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PubMed1976

Yeast aminopeptidase I. Chemical composition and catalytic properties.

An aminopeptidase (alpha-aminoacyl L-peptide hydrolase, EC 3.4.11.1) was purified to homogeneity from autolysates of brewer's yeast. The enzyme which is responsible for most of the yeast cell's aminopeptidase activity is a glycoprotein containing about 12% of conjugated carbohydrate and 0.02% Zn2+ and having a complex quaternary structure. The active species has a molecular weight of approx. 600000 and an isoelectric point of 4.7. The enzyme is remarkably stable, even in dilute solutions. All types of L-amino acid and peptide derivatives containing a free amino terminus are attacked, including amino acid amides and esters. As to its substrate specificity, the enzyme belongs to the so called leucine-aminopeptidases. It is strongly and specifically activated by Zn2+ and Cl- (or Br-) and inactivated by metal-chelating agents. The activation by Zn2+ seems to be mediated by a conformational transition which affects exclusively V and leads to a form of the enzyme which enhanced stability against heat. Halide anions, on the other hand, are acting as positive allosteric effectors, modulating both V and Km.

Amino Acids↗

PubMed1981

Purification and enzymatic properties of an L-leucine aminopeptidase from swine liver.

An L-leucine aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1), having a specificity toward the substrate L-leucine amide, but not toward L-leucyl beta-naphthylamide or L-leucyl p-nitroanilide, has been purified 332-fold from swine liver, with a yield of 8.6%. This is the first purification of this enzyme from hepatic tissue. The purified enzyme submitted to analytical electrophoresis on cellulose acetate strips or in polyacrylamide gel showed a single band after straining with Ponceau S Red dye or Amido black, respectively. Purified swine liver L-leucine aminopeptidase, a cytosol enzyme, exhibited a molecular weight of 268 000 +/- 50 000 by gel filtration. It hydrolyzed L-leucine amide substrate and L-leucyl peptides. It was activated by Mg2+ and Mn2+ and inhibited by Co2+ and Zn2+. The optimum pH was 10. It was rather sensitive to heat elevation. Swine liver L-leucine aminopeptidase was inhibited by EDTA, citric acid, isocaproic acid, dodecylamine, aliphatic alcohols and p-chloromercuribenzoate but unaffected by monoiodoacetic acid and diisopropyl fluorophosphate.

Animals↗

PubMed1976

Isolation of aminopeptidase from Aspergillus flavus.

A mixture of aminopeptidase and neutral protease from the Aspergillus flavus mold obtained by chromatography on DEAE-Sephadex was fractionated by chromatography on the hydroxyalkyl methacrylate gel with chemically bonded 1,6 hexamethylene diamine and D-leucine. Aminopeptidase thus obtained was electrophoretically homogeneous. Conditions for chromatography were worked out allowing a one stage isolation of a highly active aminopeptidase sample directly from the alcoholic precipitate of the culture medium of the Aspergillus flavus mold.

Aminopeptidases↗

PubMed1983

L-leucinthiol - a potent inhibitor of leucine aminopeptidase.

L-leucinthiol (2-amino-4-methyl-1-pentanethiol) was designed as an inhibitor of leucine aminopeptidase by analogy with sulfhydryl inhibitors of other zinc-containing peptidases. It was synthesized from L-leucinol and shown to be a potent competitive inhibitor of the microsomal aminopeptidase from porcine kidney (Ki = 2.2 X 10(-8) M). The results suggest that the mechanism of aminopeptidase may be similar to that of other metalloproteases.

Animals↗

PubMed1987

The effects of the aminopeptidase inhibitors amastatin and bestatin on angiotensin-evoked neuronal activity in rat brain.

During a recent comparison of iontophoretically applied angiotensin II (AII) and angiotensin III (AIII) in the paraventricular nucleus of the rat, we observed that the response latency for AIII was much shorter than that for AII. This suggested that AII may have to be converted to AIII before it becomes active. To test this hypothesis we performed 3 experiments. (1) We examined the effects of bestatin, an aminopeptidase B inhibitor, on the activity of applied AII and AIII. (2) Next, we monitored the effects of amastatin, a specific aminopeptidase A inhibitor, on the action of co-applied AII or AIII. (3) And, finally, we examined the response to the aminopeptidase-resistant analog Sar1-AII, both applied alone and in combination with AII or AIII. Bestatin, while having no activity of its own, dramatically enhanced the actions of both AII and AIII. Amastatin, on the other hand, had little effect on AII's action and diminished or totally blocked AII-dependent activity. Like bestatin, amastatin had no effect alone. Sar1-AII reduced spontaneous activity of angiotensin-sensitive neurons and inhibited the actions of AII and AIII in a reversible manner. The same cells were also blocked by the recognized angiotensin antagonist Sar1, Ile8-AII. In total these results strongly support the notion that AII must be converted to AIII in the brain before it is activated.

