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At least 919 records · Page 51Linked to original sources

PubMed1999

Cardioprotective effects of the aminopeptidase P inhibitor apstatin: studies on ischemia/reperfusion injury in the isolated rat heart.

Aminopeptidase P and angiotensin-converting enzyme (ACE) are responsible for the metabolism of exogenously administered bradykinin in the coronary circulation of the rat. It has been shown that ACE inhibitors decrease cytosolic enzyme release from the ischemic rat heart and reduce reperfusion-induced ventricular arrhythmias by increasing endogenous levels of bradykinin. It was hypothesized that the aminopeptidase P inhibitor apstatin could do the same. In an isolated perfused rat heart preparation subjected to global ischemia and reperfusion, both apstatin and ramiprilat (an ACE inhibitor) significantly decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release. The difference between the postischemia and preischemia levels of released CK was reduced 68% by apstatin and 68% by ramiprilat compared with control. The corresponding reductions in LDH release were 74% for apstatin and 81% for ramiprilat. A combination of the inhibitors was not significantly better than either one alone. Apstatin and ramiprilat also significantly reduced the duration of reperfusion-induced ventricular fibrillation by 69 and 61%, respectively. The antiarrhythmic effect of apstatin was reversed by HOE140, a bradykinin B2-receptor antagonist, suggesting that apstatin is acting by potentiating endogenously formed bradykinin. The results demonstrate that the aminopeptidase P inhibitor apstatin is cardioprotective in this model of cardiac ischemia/ reperfusion injury.

Adrenergic beta-Antagonists↗

PubMed1990

Localization of dipeptidyl aminopeptidase yscIV in the plasma membrane of Saccharomyces cerevisiae.

The subcellular distribution of dipeptidyl aminopeptidase activity was studied in protoplast lysates of Saccharomyces cerevisiae that were virtually free from vacuolar contamination. Dipeptidyl aminopeptidase yscIV, the STE13 gene product, was found to be associated with plasma membrane vesicles after sucrose gradient isopycnic centrifugation. Another dipeptidyl aminopeptidase activity, not yet fully characterized, was localized in a microvesicular population co-sedimenting with chitosomes.

Cell Membrane↗

PubMed1994

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings (Identification of Etiolation-Related Plastidial Aminopeptidase Activities).

We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris L.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greening capacity. However, this situation of efficient greening capacity was short-lived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation.

Journal Article↗

PubMed1985

Aminopeptidase activity from germinated jojoba cotyledons.

One major and two minor aminopeptidase activities from germinated jojoba (Simmondsia chinensis) cotyledon extracts were separated by ammonium sulfate precipitation and chromatofocusing. None of the activities were inhibited by 1,10 phenanthroline.The major aminopeptidase, purified 260-fold, showed a pH optimum of 6.9 with leucine-p-nitroanilide as substrate, a molecular weight estimated at 14,200 by electrophoretic analysis, and an isoelectric point of 4.5 according to the chromatofocusing pattern. Activity was inhibited by p-chloromercuribenzoate, slightly stimulated by 1,10 phenanthroline and 2-mercaptoethanol, and not influenced by Mg(2+) or diethyl pyrocarbonate. Inhibition by p-chloromercuribenzoate was prevented by the presence of cysteine in the assay. Leucine-p-nitroanilide and leucine-beta-naphthylamide were the most rapidly hydrolyzed of 11 carboxy-terminal end blocked synthetic substrates tested. No activity on endopeptidase or carboxypeptidase specific substrates was detected. The major aminopeptidase showed activity on a saline soluble, jojoba seed protein preparation and we suggest a possible physiological role for the enzyme in the concerted degradation of globulin reserve proteins during cotyledon senescence.

Journal Article↗

PubMed1994

Dipeptidyl aminopeptidase yspI mutants of Schizosaccharomyces pombe: genetic mapping of dpa1+ on chromosome III.

