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PubMed1984

Assay of prekallikrein as plasminogen proactivator in plasma specimens from reactors to dextran or to contrast media.

In a previous study of plasma specimens from reactors to clinical dextran, it was concluded that a factor activated by acetone converted the high-molecular-weight kininogen (HMrK) into a non-functional state. We recently identified the HMrK-destroying factor as a plasma kallikrein modification with high plasminogen activator (PGA) activity. The aim of the present work was to investigate whether the level of plasma kallikrein, assayed as PGA on fibrin plates, was higher than normal in plasma from patients reacting to dextran or radiographic contrast media. Plasma specimens from 10 reactors and 16 controls were examined. No difference in PGA level could be detected between reactors and controls in citrated plasma stabilized with benzamidine 9 mM, whereas a significant loss of PGA activity took place in citrated reactor plasma during the acetone activation procedure. The loss of PGA activity could not be due to known inhibitors of plasma kallikrein. It was only partial, and did not influence the amidase effect against the tripeptide substrate H-D-Pro-Phe-Arg-pNA. The results might indicate the presence in plasma of different amounts of an unknown constituent important for the obtainable level of plasma kallikrein with high PGA activity. The significant loss of PGA activity in reactor plasma might indicate an abnormally high level of the unknown plasma factor.

Acetone↗

PubMed1983

Investigation of intercellular matrix macromolecules involved in lichen sclerosus.

Dermal changes of the vulva in lichen sclerosus were compared with control vulvar samples using ultrastructural and immunofluorescence techniques. Collagen degeneration and regeneration were observed ultrastructurally in the superficial dermis of lichen sclerosus with increased amounts of ground substance. These processes appeared to alter the affinity of collagen fibres for the anticollagen antisera types I, III, IV. A decrease in elastin content was observed by electron microscopy. A loss of fibronectin was discovered at the dermo-epidermal junction, which looked normal ultrastructurally. The linear laminin pattern at the dermo-epidermal junction was also altered. These results suggested an enzymatic process in the pathogenesis of lichen sclerosus. Amidase activity could be determined in 6 normal and 6 pathological biopsies, though higher in the pathological samples (p less than 0.01).

Atrophy↗

PubMed1983

A functionally active heavy chain derived from human high molecular weight urokinase.

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.

Amino Acids↗

PubMed1984

[Identity of kininogenase and plasminogen activators in human granulocytes].

It was shown that the plasminogen activator inhibitor, ZGlyGlyArgCH2Cl, inactivates the kininogenase and plasminogen activator activities in the whole human granulocyte lysate and human granulocyte proteinase fractions isolated by isoelectrofocusing from the granulocyte lysate (pH 3-10). The kinetics of irreversible inhibition of the ZGlyGlyArgpNA-amidase activity in granulocyte proteinase fractions (pI 10.75, 8.9 and 8.3) by ZGlyGlyArgCH2Cl was measured. These data confirm the earlier obtained results on the trypsin-like nature of the human granulocyte plasminogen activator and its identity to this cell kininogenase.

Amino Acid Chloromethyl Ketones↗

PubMed1984

Metabolism of [14C]acetylisoniazid and [14C]acetylhydrazine by the rat and rabbit.

Male rats and rabbits were singly dosed with either 1-[14C]acetyl isoniazid (acetylisonicotinoylhydrazine, acetyl-INH, 200 mg/kg po) or 1-[14C]acetylhydrazine (50 or 100 mg/kg ip). Urine and expired 14CO2 were collected, and after 6 hr the animals were killed for the analysis of tissue 14C concentrations and covalent binding of 14C to hepatic protein. Rats excreted proportionately more 14C in urine and had lower 14C levels in their tissues compared to rabbits. When acetyl-INH was administered, covalent hepatic protein binding of the acetyl moiety was greater in the rabbit than the rat, but the opposite was observed when acetylhydrazine was administered. Analysis of blood and urine by TLC revealed that the rabbit more rapidly metabolized both acetyl-INH to acetylhydrazine, and acetylhydrazine to diacetylhydrazine than did the rat. These observations suggest that higher amidase activity in the rabbit compared to the rat leads to faster conversion of acetyl-INH to acetylhydrazine which in turn leads to greater covalent binding and hepatotoxicity.