Aminopeptidases↗

PubMed1988

Effects of aminopeptidase inhibition on the half-lives of [125I]angiotensins in the cerebroventricles of the rat.

Anesthetized Sprague-Dawley rats fitted with intracerebroventricular (i.c.v.) cannulas were infused with one of the aminopeptidase inhibitors, amastatin or bestatin, over a 5-min period. After infusion, 1-2 X 10(6) cpm of [125I]angiotensin II ([125I]AII) or [125I]angiotensin III ([125I]AIII) was injected through the same cannula. The rats were subsequently killed 60 s later by focused microwave irradiation which instantaneously terminated further [125I]angiotensin metabolism. HPLC analysis of the extracted [125I]angiotensin and metabolic products allowed for the calculation of t1/2s of disappearance for the parent peptides. Both inhibitors effectively lengthened the half-lives of [125I]AII and [125I]AIII. Bestatin, which is considered a selective aminopeptidase B blocker, had a more pronounced effect on [125I]AIII metabolism, while amastatin, a selective aminopeptidase A inhibitor, was better at slowing [125I]AII degradation. The results indicate that amastatin and bestatin are very effective blockers of the cerebroventricular metabolism of angiotensins but are only marginally selective with regard to AII and AIII.

Aminopeptidases↗

PubMed1988

Influence of aminopeptidase inhibitors on brain angiotensin metabolism and drinking in rats.

The metabolism of [125I]angiotensin II (AII, octopeptide) and [125I]angiotensin III (AIII, heptapeptide) was determined in the cerebroventricular compartment following intracerebroventricular (i.c.v.) pretreatment with artificial cerebrospinal fluid (aCSF) or the aminopeptidase B inhibitor, bestatin (BE). Microwave fixation was used to stop brain aminopeptidase activity followed by high-performance liquid chromatography methods to measure degradation. The resulting mean t 1/2 values were: aCSF/AII = 23.5 s, BE/AII = 32.0 s; aCSF/AIII = 8.2 s, BE/AIII = 16.0 s. A second experiment indicated that i.c.v. administered BE induced considerable water consumption, while amastatin (AM, an aminopeptidase A inhibitor) did not. Combined i.c.v. treatment with BE/AII or BE/AIII produced water consumptions that were equivalent with the sum of the water intakes due to independent treatments. Unexpectedly, combined treatment with AM/AII also significantly facilitated water consumption as compared with the independent treatments. These results suggest that i.c.v. BE-induced drinking is due to the extension of the half-life of endogenous AIII, and that both AM and BE facilitate drinking to i.c.v. AII and AIII by inhibiting the degradation of AII and AIII. This delay in degradation prolongs the action of AIII at central angiotensin receptors while also slowing the conversion of AII to AIII, thus providing a depot of available AII to be converted to AIII in an obligatory manner.

Aminopeptidases↗

PubMed1993

Brain soluble and membrane-bound Tyr-aminopeptidase activities during the stages of estrous and proestrous in the female rat.

Brain Tyr-aminopeptidase activity levels during two stages of the rat estrous cycle (estrous and proestrous), are described in this research. The study includes the activities of the soluble and the membrane-bound forms. Soluble Tyr-aminopeptidase activity shows a significant increase during the proestrous stage in the hypothalamus. The complete membrane-bound activity did not significantly change at any point. However, a significant rise was found in the membrane-bound puromycin insensitive aminopeptidase activity in the hypothalamus and the pituitary gland during the proestrous phase.

Aminopeptidases↗

PubMed1976

Aminopeptidase B in human serum.

The activity of arginine aminopeptidases in human serum was studied with special reference to the possible involvement of enzymes resembling aminopeptidase B (APB). The serum samples were obtained from 10 patients with various acute inflammatory processes, both during the acute phase and after treatment with penicillin. The administration of penicillin to the patients did not result in noticable changes in the chromatographic pattern of the APB-like enzyme. The disappearance of the acute symptoms did not significantly affect the overall specific activity of arginine aminopeptidases in serum. The results showed that human serum does contain, contrary to earlier reports, an enzyme corresponding to APB, even though in very low concentrations. The low enzyme activity was revealed by prolonged reaction times and by the use of larger samples in chromatography than earlier. The APB-like enzyme in serum may have derived from blood cells.