A mutant strain of Schizosaccharomyces pombe lacking dipeptidyl aminopeptidase yspI was isolated from a strain already defective in aminopeptidase activity by means of a staining technique with the chromogenic substrate ala-pro-4-methoxy-beta-naph-thylamide to screen colonies for the absence of the enzyme. The defect segregated 2+:2- in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Gene dosage experiments indicated that the mutation resides in the structural gene of dipeptidyl aminopeptidase yspI, dpa1+. The dpa1+ gene was located on chromosome III by using m-fluorophenylalanine-induced haploidization and mitotic analysis. dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.

Chromosome Mapping↗

PubMed2006

Bacterial beta-peptidyl aminopeptidases with unique substrate specificities for beta-oligopeptides and mixed beta,alpha-oligopeptides.

We previously discovered that BapA, a bacterial beta-peptidyl aminopeptidase, is able to hydrolyze two otherwise metabolically inert beta-peptides [Geueke B, Namoto K, Seebach D and Kohler H-PE (2005) J Bacteriol 187, 5910-5917]. Here, we describe the purification and characterization of two distinct bacterial beta-peptidyl aminopeptidases that originated from different environmental isolates. Both bapA genes encode a preprotein with a signal sequence and were flanked by ORFs that code for enzymes with similar predicted functions. To form the active enzymes, which had an (alphabeta)(4) quaternary structure, the preproteins needed to be cleaved into two subunits. The two beta-peptidyl aminopeptidases had 86% amino acid sequence identity, hydrolyzed a variety of beta-peptides and mixed beta/alpha-peptides, and exhibited unique substrate specificities. The prerequisite for peptides being accepted as substrates was the presence of a beta-amino acid at the N-terminus; peptide substrates with an N-terminal alpha-amino acid were not hydrolyzed at all. Both enzymes cleaved the peptide bond between the N-terminal beta-amino acid and the amino acid at the second position of tripeptidic substrates of the general structure H-betahXaa-Ile-betahTyr-OH according to the following preferences with regard to the side chain of the N-terminal beta-amino acid: aliphatic and aromatic > OH-containing > hydrogen, basic and polar. Experiments with the tripeptides H-d-betahVal-Ile-betahTyr-OH and H-betahVal-Ile-betahTyr-OH demonstrated that the two BapA enzymes preferred the peptide with the l-configuration of the N-terminal beta-homovaline residue as a substrate.

Amino Acid Sequence↗

PubMed2005

Endogenous aminopeptidase N decreases the potency of peptide agonists and antagonists of the kinin B1 receptors in the rabbit aorta.

The B(1) receptor for kinins is selectively stimulated by bradykinin-related fragments lacking the C-terminal arginine, des-arginine(9)-bradykinin (des-Arg(9)-BK), and Lys-des-Arg(9)-BK. The latter peptide is the optimal agonist at the human and rabbit receptor. The B(1) receptor is inducible as a function of inflammatory conditions in the vasculature. We studied the effect of endogenously expressed peptidases on the potency of ligands of this receptor in an established bioassay, the rabbit aorta contractility. The potency measured for agonists (EC(50)) or antagonists (pA(2) scale) in this assay was compared with the affinity of each agent determined using [(3)H]Lys-des-Arg(9)-BK binding competition in cultured aortic smooth muscle cells and with the competition K(i) for the hydrolysis of the aminopeptidase chromogenic substrate L-Ala-p-nitroanilide by smooth muscle cell membranes. The contractile potency of the agonist Lys-des-Arg(9)-BK is decreased by in situ metabolism, and aminopeptidase N mediates most of the distortion (inhibited by amastatin but not efficiently by puromycin). At the other end of the spectrum, the fully protected agonist Sar-[D-Phe(8)]des-Arg(9)-BK is not significantly potentiated by peptidase inhibitors. A similar distortion of apparent potency was observed for some peptide antagonists used in the contractility assay, B-10350 (Lys-Lys-[Hyp(3), Igl(5), d-Tic(7), CpG(8)]des-Arg(9)-BK) and Lys-[Leu(8)]des-Arg(9)-BK being intensely potentiated by amastatin treatment and effective L-Ala-p-nitroanilide competitors. N-Protected peptide antagonists or a nonpeptide antagonist of the B(1) receptor were not potentiated by amastatin. The coexpression of aminopeptidase N and the kinin B(1) receptor in rabbit arterial tissue is of interest for the inactivation of the high-affinity agonist Lys-des-Arg(9)-BK and for the design of hydrosoluble antagonist drugs.

