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PubMed1984

Soluble and immobilized clostridial aminopeptidase and aminopeptidase P as metal-requiring enzymes.

The dependence of enzymatic activity on Co2+ concentration was found to be bell-shaped for the soluble and immobilized clostridial aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.13) and aminopeptidase P (aminoacylpropyl-peptide hydrolase, EC 3.4.11.9), with maxima in the 3-18 microM range of Co2+ concentration. The Co2+-enzyme association constants derived from the activation of soluble, glass- and cellulose-bound clostridial aminopeptidase by Co2+ were KE-Co = 5.2 X 10(5), 4.5 X 10(6) and 2.0 X 10(5) M-1, respectively; for soluble and glass-bound aminopeptidase P, the KE-Co were 1.5 X 10(5) and 8.2 X 10(5) M-1, respectively. Kinetic measurements indicate the involvement of Co2+ in the enzyme-substrate binding. Cobalt-citrate (Co-cit) acted as a useful metallobuffer and protected both enzymes against inhibition by high concentrations of CoSO4. For association of citrate with Co2+ under the assay conditions, KCo-cit was determined as (5.3 +/- 1.4) X 10(3) M-1 by anodic stripping polarography. In contrast to the rapid association of Co2+ with soluble and glass-bound clostridial aminopeptidase (less than 1 min at 4 degrees C), the dissociation process was very slow (hours to days), being slower for the glass-bound than for the soluble and cellulose-bound enzyme. For aminopeptidase P, both processes were rapid. All the interactions were shown to be reversible.

Aminopeptidases↗
PubMed1999

4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules.

Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for these effects because of their ability to damage cellular components such as membranes, proteins, and DNA. In the present study, we provide evidence that oxidative stress causes a transient impairment of intracellular proteolysis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment with the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress, as monitored by accumulation of lipid peroxidation products and 8-hydroxy-2'-deoxyguanosine, in the kidney of mice. In addition, transient accumulation of HNE-modified proteins in the kidney was also found by competitive enzyme-linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitinated suggested a crucial role of proteasomes in the metabolism of HNE-modified proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent elimination of HNE-modified proteins. We found that, among three proteolytic activities (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase activities in the kidney were transiently diminished in accordance with the accumulation of HNE-modified proteins during oxidative stress. The loss of proteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an immunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impairment of ubiquitin/proteasome-dependent intracellular proteolysis.

Adenosine Triphosphate↗
PubMedDate not supplied

[Heterogeneity of protein metabolism and activities of certain enzymes in functionally different structures of nerve tissue].

The article deals with a review of the results of studies conducted at the department which evidence for inhomogeneity of localization, composition and different intensity of intravital renewal of proteins as well as for different activity of some enzymes most important for the nervous system functions in the macro- and microstructural formation of the nerve tissue. On the basis of the experimental data concerning the presence in the nerve tissue of a wide range of rates for intravital renewal of proteins in the structures of different functions as well as of those concerning subcellular and suborganoid localization of intracellular peptid-hydrolases, a conclusion is drawn that catabolism of the protein molecules occurs mainly on the cell membrane structures. The effect of acid peptide-hydrolases is chiefly manifested in the died and damaged cells and neutral ones in the intact functioning cells. As among the proteins most important functionally for the nervous activity ATP-hydrolyzing enzymes occupy a special place, the article provides basic data of studies in the ATPase activity in the nervous tissue, intracellular localization of the enzyme, its sensitivity to uni- and bivalent ions, as well as in the mechanism of interaction with ouabain--specific inhibitor and with melipramine--neurotropic substance.

Adenosine Triphosphatases↗
PubMed2005

Proteolytic activity and reduction of gliadin-like fractions by sourdough lactobacilli.

AIMS: To characterize the peptide hydrolase system of Lactobacillus plantarum CRL 759 and CRL 778 and evaluate their proteolytic activity in reducing gliadin-like fractions. METHODS AND RESULTS: The intracellular peptide hydrolase system of Lact. plantarum CRL 759 and CRL 778 involves amino-, di- (DP), tri- (TP) and endopeptidase activities. These peptidases are metalloenzymes inhibited by EDTA and 1,10-phenanthroline and stimulated by Co2+. DP and TP activities of Lact. plantarum CRL 759 and CRL 778, respectively, were completely inhibited by Cu2+. Lactobacillus plantarum CRL 778 showed the highest proteolytic activity and amino acids release in fermented dough. The synthetic 31-43 alpha-gliadin fragment was hydrolysed to 36% and 73% by Lact. plantarum CRL 778 and CRL 759 respectively. CONCLUSIONS: Lactobacillus plantarum CRL 759 and CRL 778 have an active proteolytic system, which is responsible for the high amino acid release during sourdough fermentation and the hydrolysis of the 31-43 alpha-gliadin-like fragment. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new information of use when obtaining sourdough starters for bread making. Moreover, knowledge regarding lactobacilli capable of reducing the level of gliadin-like fractions, a toxic peptide for coeliac patients, has a beneficial health impact.