Abscess↗

PubMed1980

Serum and liver glycylproline dipeptidyl aminopeptidase activity in rats with experimental hepatic cancer.

Changes of glycylproline dipeptidyl aminopeptidase (GPDA) and gamma-glutamyl transpeptidase (gamma-GTP) activities were compared in the serum and liver tissue of rats with hepatic cancer induced by 3'-methyl DAB. Serum glycylproline dipeptidyl aminopeptidase activity in rats with the azo dye-induced hepatic cancer was significantly higher than that in healthy rats, but the increase was not so extensive compared with that of gamma-glutamyl transpeptidase. The specific activity of glycylproline dipeptidyl aminopeptidase was decrease in the microsomal fraction and increased in the supernatant fraction of hepatic cancer tissue, whereas that of gamma-glutamyl transpeptidase was increased in both microsomal and supernatant fractions. These results suggest that the mechanisms, whereby serum activities of these two enzymes were increased in rats with hepatic cancer, were different from each other.

Aminopeptidases↗

PubMed1981

Urinary excretion of N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase in patients receiving amikacin or cis-platinum.

Urinary excretion of alanine aminopeptidase (EC 3.4.11.2) and N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) was determined for 25-70 days in five patients receiving cis-platinum and for 8-53 days in six patients receiving amikacin. This study was performed to investigate if the excretion of urinary enzymes represents a sensitive parameter for the early detection of toxic kidney damage. The determination of N-acetyl-beta-D-glucosaminidase was carried out by the method of Knoll et al. [13]. The procedure of Mondorf et al. [14] for the estimation of alanine aminopeptidase activity was adapted to the Gemsaec Fast-Analyzer. In both patient groups an increase in the excretion of the two enzyme activities could be demonstrated. In patients receiving amikacin, the excretion of alanine aminopeptidase was always higher than that of N-acetyl-beta-D-glucosaminidase, whereas in three patients receiving cis-platinum it was the opposite. In two cis-platinum patients the excretion of both enzymes was of the same size. The changes during amikacin therapy seem to be reversible, whereas in four cis-platinum patients these changes seemed to be partly irreversible. Serum creatinine concentration was less sensitive than the urinary enzyme excretion for detection of kidney damage.

Acetylglucosaminidase↗

PubMed1991

Aminopeptidase P and dipeptidyl peptidase IV activity in human leukocytes and in stimulated lymphocytes.

Human white blood cells were shown to contain high aminopeptidase P activity. The specific activities found in the high-speed supernatant of the extracts of granulocytes, lymphocytes and monocytes ranged from 30 to 70 units per mg protein. Culturing lymphocytes during 7 days in the presence of phytohaemagglutinin resulted in a 70-200% increase in the specific aminopeptidase P activity and a 200% increase in the specific activity of dipeptidyl peptidase IV. The time-course of the activity of both aminopeptidase P and dipeptidyl peptidase IV during the stimulation of human T-lymphocytes by phytohaemagglutinin indicates an involvement of these two enzymes in the proliferative process of these immunocompetent cells. Due to their substrate specificity their potential substrates must have the N-terminal Xaa-Pro sequence known to be present in several immunologically important polypeptides.

Aminopeptidases↗

PubMed1984

Comparison of leucine aminopeptidase from human lens, beef lens and kidney, and hog lens and kidney.

Antisera specific for leucine aminopeptidase (LAP) were prepared. Using these antisera in immunodiffusion tests, the identity of LAP isolated from beef lens and kidney is demonstrated. The same pertains to hog lens and kidney LAP. As indicated by only partial fusion of the immunoprecipitates in immunodiffusion plates, leucine aminopeptidases isolated from hog and beef are antigenically similar but not identical. These tests also indicate substantial similarity between a component in human lens homogenates and bovine or hog LAP. Microcomplement fixation tests corroborate these observations and indicate that, under these conditions, LAP from beef lens and kidney, or hog lens and kidney, are indistinguishable. However, there is an approximate 8.5% amino acid sequence difference between beef and hog LAP. Microcomplement fixation tests with human lens homogenate also corroborate immunodiffusion results and indicate an approximate 19% amino acid sequence difference between beef and human LAP. These data establish that LAP is a species-specific enzyme and they indicate that it is not organ specific. Maximal complement fixation occurs at approximately 0.1 microgram antigen per tube in those assays in which pure aminopeptidases were tested. This permits standardization of the microcomplement fixation assay for LAP under these conditions. Maximal complement fixation occurs at 160-200 micrograms human lens homogenate per tube. Assuming that in this quantity of homogenate there is 0.1 microgram LAP, then it can be calculated that LAP comprises about 0.05% of the lens protein. This agrees closely with the percentage of LAP in hog and beef lenses. Thus, the reduced LAP activity reported for human lens tissue appears not to result from an absence of the enzyme but rather, may be due to diminished catalytic competency of the enzyme in aged human lens tissue (see Taylor and Juhngen , 1984). The unit evolutionary period, 4.7-5.8 X 10(6) years, indicates that LAP has been highly conserved during evolution.