Animals↗

PubMed1990

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2.

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40 degrees C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu and Cd. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or beta-mercapto-ethanol, while Zn or Co restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K(m), 0.55 mM) but that it can hydrolyze this substrate at a high rate (V(max), 30 mumol/min per mg of protein).

Journal Article↗

PubMed2002

Evaluation of the inhibition effect of thiolated poly(acrylates) on vaginal membrane bound aminopeptidase N and release of the model drug LH-RH.

The purpose of this study was to evaluate the inhibitory effect of thiolated carbopol 974P (carb-cys) on the enzymatic activity of vaginal aminopeptidase N in-vitro. Mediated by a carbodiimide, L-cysteine was covalently linked to carbopol 974P. Depending on the weight ratio of polymer to cysteine during the coupling reaction, resulting conjugates displayed 31.3-54.4 micromol thiol groups per g polymer. The inhibitory effect of carb-cys conjugates was evaluated towards isolated aminopeptidase N and aminopeptidase-N-like activity of excised vaginal mucosa covered with native mucus, respectively. Enzymatic activity was assayed spectrophotometrically using L-leucine-p-nitroanilide (L-leu-pNA) as a synthetic substrate. Carb-cys thereby showed a significantly higher inhibitory effect than unmodified polymer towards both isolated enzyme and vaginal mucosa. Moreover, enzyme inhibition was strongly dependent on the amount of thiol groups being immobilised. The more thiol groups available the higher was the inhibitory effect. Due to its additional high cohesive properties and the possibility of a sustained drug release, which could be shown for the model drug LH-RH, carb-cys appears interesting for the development of vaginal peptide drug-delivery systems.

Acrylates↗

PubMed1994

Isolation and characterization of a dipeptidyl aminopeptidase from Streptomyces sp. WM-23.

A screening test was undertaken to isolate microorganisms that produced dipeptidyl aminopeptidase. The hydrolytic activity toward alanyl-phenylalanine p-nitroanilide was found in a culture filtrate of a actinomyces strain (WM-23), newly isolated from a soil sample. The enzyme (WM-23 dipeptidyl aminopeptidase) was isolated from the culture filtrate as a homogeneous preparation. The WM-23 enzyme, inhibited by phenylmethylsulfonyl fluoride, may be classified to mammalian dipeptidyl aminopeptidase II. The enzymatic characteristics were investigated.

Amino Acid Sequence↗

PubMed1995

Immunocytochemical studies of an antigen in a human T-cell lymphoma line (Jurkat) recognized by certain monoclonal antibodies to CD-13 (aminopeptidase-N).

The immunofluorescent staining of Jurkat cells by a panel of eight CD-13 monoclonal antibodies has been investigated. With unfixed cells all antibodies were negative, in contrast to the CD-13-positive cell line, HL60, which gave staining in each case. These results were in line with our previous enzymological studies in which we showed that the aminopeptidase activity observed with intact Jurkat cells was located in the cytoplasm and was distinct in properties from aminopeptidase-N (CD-13), which is expressed by HL60 cells. After fixation of Jurkat cells, four antibodies, 22A5; MCS-2, 72a and WM-15, gave consistent positive staining, while the other four, WM-47, RMAG6, MY7 and CLB-mon-gran/2 were consistently negative. Prior fixation was an absolute requirement for staining, but was observed over a range of concentrations of paraformaldehyde from 0.4% to 4%, as well as with acetone or methanol/acetic acid. Confocal fluorescence microscopy confirmed that the positive fluorescence given by 22A5 was located in the cytoplasm, contrasting with that given by an antibody to a cell-surface marker, HLA-DR, which was associated with the plasma membrane. The fixatives thus appeared to have rendered the cells permeable to the antibodies, but the molecular size of the antibodies could not explain the different behaviour of the two classes of antibodies, since IgG1 isotypes were present in both positive and negative groups. We exclude the possibility that Jurkat cells express an intracellular form of aminopeptidase-N; the identity of the protein(s) bearing the epitope(s) to which four of the antibodies bound has yet to be determined.