Amino Acids↗
PubMed1978

Agonist-induced secretions and potassium release from rat submandibular gland slices.

Secretory activity induced by stimulation of alpha-adrenergic, beta-adrenergic, and muscarinic cholingeric receptors and by dibutyryl cAMP and 8-bromo cGMP has been investigated in rat submandibular tissue slices. Isoproterenol produced a sialic acid secretion from the acinar cells that was inhibited by propranolol, but not by phenoxybenzamine or atropine. Dibutyryl cAMP produced a sialic acid secretion from the acinar cells that was not inhibited by propranolol, phenoxybenzamine, or atropine. Both norepinephrine and acetylcholine produced significant secretion of sialic acid but at a reduced efficacy. Norepinephrine stimulated both peptide hydrolase secretion from the granular duct cells and a release of K+ from the acinar cells. Both actions were inhibited by phenoxybenzamine, but not by propranolol or atropine. Acetylcholine stimulated a minimal secretion of peptide hydrolase from the granular duct cells and a significant release of K+ from the acinar cells. The norepinephrine- and acetylcholine-stimulated release of K+ was increased after the addition of 1 mM ouabain. High concentrations of 8-bromo cGMP induced a K+ efflux that was not inhibited by phenoxybenzamine or atropine. Vacuolation of the acinar cells was correlated with K+ release.

Animals↗
PubMed1980

Intestinal enzyme adaptation to normal diets of different composition.

Groups of rats were fed isocaloric amounts of diets rich in starch, protein, or fat. After 7 days, activities of sucrase, maltase, lactase, alkaline phosphatase, L-phenylalanylglycine hydrolase, and L-leucyl-L-alanyl hydrolase were measured in the duodenal, jejunal, and ileal mucosa of fed rats. Peptide hydrolase and alkaline phosphatase activities were significantly higher in rats fed high-protein diets, particularly in the ileum. Disaccharidases were highest in rats fed high-starch diets, especially in jejunum; the lowest activities of peptide hydrolase were seen with this diet. The high-fat diet was generally associated with intermediate activities of most enzymes, except disaccharidases, which were lowest on this diet. The different macromolecular nutrients were associated with adaptive differences in intestinal enzyme activities, which were biologically appropriate and coordinated with similar known changes in pancreatic and other enzyme activities concerned with assimilation of the particular food. However, other less interpretable changes occurred, which may have been due to effects on pancreatic secretion or on the metabolism, growth, and differentiation of intestinal cells.

Alkaline Phosphatase↗
PubMed2001

Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion.

Restoration of blood flow to ischemic myocardial tissue results in an increase in the production of oxygen radicals. Highly reactive, free radical species have the potential to damage cellular components. Clearly, maintenance of cellular viability is dependent, in part, on the removal of altered protein. The proteasome is a major intracellular proteolytic system which degrades oxidized and ubiquitinated forms of protein. Utilizing an in vivo rat model, we demonstrate that coronary occlusion/reperfusion resulted in declines in chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-like activities of the proteasome as assayed in cytosolic extracts. Analysis of purified 20 S proteasome revealed that declines in peptidase activities were accompanied by oxidative modification of the protein. We provide conclusive evidence that, upon coronary occlusion/reperfusion, the lipid peroxidation product 4-hydroxy-2-nonenal selectively modifies 20 S proteasome alpha-like subunits iota, C3, and an isoform of XAPC7. Occlusion/reperfusion-induced declines in trypsin-like activity were largely preserved upon proteasome purification. In contrast, loss in chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities observed in cytosolic extracts were not evident upon purification. Thus, decreases in proteasome activity are likely due to both direct oxidative modification of the enzyme and inhibition of fluorogenic peptide hydrolysis by endogenous cytosolic inhibitory protein(s) and/or substrate(s). Along with inhibition of the proteasome, increases in cytosolic levels of oxidized and ubiquitinated protein(s) were observed. Taken together, our findings provide insight into potential mechanisms of coronary occlusion/reperfusion-induced proteasome inactivation and cellular consequences of these events.

Aldehydes↗
PubMed1975

Influence of exocrine and endocrine pancreatic function on intestinal brush border enaymatic activities.