Animals↗

PubMed1984

In vitro deacetylation studies of acetamidophenolic compounds in rat brain, liver and kidney.

The in vitro deacetylation of ortho-, meta- and para-substituted acetophenetidines and acetamidophenols including acetanilide by arylacylamidases in rat brain, liver and kidney was investigated. In general, deacetylation rates were highest in liver and kidney preparations, whereas brain exhibited lower enzyme activities. Acetophenetidines were more suitable N-deacetylation substrates in comparison to acetamidophenols, whereby o-substituted compounds were split more easily than their corresponding m- or p-analogues. Among the arylacylamides tested, o-acetophenetidine was the predominant substrate in all tissues. It was deacetylated by far more rapidly than phenacetin. The enzymic deacetylation of acetophenetidines in kidney and brain was inhibited by p-nitrophenylacetate revealing that N-acetylamides as well as O-acetyl esters undergo similar degradation by carboxylesterase-amidases. The organophosphorous diester bis(p-nitrophenyl)-phosphate which is known to be a rather selective inhibitor of acetanilide-cleaving hydrolases in rat liver suppressed the deacetylation of acetanilide in all tissues investigated. In contrast to acetanilide, the deacetylation of acetophenetidines was inhibited by bis(p-nitrophenyl)-phosphate in liver and kidney, but bis(p-nitrophenyl)-phosphate was by far less active against acetophenetidine hydrolases in brain.

Acetamides↗

PubMed1983

[Clinical evaluation of kininogenesis indices].

The discrepancies between kininogenase, BAEE-esterase and BAPNA-amidase activity in plasma kallikrein were shown in experiments in vitro. The same discrepancies were detected during examination of children aged under 3 years suffering from bronchopulmonary diseases (acute and lingering pneumonia, bronchial asthma). In these children, prekallikrein, kallikrein and kallikrein inhibitor were determined at a time by the 3 methods (esterase, test-tube chromatographic and kininogenase ones). It has been shown that the kininogenesis intensity could be assessed objectively only in the measurement of the kininogenase activity of kallikrein contained by the whole plasma or its fractions. The esterase technique characterizes total factors of contact activation and does not reflect the activity of kallikrein, prekallikrein and kallikrein inhibitor. The test-tube chromatographic method might be regarded as objective only in the measurement of the kininogenase activity of the fractions that contain kallikrein or prekallikrein.

Asthma↗

PubMed1983

[Prothrombin--substrate for blood plasma kallikrein and factor XIIa].

A study was made of the interaction between prothrombin and enzymes: blood plasma kallikrein and factors alpha-XIIa and beta-XIIa immobilized on enzacryl-AH. Kallikrein-induced prothrombin proteolysis was accompanied by a decrease in prothrombin activity, appearance of BAME-esterase and poor clotting activity. As a result of fractionation of products on the column with DEAE-Sephadex A-50, some fractions that have thrombin amidase activity (splitting of the substrate S-2238) and high antithrombin activity were obtained. Antithrombin activity manifested in the inhibition of fibrinmonomer aggregation during fibrin formation. During incubation with prothrombin, factors alpha-XIIa and beta-XIIa also stimulated the appearance of BAME-esterase activity. None of the immobilized enzymes activated factor X.

Acrylic Resins↗

PubMed1983

[Cultural, biochemical and serological characteristics of Mycobacteria, avium and atypical, isolated from pigs slaughtered in the Białystok abattoirs].

