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At least 37 records · Page 2Linked to original sources
PubMed1987

Acyl-peptide hydrolase from rat liver. Characterization of enzyme reaction.

Acyl-peptide hydrolase, which catalyzes the hydrolysis of an N-terminally acetylated peptide to release an N-acetylamino acid, was isolated from rat liver and found to be N-terminally blocked. The kinetics of the hydrolysis of acetyl (Ac)-Ala-Ala, Ac-Ala-Ala-Ala, acetylalanine p-nitroanilide, and acetylalanine beta-naphthylamide were investigated. The Km values were between 1 and 9 mM, and the Vmax values were between 100 and 500 nmol/min/micrograms of enzyme. The enzyme activity toward acetylalanine p-nitroanilide and acetylalanine beta-naphthylamide was activated by the presence of Cl- and SCN- at concentrations between 0.1 and 0.5 M. By contrast, the activity toward Ac-Ala-Ala and Ac-Ala-Ala-Ala was inhibited by these anions. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by the addition of diisopropyl fluorophosphate, diethyl pyrocarbonate. Woodward's Reagent K, and glycine methyl ester/carbodiimide. Titration by diisopropyl fluorophosphate showed 0.7 mol of active serine/mol of enzyme subunit, which was confirmed by the incorporation of [3H]diisopropyl fluorophosphate into the enzyme. Acetylalanine chloromethyl ketone inactivated the enzyme following pseudo-first order kinetics; and Ac-Ala, a competitive inhibitor, protected the enzyme from this inactivation. Acyl-peptide hydrolase appears to be a serine protease utilizing a charge relay system involving serine, histidine, and, probably, a carboxyl group(s). Two series of acetyl dipeptides, acetylamino acid p-nitroanilides and acetylamino acid beta-naphthylamides, were prepared in order to determine enzyme specificity. The enzyme preferentially removed Ac-Ala, Ac-Met, and Ac-Ser, the most common acetylated N-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jörnvall, H. (1985) Eur. J. Biochem. 152, 523-527). The enzyme was shown to be useful for deblocking peptides (e.g. alpha-melanocyte-stimulating hormone and acetyl-renin substrate), and the crude enzyme/substrate mixtures were amenable to direct protein sequence analysis.

Affinity Labels↗
PubMed1976

Release of peptide hydrolases during incubation of intact intestinal segments in vitro.

1. Rat intestinal segments have been incubated in isotonic saline in vitro and release of peptide hydrolase enzymes into the incubation media examined over a 90 min study period. 2. Chemical assay data, as well as analysis of electrophoretic mobilities of release enzymes on starch gel, indicate that peptide hydrolase enzymes in the incubation media originate predominantly from the cytoplasm of the mucosal cells. 3. Peptide hydrolases were released rapidly from intact intestinal segments. Release occurred from the start of the in vitro incubations and was not affected by temperature and shaking. 4. It is concluded that compared to the in vivo situation, cytoplasmic peptide hydrolases are released from intestinal tissue very rapidly in vitro. Caution is therefore required when comparing results of in vivo and in vitro peptide absorption experiments.

Animals↗
PubMedDate not supplied

[Thiol peptide hydrolases from animal tissues, their structure and function].

Data on properties, structure and biological functions of a variety of thiol (cysteine) peptide hydrolases from animal tissues have been summarized. This large group of diverse intracellular enzymes involves both endo- and exopeptidases. Best studied are lysosomal thiol peptide hydrolases: cathepsins B, H and L, the primary structure of which is deciphered. They present a family of homologous proteins, structurally similar to papain. Ca2+-dependent neutral proteinases is another family of related proteins. The biological functions of various thiol peptide hydrolases are considered: their participation in protein turnover, post-translational processing, regulation of unidirectional biological processes and metabolic refolding. Data on endogenous inhibitors of thiol peptide hydrolases and on regulation of enzymic activity are presented.

Amino Acid Sequence↗
PubMedDate not supplied

[Peptide-hydrolase activity in different areas of brain with introduction of hydrocortisone and ACTH].

