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PubMed1984

Evaluation of cytosolic aminopeptidase in human sera. Evaluation in hepatic disorders.

Employing the quantitative electrophoretic separation of cytosolic "LAP" activity (EC. 3.4.11.1), the authors investigated the clinical significances of cytosolic and total "LAP" activities in the sera of patients with several different hepatic disorders and elucidated the previously confusing results of aminopeptidase measurements in clinical laboratories. The dominant aminopeptidases measured in human sera by L-leucinamide as substrate are both cytosolic "LAP" and microsomal aminopeptidase (EC. 3.4.11.2). Total "LAP" activity means the sum of these two aminopeptidases. Different substrate specificities are observed in these two enzymes located in other subcellular fractions and different increasing rates of these enzymes are observed in various hepatic diseases. In cases of acute hepatic cell damage, cytosolic "LAP" accounts for a large percentage of total "LAP" of a patient's serum. On the other hand, in cases of elevation of hepatobiliary enzymes, microsomal aminopeptidase constitutes a large part of total "LAP" activity, and the ratio between cytosolic and total "LAP" activity is very low.

Aminopeptidases↗

PubMed2001

Serum aminopeptidase A activity of mice is related to dietary fat saturation.

A high intake of monounsaturated fat has been proposed to be a dietary factor that can decrease the incidence of cardiovascular disease and hypertension. In addition, increasing dietary fat saturation has been shown to increase plasma total cholesterol and elevate systolic and diastolic blood pressures. We demonstrated previously that cholesterol selectively increases in vitro aminopeptidase A activity, which is related to angiotensin metabolism. In this study, we investigated the effect of different degrees of dietary fatty acid saturation on serum aminopeptidase activities in vivo. Serum total cholesterol concentrations were also measured. Five groups of male Balb/C mice were fed for 10 wk diets containing 2.4 g/100 g of sunflower oil, fish oil, olive oil, lard or coconut oil. We measured alanyl-, arginyl-, cystinyl-, pyroglutamyl-, aspartyl- and glutamyl-specific aminopeptidase activities using arylamides as substrates. Serum total cholesterol levels were higher in mice fed diets containing saturated oils (lard and coconut) than in those consuming sunflower oil, which is unsaturated. Two of the serum aminopeptidase A activities (aspartyl and glutamyl aminopeptidase) increased progressively with the degree of saturation of the dietary fatty acids; activities were significantly greater in mice fed coconut oil than in those fed sunflower or fish oil. Therefore, the substrates hydrolyzed by this activity as well as their functions may be similarly affected. These results may have some implication for the treatment of cardiovascular disease.

Aminopeptidases↗

PubMed1988

Purification and characterization of human placental aminopeptidase A.

Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.

Aminopeptidases↗

PubMedDate not supplied

Lys- and Leu-aminopeptidase activity after acute toluene exposure in the rat brain.

Changes in Lys- and Leu-aminopeptidase activities in several brain regions of the rat, after acute toluene administration, are described in this research. Aminopeptidase activity has been suggested as a candidate regulator of the degradation of several neuroactive peptides. Lys-aminopeptidase activity was significantly decreased in the thalamus, amygdala, and medulla oblongata. Leu-aminopeptidase activity was significantly decreased in the thalamus and cerebellum. It is suggested that these aminopeptidase activities could play a part in the mechanism of toluene neurotoxicity.

Aminopeptidases↗

PubMed1999

Co-expression of glutathione S-transferase with methionine aminopeptidase: a system of producing enriched N-terminal processed proteins in Escherichia coli.