Two hundred and thirty-eight strains of acid-fast bacilli isolated from lymph nodes of pigs were examined. Among these strains 198 were classified as Myc. avium and 40 - as atypical bacilli belonging to group II, III and IV of Runyon's classification. Only 118 strains of Myc. avium showed cultural and biochemical properties typical for these species. Some of the remaining strains were positive in the arylsulphatase test, in the reduction nitrate test or in the hydrolysis of Tween 80 test, but were negative in all amidase tests. The serological study of the bacilli belonging to Myc. avium - intracellulare group was performed according to Schaefer's method. It was found that among Myc. avium strains serotype 2 dominated (187 strains). Myc. avium serotypes 1 and 3 appeared sporadically. The majority of atypical bacilli were classified as Myc. fortuitum, and only some strains were identified as Myc. intracellulare and Myc. vaccae. Some strains remained unclassified. The results showed that identification of Myc. avium and some atypical strains isolated from pigs by means of cultural and biochemical tests is not sufficient. The agglutination test with the use of sera specific for individual serotypes of Myc. avium - intracellulare group is very useful for identification of bacilli belonging to these groups. If we have a full set of specific sera we can identify Myc. avium and Myc. intracellulare more quickly and better than with use of cultural and biochemical tests.

Abattoirs↗

PubMed1984

Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.

Amidohydrolases↗

PubMed1980

Isoniazid hepatoxicity: the relationship between covalent binding and metabolism in vivo.

The relationship between the hepatotoxicity and metabolism of isoniazid and its metabolites, acetylisoniazid and acetylhydrazine, has been investigated. Toxic doses of acetylisoniazid and acetylhydrazine, radiolabeled in the acetyl group, were found to bind covalently to liver protein in vivo. This binding was mediated by the microsomal enzyme system as indicated by the effects of pretreatments altering the activity of these enzymes. Metabolic studies revealed that the pretreatments increased the metabolism of the acetylhydrazine moiety of acetyl-labeled acetylisoniazid and of acetylhydrazine itself by the microsomal enzyme system. Pretreatment with the acyl amidase inhibitor, bis-p-nitrophenyl phosphate, inhibited the hydrolysis of acetylisoniazid to isonicotinic acid plus acetylhydrazine and concomitantly decreased the covalent binding of acetyl-labeled acetylisoniazid. The changes in the metabolism of isoniazid, acetylisoniazid and acetylhydrazine effected by various pretreatments paralleled changes in the severity of the hepatic necrosis caused by the compounds. These results strongly suggest that acetylhydrazine is the metabolite responsible for the hepatic necrosis caused by isoniazid and that mirosomal metabolism of acetylhydrazine in vivo leads to the production of a reactive acylating species capable of reacting covalently with tissue macromolecules.

Acetylation↗

PubMed1978

Hepatotoxicity and metabolism of iproniazid and isopropylhydrazine.

Iproniazid (1-isonicotinoyl-2-isopropylhydrazine), an antidepressant drug removed from clinical use because of hepatic injury, and isopropylhydrazine, a metabolite of iproniazid, were found to be potent hepatotoxins in rats. This animal model was used in studies in vivo and in vitro to define better the biochemical and chemical mechanism(s) by which iproniazid and isopropylhydrazine mediate hepatotoxicity. Phenobarbital, an inducer of a class of hepatic microsomal cytochrome P-450 enzymes, greatly potentiated the necrosis, whereas inhibitors of these microsomal enzymes such as cobalt chloride, piperonyl butoxide and alpha-naphthylisothiocyanate, prevented the necrosis. Bis-para-nitrophenyl phosphate, an inhibitor of esterase and amidase enzymes, prevented the necrosis caused by iproniazid but had no effect on the necrosis caused by isopropylhydrazine. Iproniazid and isopropylhydrazine labeled with tritium or carbon-14 in the isopropyl group were found to bind covalently to hepatic tissue macromolecules, and those pretreatments that increased hepatic necrosis significantly increased covalent binding, whereas those pretreatments which prevented necrosis significantly decreased covalent binding. Iproniazid labeled with tritium in the pyridine ring or carbon-14 in the carbonyl group did not bind significantly to hepatic tissue. Rats that were given iproniazid or isopropylhydrazine, labeled specifically with tritium and carbon-14 on the c-2 methine position of the isopropyl group, expired acetone and carbon dioxide labeled with carbon-14. More importantly, propane was expired and contained a ratio of 3H/14C that was identical to that in the administered iproniazid or isopropylhydrazine and also identical to the 3H/14C ratio of the metabolite that was covalently bound to hepatic tissue macromolecules. Experiments carried out with rat liver microsomes and isopropylhydrazine specifically labeled with deuterium, tritium and carbon-14 support the view that isopropylhydrazine is the metabolite of iproniazid that is oxidized by a microsomal P-450 enzyme to a species that alkylates tissue macromolecules. Some of the urinary metabolites excreted by rats that were administered hepatotoxic doses of iproniazid and isopropylhydrazine have been identified by cochromatography and isotope dilution with synthetic standards and by comparative mass spectra. Compounds excreted into the urine of rats dosed with iproniazid include iproniazid, iproniazid-1-oxide, isonicotinic acid, isonicotinoyl glycine, acetylisoniazid, isopropylhydrazine, 1-acetyl-2-isopropylhydrazine and acetone. Isopropylhydrazine, 1-acetyl-2-isopropylhydrazine, and acetone have been found in the urine of animals administered toxic doses of isopropylhydrazine.