A single introduction of hydrocortisone is established to evoke an increase in the total activity of acid peptide-hydrolase in most areas of the brain. A single administration of ACTH single and long term administration of hydrocortisone was found to increase the specific activity of acid peptide-hydrolase of the soluble fraction. The specific activity of neutral peptide-hydrolase of the soluble fraction increases with a single administration of ACTH and long term administration of hydrocortisone. A single and long term administration of hydrocortisone and ACTH cause an increase in the percentage of the soluble form of peptide-hydrolases of the total activity.

Adrenocorticotropic Hormone↗
PubMedDate not supplied

[Effects of oxygen deficiency on rat brain peptide-hydrolase activity with and without hypothermia].

The effect of acute oxygen deficiency on the rat brain peptide-hydrolases (EC 3.4) activity was studied under conditions of acute hypoxic hypoxia (the pressure in altitude chamber 240 mm Hg) with concomitant hypothermia (--5.8 degree C) and without it. The activity of neutral peptide hydrolases decreases by 29% in rats with hypothermia, in animals with normothermia remains unchanged. The activity of acid peptide hydrolases was the control level. The data obtained are compared with those relative to preservation of the content of soluble proteins under hypoxia with hypothermia.

Animals↗
PubMed1976

Partial purification and characterization of two Peptide hydrolases from pea seeds.

Two peptide hydrolases have been found in pea seeds (Pisum sativum var. Greenfeast) and extensively purified by ion exchange chromatography using benzoyl-dl-arginine-p-nitroanilide as substrate. The enzymes which both have molecular weights of 65,000 can be separated by anion exchange chromatography but are otherwise virtually identical in the properties tested. They did not hydrolyze several common protease substrates but readily hydrolyzed small peptides containing basic amino acids on the carboxyl side of these residues. They are completely inhibited by diisopropylfluorophosphate and are inhibited to varying extents by thiol reagents.

Journal Article↗
PubMedDate not supplied

[The use of various fluorogenic substrates for determining peptide hydrolase activity of the blood serum].

4-methoxy-beta-naphthylamide and 7-amino-4-methyl coumarin, derivatives of Z-Ala-Arg-Arg, Leu- and Gly-Phe-beta-naphthylamides were used as substrates in estimation of peptide hydrolases activity in blood serum of patients with malignant tumors and glomerulonephritis in order to ascertain their efficiency for diagnostic purposes in clinic. Each of the fluorogenic substrates studied was hydrolyzed by various peptide hydrolases from blood serum both under normal and pathological conditions: metallopeptidases, cysteine- and serine-dependent peptide hydrolases. The rate of Z-Ala-Arg-Arg-MNA hydrolysis was decreased in lung, kidney and ileum cancer as well as in glomerulonephritis as compared with normal state. The "alkaline-resistant" cysteine-dependent cathepsin B-like proteinase, hydrolyzing this peptide, was not detected in blood serum neither in normal state nor in these diseases studied. Leu-NA and Gly-Phe-NA were hydrolyzed most effectively in blood serum of patients with lung cancer and glomerulonephritis as compared with normal state; cysteine-dependent peptide hydrolases were most markedly activated. Alterations in the enzymatic activity, detected in blood serum, did not exhibit any specificity for definite diseases, they were observed both in malignant and inflammatory impairments. The data obtained suggest that the fluorogenic substrates studied could not be suitable for clinico-diagnostic purposes.

Clinical Enzyme Tests↗
PubMed1976

[Peptide hydrolase activity of the soluble protein fractions of cattle and rabbit cerebral hemispheric gray and white matter].

The neutral peptide-hydrolase activity of the soluble protein fraction of homogenates of whole brain and white and grey matters of bovine and rabbit cerebral hemispheres are significantly reduced on dialysis against water. In the evaporated dialysate the enzymatic activity of the soluble protein fraction is completely recovered. The reduction of peptide hydrolase activity following dialysis is probably due to the decrease of ionic strengths. The peptide hydrolase system of bovine brain is less stable on storage than that of rabbit brain.

Animals↗
PubMed1972

Peptide hydrolases in the bruch border and soluble fractions of small intestinal mucosa of rat and man.