Antibodies, Monoclonal↗

PubMed2002

Debittering of enzymatic hydrolysates using an aminopeptidase from the edible basidiomycete Grifola frondosa.

Bitter peptide solutions, prepared by the enzymatic hydrolysis of soy protein and milk casein, were treated with an aminopeptidase from the edible basidiomycete Grifola frondosa. As the incubation time elapsed, the amount of free amino acids released increased and the bitterness of the enzyme reaction mixtures decreased. However, the debittering of the milk casein hydrolysate by the aminopeptidase was less effective than that observed for the soy protein hydrolysate. Hydrophobic amino acids such as valine, leucine, phenylalanine, tyrosine, and isoleucine were preferentially released from the bitter solutions by the action of the aminopeptidase.

Journal Article↗

PubMed1989

Cell cycle phases of two human lung tumor cell lines derived from squamous cell carcinoma and pulmonary metastasis of a rhabdomyosarcoma (HS 24 and HS 57) and N-acetylalanine aminopeptidase activity.

The cell cycle phase distribution of two human lung cancer cell lines (HS 24 and HS 57) grown both to half-confluency and confluency was determined. Both cell lines were then synchronized by applying a thymidine block forcing them to stay in the S-phase. After removal of the thymidine block, which allows the cells to go then through their cell cycle phases, N-acylamino acylpeptide hydrolase (EC 3.4.19.1) activity was measured using N-acetylalanine-p-nitroanilide as substrate (N-acetylalanine aminopeptidase). At the same times that the enzymatic activity was measured, the cell cycle phase distribution was analyzed, in order to determine the cell cycle phase of N-acetylalanine aminopeptidase synthesis. However, the cell cycle phase of N-acetylalanine aminopeptidase synthesis could not be determined. This result was caused by the fact that the cells remained synchronous only for a short period of time.

Carcinoma, Squamous Cell↗

PubMed1995

The use of FAB mass spectrometry and pyroglutamate aminopeptidase digestion for the structure determination of pyroglutamate in modified peptides.

A new method for the determination of the modified amino acid structures of peptides containing pyroglutamyl residue in their N-terminal position is reported. The combination of fast atom bombardment (FAB) mass spectrometry and pyroglutamate aminopeptidase digestion provides a convenient and sensitive method for the identification of pyroglutamate. In order to investigate the nature of the amino terminal region of the follow post-translationally modified peptides containing pyroglutamyl residues, Neurotensin, Hypertrehalosaemic neuropeptide, and Luteinizing Hormone Releasing Hormone (LH-RH) were digested with pyroglutamate aminopeptidase and analyzed by FAB mass spectrometry. The method provided direct information about the N-terminal structure. The N-terminal pyroglutamates of these peptides are unequivocally determined at a level of 0.9-2.8 nmol per peptide. Several of the complicated procedures and controversies, which accompany the application of traditional methods, are eliminated. We describe the advantages of the combination procedure of FAB mass spectrometry and pyroglutamate aminopeptidase digestion for modified amino acid determination.

Amino Acid Sequence↗

PubMed2005

Methionine aminopeptidases type I and type II are essential to control cell proliferation.