We describe here an Escherichia coli expression system that produces recombinant proteins enriched in the N-terminal processed form, by using glutathione S-transferase cGSTM1-1 and rGSTT1-1 as models, where c and r refer to chick and rat respectively. Approximately 90% of the cGSTM1-1 or rGSTT1-1 overexpressed in E. coli under the control of a phoA promoter retained the initiator methionine residue that was absent from the mature isoenzymes isolated from tissues. The amount of initiator methionine was decreased to 40% of the expressed cGSTM1-1 when the isoenzyme was co-expressed with an exogenous methionine aminopeptidase gene under the control of a separate phoA promoter. The recombinant proteins expressed were mainly methionine aminopeptidase. The yield of cGSTM1-1 was decreased to 10% of that expressed in the absence of the exogenous methionine aminopeptidase gene. By replacing the phoA with its natural promoter, the expression of methionine aminopeptidase decreased drastically. The yield of the co-expressed cGSTM1-1 was approx. 60% of that in the absence of the exogenous methionine aminopeptidase gene; approx. 65% of the initiator methionine residues were removed from the enzyme. Under similar conditions, N-terminal processing was observed in approx. 70% of the recombinant rGSTT1-1 expressed. By increasing the concentration of phosphate in the growth medium, the amount of initiator methionine on cGSTM1-1 was decreased to 14% of the overexpressed isoenzymes, whereas no further improvement could be observed for rGSTT1-1. The initiator methionine residue does not affect the enzymic activities of either cGSTM1-1 or rGSTT1-1. However, the epoxidase activity and the 4-nitrobenzyl chloride-conjugating activity of the purified recombinant rGSTT1-1 are markedly higher that those reported recently for the same isoenzyme isolated from rat livers.

Aminopeptidases↗

PubMedDate not supplied

Insulin-regulated aminopeptidase/placental leucil Aminopeptidase (IRAP/P-lAP) and angiotensin IV-forming activities are modified in serum of rats with breast cancer induced by N-methyl-nitrosourea.

BACKGROUND: In previous reports, changes in oxytocinase activity in human breast cancer tissue and in the serum of N-methyl-nitrosourea (NMU)-induced rat mammary tumors were described. Insulin-regulated aminopeptidase (IRAP) has been identified with oxytocinase and has also been referred to as placental leucine aminopeptidase (P-LAP). MATERIALS AND METHODS: The IRAP/P-LAP activity in rat serum was assayed to analyze the putative role that IRAP/P-LAP may play in regulating mammary gland carcinogenesis induced by NMU. Furthermore, as it has been recently described that IRAP/P-LAP is the angiotensin IV (Ang IV) receptor AT4, the activities of Ang IV-forming aminopeptidase N (APN) and aminopeptidase B (APB) were also assayed. RESULTS: Changes in serum IRAP/P-LAP and Ang IV-forming APB activities were found in rats with mammary tumors induced by NMU. Both activities were greatly increased, although the Ang IV-forming APN activity was not modified. CONCLUSION: These changes in aminopeptidase activities may reflect the local functional status of their substrates, which can be selectively activated or inhibited in the affected tissue as a result of specific conditions brought about by the tumor. Thus, these enzymatic activities may be involved in the promotion and progression of breast cancer through oxytocin (OT), vasopressin (AVP) and/or renin-angiotensin system (RAS) misregulation.

Aminopeptidases↗

PubMed1990

Soluble and membrane-bound leucyl- and arginyl-aminopeptidase activities in subcellular fractions of young and adult rat brains.

The subcellular distribution of soluble and membrane-bound leucyl- and arginyl-aminopeptidase activities were analyzed in one and five month old rat brains using Leu- and Arg-2-naphthylamide as substrates. Both soluble leucyl- and arginyl-aminopeptidase activities showed the highest levels in the synaptosomal fraction in the two groups of rats. The highest levels of membrane-bound leucyl- and arginyl-aminopeptidase activities were found in the microsomal fraction in the two ages studied. There were no differences between the two ages in soluble leucyl- and arginyl-aminopeptidase activities. However, a significant decrease in both membrane-bound enzymatic activities was evidenced in the synaptosomal fraction of older rats. Developmental changes of these aminopeptidase activities in a determined subcellular localization, may reflect modifications in their effect on neuropeptides susceptible to be hydrolyzed in this particular location.

Aminopeptidases↗

PubMed1976

[Aminopeptidases in the serum and urine of patients with hyperthyroidism].

In patients with hyperthyroidism the serum activities of the leucine aminopeptidase (LAP) and the alanine aminopeptidase (AAP) as well as the alanine aminopeptidase excretion in the urine were determined. A significantly increased activity of the leucine aminopeptidase in the serum and an increased excretion of alanine aminopeptidase in the urine were found. The AAP in the serum did not show a significant increase of activity. On account of the changes in the serum and in the urine before and during therapy a low-grade participation of the hepatobiliary and renal systems which are clinically not uppermost is to be assumed. Increased excretion of AAP and hyperthyroidism coincide nearly without exception in out patients. Correlation-statistical investigations make it probable to regard the increased excretion of AAP in the urine as an indirect parameter of the peripheral metabolism in hyperthyroidism.