Peptide hydrolases, catalyzing the hydrolysis of 13 dipeptides and 5 tripeptides into their respective amino acids, were studied in small intestinal mucosa and other tissues, in man and in the rat. Studies on the subcellular distribution of these enzymes showed enzyme activities in both the soluble and brush border fractions of the rat small intestinal mucosa, the former constituting 80-90% and the latter 10-15% of the total activity. Zymogram studies of peptide hydrolases, in both fractions, yielded multiple bands indicating multiple zones of enzyme activity. With most substrates a rather broad range of enzyme activities was observed in the soluble fraction differing only slightly from substrate to substrate, the exception being when L-leucyl-L-proline was used: this latter led to a zymogram pattern which was quite distinct. The synthetic substrates, L-leucyl-beta-naphthylamide and L-leucinamide appeared to be hydrolyzed by two electrophoretically distinct enzymes, different from those hydrolyzing other leucyl-containing peptide substrates. Zymogram patterns of the brush border membrane fraction were quite different from those of the soluble fraction of rat small intestine indicating that enzymes from the two sources may be different. No comparable human data were obtained.Peptide hydrolases in the soluble fractions of various organs from the same species gave similar zymogram patterns, while those from the plasma membrane-bound fractions of different organs in the same species were peculiar to each organ. From these data, it is suggested that peptide hydrolases in the brush border and the soluble fractions of small intestine are distinct enzymes and may play different roles in cellular function.

Amides↗
PubMedDate not supplied

[Changes in neutral peptide hydrolase levels in the blood and catecholamine levels in the tissues during adaptation to high altitude hypoxia].

Activity of neutral peptide-hydrolases in blood plasma and content of catecholamines in the tissues of rats under conditions of their stay in mountains at different height have been studied in dynamics. Unidirectional phase changes in activity of proteolytic enzymes of blood plasma and content of catecholamines in adrenal glands are revealed. A conclusion is made on participation of neutral blood peptide-hydrolases in organism adapted to Alpine hypoxia. Relations between neutral blood peptide-hydrolases and catecholamines in the organism and possibility of their direct mutual effect are under discussion.

Adaptation, Physiological↗
PubMed1969

Peptide hydrolase activities of the mucosa of human small intestine.

Few studies have been published on peptide hydrolase activities of human small intestine mucosa. We developed methods to screen tissue extracts for such enzymes and to quantitate hydrolase activities for dipeptides containing the aromatic amino acid L-phenylalanine. The screening procedure indicated glycyl-L-proline hydrolase activity was reduced in biopsy specimens from patients with flattened intestinal mucosa. To explore this further, we established optimal assay conditions for hydrolase activities (a) glycyl-L-proline, (b) L-phenylalanyl-L-proline, (c) L-alanyl-L-phenylalanine, and (d) L-phenylalanylglycine. Biopsy specimens from patients with various intestinal disorders, but without flattened mucosa, and from three patients with flattened mucosa, showed a disproportionate reduction in activities (a) and (b), with the reduction being significantly more marked in the latter patients. We suggest that intestinal imidopeptide hydrolase activities, such as (a) and (b), are sensitive to changes in intestinal disease generally, particularly to the altered physiology associated with flattening of the mucosa, and are secondary to, rather than a cause of, the intestinal pathology. Our finding that intestinal alkaline phosphatase activity tended to parallel imidopeptide hydrolase activity, and that activity (a) was partially localized to the particulate fraction of mucosal homogenate, suggested that imidopeptide hydrolase activities may be located in the microvilli of the intestinal epithelium and that, like alkaline phosphatase activity, they may be reduced in flattened mucosae, in part at least because of the pathologic changes in the microvilli. In our studies of control subjects we did not detect peptide hydrolase activity deficiency analogous to asymptomatic disaccharidase deficiency.

Alkaline Phosphatase↗
PubMedDate not supplied

[Neutral peptide hydrolases in the blood serum and lungs in hypoxia].

In rats exposed to hypoxia (breathing 11% O2-N2 for 15 min), activity of neutral peptide hydrolases in lungs and serum was measured before and after altitude chamber training. When exposed to normal air breathing, activity of lung neutral peptide hydrolases was markedly higher in Group 1 rats, i.e. those in whom O2 consumption changed insignificantly in response to the hypoxic test, than in Group 2 rats, i.e. those in whom O2 consumption increased in response to the hypoxic test. After altitude chamber training activity of lung and serum neutral peptide hydrolases decreased in Group 1 rats and increased in Group 2 rats. The potential correlation between lung and blood neutral proteolysis is discussed. Its contribution to the biochemical control of oxygen transport via blood rheological properties and vascular tone is considered.