Animals↗

PubMed1990

Schistosoma mansoni: purification and characterization of a membrane-associated leucine aminopeptidase.

Membrane-associated leucine aminopeptidase (EC 3.4.11.1, LAP) has been purified to homogeneity from Schistosoma mansoni egg homogenates by a combination of ultracentrifugation, chromatofocusing, and molecular sieve chromatography. A 260-fold increase in specific activity was observed after purification. This is a metalloenzyme, containing carbohydrate moieties. Optimal enzyme activity was found at neutral pH. Enzyme activity was measured using L-leucine-7-amino-4-trifluoromethylcoumarin (L-Leu-AFC); in addition, schistosome egg LAP hydrolyzed a variety of other aminopeptidase substrates. Hydrolysis of L-Leu-AFC was inhibited by a number of aminopeptidase inhibitors, including 1,10-phenanthroline, bestatin, and amastatin.

Animals↗

PubMed1984

An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from beta-lipotropin60-65.

Secretory vesicles isolated from the neural and intermediate lobes of the bovine pituitary contained a membrane-bound aminopeptidase activity which cleaved arginine from beta-LPH60-65 (Arg-Tyr-Gly-Gly-Phe-Met) and Arg-MCA. Neither methionine enkephalin (Tyr-Gly-Gly-Phe-Met) nor Substance P, which has an N-terminal arginine followed by a proline, could serve as substrates for this aminopeptidase activity; nor could cathepsin B-like or chymotrypsin-like enzyme activities be detected in the vesicle preparations. Maximal enzyme activity was at pH 6.0, and the activity was inhibited by EDTA, stimulated by Co2+ and Zn2+, but was unaffected by leupeptin, pepstatin A, phenylmethylsulfonyl fluoride and p-chloromercuribenzenesulfonate, suggesting that the enzyme is a metalloaminopeptidase. The presence of this aminopeptidase activity in secretory vesicles suggests that it may be involved in peptide prohormone processing.

Aminopeptidases↗

PubMed1988

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA.

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.

Amino Acid Sequence↗

PubMed1989

Yeast vacuolar aminopeptidase yscI. Isolation and regulation of the APE1 (LAP4) structural gene.

The structural gene, APE1, (LAP4), for the vacuolar aminopeptidase I of Saccharomyces cerevisiae was cloned with the aid of a staining technique which permitted monitoring of aminopeptidase activity in yeast colonies. Genetic linkage data demonstrate that integrated copies of the cloned gene map to the APE1 locus. The nucleotide sequence of the cloned gene was determined. The open reading frame of APE1 consists of 514 codons and, therefore, encodes a larger protein (MW 57,003) than the reported mature aminopeptidase yscI (MW 44,800), suggesting that proteolytic processing must occur. A 1.75-kb mRNA, which is made in substantial amounts only when yeast cells have exhausted the glucose supply, was identified.

Amino Acid Sequence↗

PubMed1983

Characterization of two thiol-dependent aminopeptidases partially purified from human placenta.

1. Two thiol-dependent aminopeptidases (I and III) were partially purified from the soluble fraction of human placenta. 2. Aminopeptidase I preferred L-alanine-beta-naphthylamide (AlaNA) as substrate at pH 7.0-7.5. It was sensitive to heat and some divalent metal ions (Co2+, Ni2+, Zn2+) but resistant to EDTA, and some amino acids. 3. Aminopeptidase III hydrolysed equally well AlaNA and ArgNA at pH 6.5-7.0. It was markedly suppressed by EDTA and reactivated by Co2+. It was inhibited by heat, some amino acids, benzamadine and puromycin.

Aminopeptidases↗