Adult↗

PubMed1993

Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation.

We have investigated the effect of monoclonal antibodies (MAbs) specific for aminopeptidase N/CD13 on the invasion of human metastatic tumor cells into reconstituted basement membrane (Matrigel). The invasion of human metastatic tumor cells (SN12M renal-cell carcinoma, HT1080 fibrosarcoma and A375M melanoma) into Matrigel-coated filters was inhibited by an anti-CD13 MAb, WM15, in a concentration-dependent manner. However, this MAb did not have any effect on tumor-cell adhesion and migration to the extracellular matrices, which may be involved in tumor-cell invasion. MAb WM15 inhibited the degradation of type-IV collagen by tumor cells in a concentration-dependent manner. We also found that WM15 inhibited hydrolysing activities towards substrates of aminopeptidases in 3 different tumor cells. Since our previous study indicated that bestatin, an aminopeptidase inhibitor, was able to inhibit tumor-cell invasion, as well as aminopeptidase activities of murine and human metastatic tumor cells, cell-surface amino-peptidase N/CD13 may be partly involved in the activation mechanism for type-IV collagenolysis to achieve tumor-cell invasion, and anti-CD13 MAb WM15 may inhibit tumor-cell invasion through a mechanism involving its inhibitory action on the aminopeptidase N in tumor cells.

Aminopeptidases↗

PubMed2004

GPI-anchored aminopeptidase is involved in the acrosome reaction in sperm of the mussel mytilusedulis.

The sperm of the mussel Mytilus had hydrolytic activities against substrates for aminopeptidase. Acrosome reaction (AR) was suppressed in the presence of aminopeptidase substrate, Phe-4-methylcoumaryl-7-amide (MCA), and an aminopeptidase inhibitor, bestatin. Treatment of sperm with phosphatidylinositol-specific phospholipase C (PI-PLC) released aminopeptidase activity from sperm and suppressed AR. These results suggest that the enzyme is located on the sperm surface via glycosylphosphatidylinositol (GPI)-anchor and is involved in the AR. Immunoblot analysis showed that tyrosine residues of 40, 59, 68, and 72 kDa proteins were phosphorylated during induction of the AR. The 40 kDa protein was also recognized by anti-c-Src antibody by immunoblotting. The tyrosine phosphorylation of these proteins was inhibited when sperm were inseminated in the presence of Phe-MCA, and by PI-PLC treatment. Treatment of sperm with tyrosine kinase activator, 9,10-dimethyl-1,2-benzanthracene, induced AR, and its inhibitor, genistein, suppressed AR. These results suggest that tyrosine phosphorylation of 40, 59, 68, and 72 kDa proteins, induced by the interaction of GPI-anchored aminopeptidase with oocyte surface, triggers AR in Mytilus sperm.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1982

Serum aminopeptidases in pregnant vervet monkeys (Cercopithecus aethiops).

Two distinctive aminopeptidase isozymes have been identified in the serum of the vervet monkey. The L-methionine-sensitive cytosol aminopeptidase (AP; E.C. 3.4.11.1) is present in all samples, while the cystyl aminopeptidase (CAP; E.C. 3.4.11.3) isozyme, which is resistant to inhibition by 0.1 M L-methionine, is found only in the serum of pregnant females. The pregnancy-specific CAP isozyme appears in the serum between the fifth and the eleventh weeks of a nearly 22-week pregnancy. The overall aminopeptidase activity and the activity of the L-methionine-insensitive isozymes alone are both significantly greater in the serum of pregnant females than in nonpregnant females. Consequently, the presence of the cystyl aminopeptidase isozyme in the serum is a useful diagnostic indicator of pregnancy in the vervet monkey.

Aminopeptidases↗

PubMed1979

Biochemical characterization of "LAP," a polymorphic aminopeptidase from the blue mussel, Mytilus edulis.

