). Comparing the data obtained with trypsin with those observed with thrombin, elastase and subtilisin, we can establish that the homology of these enzymes can be characterized at each binding subsite by the aid of tripeptidyl-p-nitroanilide substrates. The quantities derived allow one to envisage a novel type of comparison of the proteases.

Anilides↗