Digestive enzymatic activities (disaccharidases, alkaline phosphatase, peptide hydrolases) have been determined in the mucosa of 14 patients with chronic pancreatitis. All had an abnormal secretin-pancreozymin test. Four patients had insulin-dependent diabetes mellitus, four a pathological glucose tolerance test. Nine patients had steatorrhoea. Maltase, sucrase, and alkaline phosphatase activity was significantly elevated in patients with exocrine pancreatic insufficiency, whereas those of lactase, trehalase, and peptide hydrolase were normal. Patients with steatorrhoea had higher maltase and sucrase activity than those without steatorrhoea, whereas decreased glucose tolerance had no effect on brush border enzymatic activity. It is suggested thatdecreased exocrine rather than decreased endocrine pancreatic function is responsible for the increase in intestinal disaccharidase and alkaline phosphatase activity, possible by the influence of pacreatic enzymes on the turnover of brush border enzymes from the luminal side of the mucosal membranes or by direct hormonal stimulation though cholecystokinin.

Adult↗
PubMedDate not supplied

Can elemental diets reduce the intestinal toxicity of 5-fluorouracil?

In order to test the hypothesis that elemental diets protect the intestinal mucosa against 5-fluorouracil toxicity, we have estimated water absorption, cytoplasmic peptide hydrolase activities, and mucosal DNA contents in isolated intestines from rats fed on one of three elemental diets or a standard diet and injected with 5-fluorouracil. Water absorption rates were significantly increased when rats, not injected with 5-fluorouracil, were fed on Flexical or Vivonex-HN. However, water absorption was severely impaired 3 days after administration of 5-fluorouracil and none of the elemental diets alleviated this impairment of absorption. Cytoplasmic peptide hydrolase activities and mucosal DNA contents were also severely decreased after 5-fluorouracil injection, the changes observed being independent of the diet. Also, none of the elemental diets reduced the body weight losses observed after 5-fluorouracil administration. Although the effects of the sodium salt of 5-fluorouracil on body weight and food intakes were much less severe than those of the Tris salt the two salts had identical effects on intestinal absorption even when an elemental diet was used. Although there was a smaller incidence of diarrhea after 5-fluorouracil administration when the rats had been fed on one of the elemental diets, diet B, the results do not support the suggestion that elemental diets might reduce the intestinal toxicity of 5-fluorouracil.

Animals↗
PubMed1991

Ribosomal RNA and peptidyl-tRNA hydrolase: a peptide chain termination model for lambda bar RNA inhibition.

We propose here a model to explain the inhibition of bacteriophage lambda (lambda) vegetative growth and the killing of E coli cells defective in peptidyl-tRNA hydrolase (Pth) by lambda bar RNA. The model suggests that bar RNA, which contains a characteristic UGA triplet, base-pairs in an anti-parallel fashion with the 1199-1205 region of E coli 16S rRNA. In doing so, it prevents the required functioning of that region of 16S rRNA in UGA-specific peptide chain termination. Pth is implicated in peptide chain termination because a defect in Pth is required for the achievement of the bar RNA inhibitory effects. We make certain predictions that flow from the model, predictions involving suppression of nonsense mutations, and present preliminary experimental results that demonstrate the fulfillment of those predictions.

Bacteriophage lambda↗
PubMed1971

Dipeptidase deficiency and malabsorption of glycylglycine in disease states.

The activities of jejunal mucosal peptide hydrolases for glycylglycine, glycyl-L-leucine and L-leucylglycine, were assayed in 37 patients. Eight patients, four of whom had Crohn's disease, were found to have a marked reduction in the activity of glycylglycine dipeptidase and, to a lesser extent, of the other two hydrolases. Although absorption of glycine in the two groups was similar, there was malabsorption of glycylglycine in the patients with reduced dipeptidases as shown by a flat absorption curve. The importance of peptide hydrolases of small-intestinal mucosa in the final digestion of proteins, and the implications of peptidase deficiency in disease states is discussed. It is concluded that significant peptidase deficiency may occur even when the mucosa is otherwise histologically completely normal, similar to some states of disaccharidase deficiency described in recent years.

Adolescent↗
PubMed1991

Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3.

The predicted amino acid sequence of the cDNA encoding the murine B-lymphocyte differentiation antigen BP-1/6C3 suggested that it is a member of the zinc-dependent metalloprotease family, possibly an aminopeptidase related to aminopeptidase N [microsomal aminopeptidase; alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2]. In the present studies, we examined the enzymatic activity of this antigen. From brush border preparations of the small intestine, a rich source of many endopeptidases and exopeptidases, the BP-1 antibody selectively removed aminopeptidase A [APA; L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7] activity. The APA activity of a panel of cell lines correlated in linear fashion with cell-surface levels of the BP-1/6C3 antigen. APA activity was demonstrated for the BP-1/6C3 antigen immunopurified from the pre-B-cell membrane. This activity was enhanced by alkaline earth metals such as Ca2+ and was abrogated by amastatin and angiotensin, which are known competitive inhibitors of APA. The data indicate that the murine BP-1/6C3 antigen is active APA, an enzyme that catalyzes specifically the removal of unsubstituted, N-terminal glutamic acid and aspartic acid residues from peptides.