A genetically variable naphthylamidase enzyme, previously described as "leucine aminopeptidase," was purified approximately fiftyfold, and its biochemical properties were investigated. The enzyme was renamed "aminopeptidase I." Substrate affinities demonstrate that it is an alpha-aminoacyl peptide hydrolase (E.C. 3.4.11.-). Aminopeptidase I had a monomer molecular weight of 65--68,000, average of pI of pH 4.88, and broad pH optima between 6.5 and 8.0. The enzyme was inactivated rapidly between 40 and 50 C. Antibodies from purified enzyme did not cross-react with other naphthylamidases, but aminopeptidase I activity was inhibited by immune serum. The enzyme exhibited highest naphthylamidase activity for aromatic and hydrophobic aminoacyl naphthylamides. Aminopeptidase activity was highest for aromatic and hydrophobic N-terminal residues of tripeptides. Certain divalent metal cations, p-OH-mercuribenzoate, and N-ethylmaleimide were strongly inhibitory while chelating agents activated the enzyme.

Aminopeptidases↗

PubMed1991

Characterization of dipeptidyl and tripeptidyl aminopeptidases in human kidney soluble fraction.

In an attempt to identify improved biochemical markers for the diagnosis of early kidney damage via urinary analysis of kidney derived enzymes, we have undertaken the systematic identification, quantification and characterization (following purification via anion exchange and gel filtration chromatography, and preparative electrophoresis) of the dipeptidyl and tripeptidyl aminopeptidases in normal human kidney soluble extract (with which the majority of the cellular activity of these enzymes is associated). Four chromatographically separable enzyme types were identified as follows (% relative activity in parentheses): dipeptidyl aminopeptidase I (EC 3.4.14.1; 24%); dipeptidyl aminopeptidase II (EC 3.4.14.2; 8%); dipeptidyl aminopeptidase IV (EC 3.4.14.5; 61%); tripeptidyl aminopeptidase (unclassified; 7%). Comparison of the levels of activity for the above enzyme types in normal and pathological urine may lead to an improvement upon existing procedures for the early detection of renal damage.

Amino Acid Sequence↗

PubMed1994

Aminopeptidase modulation of the pharmacological responses to synthetic thrombin receptor agonists.

Thrombin is a contractile stimulus of isolated rabbit aortic rings and apparently produces its effects through the recently characterized cleavable receptor. A synthetic hexapeptide, NAT6-NH2 (new amino terminus), was found to be the minimal active structure for full activation of this receptor. The N-terminal Ser residue of NAT6-NH2 is crucial for biological activity. In this study we examined the metabolism of NAT6-NH2 in rabbit plasma, where it was rapidly degraded by aminopeptidase M. In the presence of the aminopeptidase inhibitor amastatin, no metabolism was observed. On this basis a metabolically resistant analogue, [Sar1]NAT6-NH2, was designed. We compared the biological activity of thrombin, NAT6-NH2 and [Sar1]NAT6-NH2 in the rabbit aorta and found that [Sar1]NAT6-NH2 was more potent than NAT6-NH2; however, in the presence of amastatin the concentration-effect curve for NAT6-NH2 was shifted to the left of that for [Sar1]NAT6-NH2. The effects of [Sar1]NAT6-NH2 and of thrombin were not modified by the presence of the aminopeptidase inhibitor. We also studied the effect of amastatin on the in vivo hypotensive response to NAT6-NH2 and found that it was also influenced by aminopeptidase M inhibition. Our results show that aminopeptidase protection is important when evaluating responses to synthetic agonists of the thrombin cleavable receptor and that an in vivo model, the anesthesized and heparinized rabbit, may be useful for the development of agonists and antagonists of this receptor.

Amino Acid Sequence↗

PubMed1989

Onset of transcription of the aminopeptidase N (leukemia antigen CD 13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation.

The sequence of a cDNA clone (2.82 kbp) of rabbit intestinal aminopeptidase N (CD 13) is reported. Using the corresponding anti-sense RNA probe, the distribution of aminopeptidase N mRNA along the crypt/villus axis of the rabbit small intestine was studied by in situ hybridization. The aminopeptidase N gene is expressed along the whole length of the villus with a maximum at its base. Expression was not detected in the crypt cells. The distribution of aminopeptidase N mRNA correlates with the presence of active enzyme as monitored by histochemical staining. The results are compatible with onset of transcription of the aminopeptidase N gene at the crypt/villus transition zone during the enterocyte differentiation.