Animals↗

PubMed1981

A report on the biochemical analysis of Mycobacterium W.

A mycobacterial strain known as Mycobacterial strain W was analysed for its growth characteristics and biochemical traits. This strain was found to be a rapid grower, with luxurient growth on Lowenstein-Jensen medium, Dubos agar, Middlebrook's agar and Sauton's medium. Colonies were smooth, convex and nonpigmented. Some of the colonies which appeared rough were similar to smooth colonies at least in biochemical characteristics. This organism was tolerant to wide range of temperatures and to chemical substances like thiophene - carboxylic acid hydrazide, isoniazid, sodium chloride but not to bile salts. It was negative for niacin production, for various amidases, urease production, 3 day arylsulfatase test and also for Tween 80 hydrolysis. On the other hand this strain was found to be positive for semiquantitative catalase, heat resistant catalase, nitrate reduction, sodium salicylate degradation, tellurite reduction, 14 day arylsulfatase test and fermentation of fructose. This organism could utilize sodium nitrate and sodium nitrite as sources of nitrogen but didn't exhibit any utilization of fructose, arabinose as only sources of carbon. Significance of these findings is discussed.

Mycobacterium leprae↗

PubMed1980

[Influence of the autolytic system on the bactericidal effect induced by beta-lactamic antibiotics (author's transl)].

New experimental conditions showing the phenomena of "antibiotic tolerance" are described. In Streptococcus pneumoniae, a strong protection against the loss of viability induced by penicillin was obtained when TRIS (hydroxymethyl aminomethane), instead of potassium phosphate, was used as buffer of the growth medium. The main pneumococcal autolysin (N-acetyl-muramil amidase) present in S. pneumoniae was found to be, at least partially, responsible for the loss of viability induced by beta-lactamic molecules.

Anti-Bacterial Agents↗

PubMed1994

Functional role of N-glycosylation in alpha 5 beta 1 integrin receptor. De-N-glycosylation induces dissociation or altered association of alpha 5 and beta 1 subunits and concomitant loss of fibronectin binding activity.