Aminopeptidases↗
PubMed1991

Characterization of the DNF15S2 locus on human chromosome 3: identification of a gene coding for four kringle domains with homology to hepatocyte growth factor.

A human genomic DNA library was screened by using conditions of reduced stringency with a bovine cDNA probe coding for the kringle domains in prothrombin in order to isolate the human prothrombin gene. Twelve positives were identified, three of which coded for prothrombin (Degen & Davie, 1987). Phage L5 was characterized in more detail because of its strong hybridization to the cDNA probe and its unique restriction map compared to the gene coding for human prothrombin. The gene in L5 was sequenced and found to code for a kringle-containing protein. A human liver cDNA library was screened by using a genomic probe from the gene in L5. cDNAs were isolated that contained sequence identical with regions in the gene in L5. Comparison of the cDNA with the gene indicated that the gene in L5 was composed of 18 exons separated by 17 intervening sequences and is 4690 bp in length. Exons ranged in size from 36 to 242 bp in length while intervening sequences ranged from 77 to 697 bp in length. The putative protein encoded by the gene in L5 contains four kringle domains followed by a serine protease-like domain. This domain structure is identical with that found in hepatocyte growth factor (HGF), although the two proteins are only about 50% identical. On the basis of the similarity of the protein encoded by L5 and HGF, we propose that the putative L5 protein be tentatively called HGF-like protein until a function is identified. The DNA sequence of the gene and cDNA and its translated amino acid sequence were compared against GenBank and NBRF databases. Sequences homologous to DNF15S1 and DNF15S2, human DNF15S2 lung mRNA, and rat acyl-peptide hydrolase were identified in exon 17 to the 3' end of the characterized sequence for the gene. From our results, it is apparent that the gene coding for human HGF-like protein is located at the DNF15S2 locus on human chromosome 3 (3p21). The gene for acyl-peptide hydrolase is 444 bp downstream of the gene coding for HGF-like protein, but on the complementary strand. The DNF15S2 locus has been proposed to code for one or more tumor suppressor genes since this locus is deleted in DNA from small cell lung carcinoma, other lung cancers, renal cell carcinoma, and von Hippel-Lindau syndrome.

Amino Acid Sequence↗
PubMed1984

Peptide-degrading enzymatic activities in GH3 cells and rat anterior pituitary homogenates.

The activities of a number of peptide-degrading enzymes were compared in homogenates of GH3 cells and rat anterior pituitaries. The enzymes studied were prolyl endopeptidase (EC 3.4.21.26), a soluble metalloendopeptidase, pyroglutamyl peptide hydrolase (EC 3.4.11.8), a multicatalytic protease complex, cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), aminopeptidase (EC 3.4.11.2), and a membrane-bound neutral metalloendopeptidase (EC 3.4.24.11). Specific substrates were used to measure the activities, and active-site-directed inhibitors were used to verify the identities of the enzymes studied. Of the two lysosomal enzymes studied, cathepsin B, the enzyme with the highest activity in both preparations, had 5 times the activity in GH3 cell homogenates as in anterior pituitary homogenates. Cathespin D had a somewhat higher activity in the anterior pituitary homogenates than in the GH3 cell homogenates. Soluble metalloendopeptidase and prolyl endopeptidase, both cytoplasmic enzymes, had about twice the activity in GH3 cell homogenates as in anterior pituitary homogenates. Membrane-bound neutral metalloendopeptidase in the GH3 cell homogenates had 25% of the activity of the anterior pituitary homogenates. Of the two TRH-degrading enzymes, the activity of prolyl endopeptidase in GH3 cell homogenates was about 25 times higher than that of pyroglutamyl peptide hydrolase. Since the secretory function of the pituitary is in part controlled by neuropeptides, the knowledge of the enzyme profiles of the GH3 cells and the anterior pituitary should be of value in studying the metabolism of neuropeptides and peptide hormones in these systems.

Animals↗
PubMed2003

Aminopeptidase C of Aspergillus niger is a novel phenylalanine aminopeptidase.

A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine beta-naphthylamide (F-beta NA) and phenylalanine para-nitroanilide (F-pNA), but it also displayed activity towards other amino acids with aromatic side chains coupled to beta NA; other amino acids with non-aromatic side chains coupled to either pNA or beta NA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze N-acetylalanine-pNA, a substrate for acyl-peptide hydrolases.

Amino Acid Sequence↗
PubMed1993

Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes.

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.

Affinity Labels↗
PubMed2003

The caspase-like sites of proteasomes, their substrate specificity, new inhibitors and substrates, and allosteric interactions with the trypsin-like sites.

Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.

Aldehydes↗