The dependence of cell growth on methionine aminopeptidase (MetAP) function in bacteria and yeast is firmly established. Here we report experimental evidence that the control of cell proliferation in mammalian cells is directly linked and strictly dependent on the activity of both MetAP-1 and MetAP-2. The targeted downregulation of either methionine aminopeptidase MetAP-1 or MetAP-2 protein expression by small interfering RNA (siRNA) significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) (70%-80%), while A549 human lung carcinoma cell proliferation was less inhibited (20%-30%). The cellular levels of MetAP-2 enzyme were measured after MetAP-2 siRNA treatment and found to decrease over time from 4 to 96 h, while rapid and complete depletion of MetAP-2 enzyme activity was observed after 4 h treatment with two pharmacological inhibitors of MetAP-2, PPI-2458 and fumagillin. When HUVEC and A549 cells were treated simultaneously with MetAP-2 siRNA and PPI-2458, or fumagillin, which irreversibly inhibit MetAP-2 enzyme activity, no additive effect on maximum growth inhibition was observed. This strongly suggests that MetAP-2 is the single critical cellular enzyme affected by either MetAP-2 targeting approach. Most strikingly, despite their significantly different sensitivity to growth inhibition after targeting of either MetAP-1 or MetAP-2, HUVEC, and A549 cells, which were made functionally deficient in both MetAP-1 and MetAP-2 were completely or almost completely inhibited in their growth, respectively. This closely resembled the observed growth inhibition in genetically double-deficient map1map2 yeast strains. These results suggest that MetAP-1 and MetAP-2 have essential functions in the control of mammalian cell proliferation and that MetAP-dependent growth control is evolutionarily highly conserved.

Aminopeptidases↗

PubMed1991

Methionyl aminopeptidase from rat liver: distribution of the membrane-bound subcellular enzyme.

The selective distribution of methionyl aminopeptidase (MAP) among rat liver mitochondria (heavy and light) and microsomes is reported. Several properties of MAP from the three subcellular fractions showed that the enzyme is a typical aminopeptidase able to remove N-terminal methionine from oligopeptides and methionyl-2-naphthylamide but not from Met-Ala-Ser. MAP is a membrane-bound enzyme sensitive to SH-group oxidants and inhibitable by L-methionine but not by usual arylaminopeptidase inhibitors. It is suggested that, MAP may play an important role during protein synthesis in rat liver.

Amino Acid Sequence↗

PubMed1978

Aminopeptidase from Brevibacterium linens: activation and inhibition.

Activation and inhibition of a purified aminopeptidase from Brevibacterium linens was investigated using L-alpha-leucyl-4-nitroanilide and L-leucyl-L-leucine as substrates. The enzyme was activated by cobalt, provided that the enzyme was preincubated with the metal. Strong inhibitory effects were derived from heavy metals, metal-complexing compounds, reducing agents, the modification of aromatic amino acids, and the presence of hydrophobic substances or certain amino acids in the test mixtures. Supposing that this B. linens aminopeptidase plays a part during surface-ripening of cheeses, possible consequences of specific technological conditions for its activity are discussed.

Amino Acids↗

PubMed1975

Cystine animopeptidase and leucine aminopeptidase of choriocarcinoma cells grown in culture.

Cystine animopeptidase (CAP) and leucine aminopeptidase (LAP) in choriocarcinoma cells grown in culture were assayed and characterized electrophoretically. The specific activity of CAP in choriocarcinoma was about 1.5-fold greater than the specific activity of term placenta. The extract of choriocarcinoma contained an enzyme which is similar to one of the pregnancy-specific serum aminopeptidases in electrophoretic mobility and in resistance to inhibition by 0.1M methionine. The CAP activity was not increased in either the cells or the medium by growing the cells in the presence of oxytocin, estrogen, progesterone, or prostaglandin F-2alpha. It was tentatively concluded that choriocarcinoma cells produce CAP and that it is not regulated by any of these hormones.

Aminopeptidases↗