Fibronectin (FN)-mediated cell adhesion is controlled mainly by alpha 5 beta 1 (recognizing the RGD sequence) and alpha 4 beta 1 (recognizing the CS-1 peptide sequence of FN) integrin receptors. Integrin-dependent cell adhesion to FN is greatly promoted by optimal GM3 concentration at the surface membrane (Zheng, M., Fang, H., Tsuruoka, T., Tsuji, T., Sasaki, T., and Hakomori, S. (1993) J. Biol. Chem. 268, 2217-2222), and cell adhesion mediated by alpha 4 beta 1 (to FN) or alpha 6 beta 1 (to laminin) is inhibited by modifying N-glycosylation processing of the integrin receptor (e.g. Akiyama, S. K., Yamada, S. S., and Yamada, K. M. (1989) J. Biol. Chem. 264, 18011-18018). We therefore studied the specific role of N-glycosylation in alpha 5 beta 1 function. Key findings of the present study were as follows. (i) Adhesion of K562 cells to FN-coated plates, which is mediated solely by alpha 5 beta 1, was inhibited when cells were treated with a mixture of endo-N-acetylglucosaminidase F and peptide -N4-(N-acetylglucosaminyl)asparagine amidase F (endo-F/PNGase-F). (ii) The alpha 5 beta 1 receptor at the K562 cell surface tended to dissociate into alpha 5 and beta 1 subunits when an extract of cells treated with endo-F/PNGase-F was precipitated by integrin subunit-specific antibodies, i.e. the alpha 5 subunit was preferentially precipitated by anti-alpha 5 monoclonal antibody ZH5, and the beta 1 subunit was preferentially precipitated by anti-beta 1 monoclonal antibody ZH1. When intact cells were extracted and treated with either ZH5 or ZH1, both alpha 5 and beta 1 were coprecipitated, indicating that the two subunits are normally tightly associated with each other. (iii) Adhesion of alpha 5 beta 1-containing liposomes (phosphatidylcholine:cholesterol liposomes incorporating purified alpha 5 beta 1) to FN-coated plates was abolished by treatment of liposomes with endo-F/PNGase-F. Liposomes incorporating alpha 5 beta 1 pretreated with endo-F/PNGase-F also did not bind to FN. When purified alpha 5 beta 1 receptor was treated with endo-F/PNGase-F followed by ZH5 or ZH1, the alpha 5 or beta 1 subunit was precipitated separately, respectively. In contrast, both subunits were always coprecipitated when intact purified alpha 5 beta 1 receptor was directly treated with ZH5 or ZH1. These findings indicate that N-glycosylation of both the alpha and beta subunits of the alpha 5 beta 1 integrin receptor is essential for association of these subunits and for optimal binding to FN.

Binding Sites↗

PubMed1995

Structure of the N-linked oligosaccharides from tridacnin, a lectin found in the haemolymph of the giant clam Hippopus hippopus.

Tridacnin, a glycoprotein lectin, was isolated from the symbiotic marine clam Hippopus hippopus and the structure of its major N-glycan chains determined. Tridacnin contains only N-linked glycans which were quantitatively cleaved by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Following purification by anion-exchange HPLC, the structures of the oligosaccharides were established using a combination of electrospray ionisation mass spectrometry, 1H-NMR spectroscopy and linkage analysis. The N-glycans are primarily of the oligomannose type but, in addition, some contain a novel 6-O-Me group on the terminal mannose residue of the chain. The N-glycan chains had the following structures. [formula: see text]

Animals↗

PubMed1995

[Elastase].

Elastases are unique among the proteases in that they are capable of hydrolyzing the scleroprotein elastin. The enzymes include pancreatic elastases 1 (Protease E) and 2, and neutrophil elastase. These three elastases also have esterase and amidase activity toward synthetic substrates such as succinyl-trialanine-p-nitroanilide. Although the three enzymes are similar to each other in enzyme activity, they are quite different in immunoactivity. Therefore, each elastase has its own specific immunoassay either by RIA or EIA. Serum immunoreactive pancreatic elastases reflect disease conditions of pancreatic diseases, especially acute pancreatitis and pancreatic cancer. On the other hand, serum neutrophil elastase increases in various inflammatory diseases or conditions.

Humans↗

PubMed1994

Identification of the C-terminal amino acid amides by carboxypeptidase Y digestion and fast atom bombardment mass spectrometry.

The combination method of carboxypeptidase Y digestion and fast atom bombardment (FAB) mass spectrometry is described for the identification of C-terminal amino acid amides in peptides. Carboxypeptidase Y has amidase activity as well as exopeptidase activity in the same digestion buffer condition. Based on this concept, we develop a new technique which can definitively and easily identify the C-terminal amino acid amides. This method obviates the need for several complicated steps occurring in previous methods, but improves sensitivity, and enables exact identification of the amino acid amide by the difference of molecular mass. Analyses of carboxypeptidase Y digested peptides, not liberated free amino acid amides, were carried out by fast atom bombardment mass spectrometry. The use of truncated peptides by fast atom bombardment mass spectrometry in C-terminal amino acid amide determination gives several advantages over analyses of the liberated amino acid amides. The C-terminal amino acid amides of Allantostatin I (Leu-NH2), alpha-Melanocyte Stimulating Hormone (Val-NH2), and Ranatensin (Met-NH2) are unequivocally determined at a level of 0.90-2.3 nmol per peptide. This approach is based on entirely different principles than the previous approaches.

Amides↗