Animals↗
PubMed1977

Isolation and characterization of four peptide hydrolases from the cytosol of rat intestinal mucosa.

The high speed supernatant fluid prepared from rat intestinal mucosa was subjected to ion-exchange chromatography on diethlaminoethyl-cellulose eluted with a linear gradient of sodium chloride (0 to 0.27 M). Assay of eluted fractions for Phe-Gly hydrolase activity revealed four distinct peaks of enzyme activity. These cytosol enzymes have been designated I, II, III, and IV in order of their elution from the column. Examination of the substrate specificity of the four enzymes by use of 20 mM peptide concentrations indicated the most discriminating substrates for the four enzymes were Leu-Gly-Gly, His-Met, Ser-Phe, and leucine amide, respectively. The mean distribution of the recovered peptide hydrolase activities against these substrates among the four enzymes I, II, III, and IV was 96.1, 1.4, 1.7, and 0.8%, respectively, for Leu-Gly-Gly; 0.6, 96.4, 2.4, and 0.6% for His-Met; 0, 0, 95.8, and 4.2% for Ser-Phe; and 20.8, 19.8, 5.6, and 53.8% for leucine amide. Ion-exchange chromatography resulted in increases in specific activity of 19-, 19-, 46-, and 3.5-fold for enzymes I, II, III, and IV, respectively. The activity of all four enzymes, but especially III and IV, were stabilized by the presence of 150 muM dithioerythritol. Activity of each of the four enzymes was decreased 79 to 100% by 1mM ethylenediaminetetraacetate, HgCl2, 1, 10-phenanthroline, or 0.5 mM p-hydroxymercuribenzoate, except that the activity of enzyme I was decreased only 15% by ethylenediaminetetraacetate. No significant activation of the partially purified enzymes occurred in the presence of 500 muM Zn++, Co++, or Mg++. The four enzymes exhibited distinct pH profiles with optima at 7.5, 7.5, 8.5, and 8.0 for enzymes I, II, III, and IV, respectively. Molecular weights of the four enzymes determined by gel filtration on Sephadex G-200 were 58,500, 74,000, 97,500, and 113,000, respectively. All four enzymes lost more than 85% of their activity after 1 hr at temperatures of 50 degrees C or higher in sodium phosphate buffer, pH 7.0. The Km values determined with the most specific substrates for each enzyme were 0.76, 0.44, 3.82, and 8.3 mM for enzymes I, II, III, and IV, respectively. Recent evidence suggests that a significant amount of some small peptides are absorbed intact and hydrolyzed by cytosol peptide hydrolases. Adequate understanding of the function and control of these intracellular enzymes requires knowledge of the characteristics and substrates specificity of individual enzymes. The study described here demonstrates the presence of at least four cytosol peptide hydrolases with distinct substrate specificities. Substrates almost exclusively hydrolyzed by each of three of the enzymes, and therefore suitable for assay of each of these enzymes in the presence of the others, have been identified.

Animals↗
PubMed1979

Adjuvant-induced inflammatory disease in the rat: plasma levels of peptide hydrolases and protease inhibitors reflect disease activity.

Both during the primary (localized) inflammation and the development of the secondary (generalized) inflammation in adjuvant-treated rats, the plasma level of functional alpha-macroglobulins increases while proteases (measured as peptide hydrolases) sharply decrease. The decreased peptide hydrolase levels during episodes when protease 'spillage' into the bloodstream is elevated, suggests a more rapid clearance of alpha-macroglobulin-protease complexes associated with inflammation.

Adjuvants, Immunologic↗
PubMed1986

Delineation of a particulate thyrotropin-releasing hormone-degrading enzyme in rat brain by the use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase.

The degradation of thyrotropin-releasing hormone in rat brain homogenates was studied in the presence of N-benzyloxycarbonyl-prolyl-prolinal and pyroglutamyl diazomethyl ketone, specific and potent active-site-directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin-releasing hormone-degrading enzyme(s). Reports of a thyrotropin-releasing hormone-degrading enzyme with narrow specificity that cleaves the pGlu-His bond of this tripeptide led us to develop a coupled assay using pGlu-His-Pro-2NA as the substrate to measure this activity. Cleavage of the pGlu-His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl-peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o-phenanthroline. The particulate pyroglutamyl-peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin-releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu-His bond of thyrotropin-releasing hormone but is distinct from pyroglutamyl peptide hydrolase.