Amino Acid Sequence↗

PubMed1992

The apical sorting signal on human aminopeptidase N is not located in the stalk but in the catalytic head group.

Human aminopeptidase N carries an apical sorting signal on its ectodomain necessary for its correct transport to the apical membrane in Madin-Darby canine kidney cells. To determine whether the apical sorting signal is localized in the serine/threonine rich stalk or in the catalytic head group, anchor/stalk-minus aminopeptidase N, consisting of the hemagglutinin signal peptide and the catalytic head group of human aminopeptidase N, was expressed in MDCK cells. Anchor/stalk-minus aminopeptidase N was secreted mainly to the apical side. The catalytic head group of human aminopeptidase N thus carries an apical sorting signal.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of a ubenimex (Bestatin)-sensitive aminopeptidase B-like enzyme from K562 human chronic myeloid leukemia cells.

A ubenimex-sensitive aminopeptidase B-like enzyme was purified from the non-membrane-bound fraction of K562 cells by a series of chromatographic procedures and slab-gel electrophoresis. The apparent molecular mass of the enzyme was estimated to be 73 kDa by SDS-PAGE. The aminopeptidase activity was activated by chloride ions and inhibited by Zn2+, Cu2+, Cd2+, and p-chloromercuribenzoic acid. Ubenimex was a potent inhibitor of this aminopeptidase in the nanomolar range. The sequence of the N-terminus of the protein was not determined. Partial amino acid sequencing revealed that the N-terminus of this aminopeptidase B-like enzyme was blocked by acylation. The partial sequences of the two fragments produced by CNBr cleavage and an acylamino acid-releasing reaction showed this enzyme to be a new aminopeptidase.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of an aminopeptidase from tuna (Thunnus albacares) pyloric caeca.

An aminopeptidase was purified from a water soluble fraction of tuna pyloric caeca by heat treatment, Zn2+ fractionation, ion exchange on a DEAE cellulose column, gel filtration on Fractogel TSK-55, and immobilized metal ion affinity chromatography (IMAC) on IDA(Cu2+/Zn2+)-Sepharose 6B. The molecular mass of the enzyme was estimated to be 150,000 on Sephacryl S-300 HR, and was found to be near 72,000 by SDS-PAGE. The aminopeptidase, which is a glycoprotein rich in acidic amino acids, is optimally active at pH 8.8 and 65 degrees C. The enzyme activity was not affected by Mg2+, Zn2+, Ca2+, Mn2+, Co2+, PMSF, iPr2FP, 4-hydroxymercuribenzoic acid, iodoacetamide, puromycin, and cysteine but it was strongly inhibited by metal chelators (EDTA and o-phenanthroline), amastatin, Hg2+, Cd2+, and Cu2+. The enzyme was also inhibited by some L-amino acids. Kinetic parameters of the enzyme were determined with some aminoacyl-p-nitroanilides and aminoacyl-beta-naphthylamides. L-Alanine-p-nitroanilide and L-alanine-beta-naphthylamide were hydrolysed most rapidly while the highest hydrolytic coefficient (kcat/Km) value was obtained with L-methionine-p-nitroanilide. The apoaminopeptidase was prepared and reconstitution of an active enzyme was carried out using metal chelating interaction chromatography on an IDA-Sepharose 6B column charged with a metal ion. Full activity was restored with Zn2+, Co2+, Cu2+ and Al3+. Zn(2+)-Enzyme was the most thermostable form of the aminopeptidase. Reversal inhibition by Cu2+ and Cd2+ was also examined. When the aminopeptidase was partially deglycosylated by a treatment with N-glycosidase F some of its physical properties differed from that of the native enzyme: its electrophoretic mobility was reduced and its stability to denaturation by SDS and by ionic strength were lower than those of the untreated enzyme. All together, our results indicate that the tuna pyloric caeca aminopeptidase is distinct from the peptide hydrolases characterized in the literature.

Amino Acids↗