Aminopeptidases↗
PubMedDate not supplied

[Brain peptide-hydrolase activity in hypothermia].

The cooling of rats to the body temperature of 30 and 25 degrees C does not change acidic peptide-hydrolase activity in tissue homogenates of cerebral cortex, hypothalamus, thalamus, hippocampus, midbrain, cerebellum and medulla oblongata. In deep hypothermia (to 20 degrees C) there occurs a significant increase in the peptide-hydrolase activity in tissue of the studied brain areas both in an incubation sample at 37 degrees C, and at temperature corresponding to the body temperature of cooled animals (20 degrees C). But the enzyme activity remains unchanged in homogenates of medulla oblongata incubation at 37 degrees C.

Animals↗
PubMed1989

Purification and characterization of an N-acylaminoacyl-peptide hydrolase from rabbit muscle.

An N-acylaminoacyl-peptide hydrolase has been purified to homogeneity (7,000-fold with 20% yield) from rabbit muscle. This overall enrichment and its general properties as a soluble protein suggest that it is of cytosolic origin and not a component of ribosomes or other cellular organelles. The enzyme has an Mr of 230,000-245,000 and a subunit Mr of 76,000-80,000. An extensive survey of the substrate specificity of the pure enzyme reveals that our earlier conclusions (Radhakrishna, G., and Wold, F. (1986) J. Biol. Chem. 261, 9572-9575) that the enzyme is specific for Ac-Met-peptides are wrong. The enzyme catalyzes the rapid removal of Ac-Thr, Ac-Ala, Ac-Met, Ac-Ser, and more slowly Ac-Gly from peptides of different lengths. Other acetylated amino acids (Cys, Tyr, Asp, Val, Phe, Ile, Leu) may be removed at 1% or less of the rate of the above good substrates from some peptide substrates. The nature of the amino acid in the second position of the acetylated peptide generally has only a minor effect on the reaction rate; however, with charged amino acids (Arg, Asp) in the second position the reaction is retarded, and with proline it is virtually abolished. Except for slow rate of hydrolysis of acetylated dipeptides, the hydrolase does not appear to be severely affected by the peptide length in the range studied (from 2 to 11 amino acid residues). The hydrolase also cleaves formylamino acids from formylated peptides. The biological function of the enzyme is not clear.

Acetylation↗
PubMed1987

Characterization of a neutral aminoacyl-peptide hydrolase from Naegleria fowleri.

An intracellular alpha-aminoacyl-peptide hydrolase (EC 3.4.11.-) from Naegleria fowleri nN68 (ATCC 30894) has been characterized. The enzyme preparation hydrolyzed phenylalanyl-, tyrosyl-, leucyl-, arginyl-, alanyl-, tryptophanyl-, histidyl-, methionyl-, and lysyl-naphthylamide but not benzoylleucyl-, leucylglycyl-, glycylprolylleucyl-, glycyl-, threonyl-, aspartyl-, or glutamyl-naphthylamide. The aminopeptidase activity was inhibited by the cysteine-protease inhibitors--hydroxymercuribenzoate, chloromercurisulfate, and iodoacetate--by the aminopeptidase inhibitors--bestatin and trans-epoxysuccinyl-leucyl-agmatine--by an inhibitor of soluble alanyl aminopeptidase EC 3.4.11.14, puromycin, and by the metalloprotease inhibitor, o-phenanthroline. The exopeptidase activity was not inhibited by the chelator, ethylenediaminetetraacetate, or the serine-protease inhibitor, phenylmethylsulfonylfluoride. The pH optimum of the exopeptidase was between 7.0 and 8.0. Enzyme activity was stable at 55 degrees C for 30 min, but all activity was lost after 15 min at 80 degrees C. Enzyme activity was inhibited by 100 microM HgCl2 and CdCl2 but not by 1 mM CoCl2, CuCl2, MnCl2, NiCl2, FeCl3, or ZnCl2. Enzyme activity was inhibited by 0.1% sodium dodecyl sulfate but not by 0.2% Brij 35, Tween 20, Tween 80, or Triton X-100.

Amoeba↗