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PubMed1986

Enhanced instrumental sensitivity and selectivity for aminopeptidase profiling.

Laser-excited fluorimetry has been applied to the identification of bacteria and fungus. The instrumental sensitivity and selectivity of the aminopeptidase profiling method has been enhanced by the use of laser excitation in conjunction with improved spectral and temporal background rejection. The linear dynamic range for the aminopeptidase technique has been increased by achieving a reduced lower limit of detection of the fluorescent tag, beta-naphthylamine. Standard aminopeptidase methodology only provides a linear dynamic range of 1.5 orders of magnitude. The laser-based method expanded the range to three orders of magnitude allowing the inherent specificity of aminopeptidase enzymes within the pathogen to be observed. The enhanced linear dynamic range was observed in profiles of Agrobacterium tumefaciens rubi and Phytophthora megasperma var. sojae.

2-Naphthylamine↗

PubMed1990

Native agarose-polyacrylamide gel electrophoresis allowing the detection of aminopeptidase, dehydrogenase, and esterase activities at the nanogram level: enzymatic patterns in some Frankia strains.

Nanogram amounts of soluble aminopeptidases, dehydrogenases, and esterases were detected by nondenaturing ultralow gelling point agarose-polyacrylamide gel electrophoresis (ULGA-PAGE). Cytosolic fractions from Frankia sp. were electrophoresed at 4 degrees C in the presence of Co2+, Zn2+, or Mg2+ ions. Then, aminopeptidases and esterases were revealed by simultaneous capture staining by using fast garnet GBC diazonium salt as the chromogenic coupling compound. Dehydrogenases were revealed by using nitro blue tetrazolium salt as electron acceptor. A variety of aminopeptidases, dehydrogenases, and esterases could be identified by their migration in ULGA-PAGE and by their sensitivities to NaCl, CoSO4, ZnSO4, and MgCl2 when assayed "ingel." The presence of agarose was essential for the detection of the complex enzyme patterns. The patterns were remarkably similar for the five Frankia strains isolated from Allocasuarina and Casuarina host plants and differed from those of Frankia strains isolated from Comptonia and Hippophaë host plants. A nomenclature is proposed for aminopeptidases and other Frankia enzymes. The richness of the Frankia amino-peptidases and esterases zymograms makes them adequate marker enzymes for taxonomical, genetic, or biochemical studies. Dehydrogenases might also be useful, although a more restricted number of bands were found with L-lactic and L-malic acid as substrates.

Actinomyces↗

PubMed1986

Substrate specificity and binding loci for inhibitors in an aminopeptidase purified from the plasma membrane of midgut cells of an insect (Rhynchosciara americana) larva.

Plasma membrane-bound aminopeptidases (EC 3.4.11.2) are found in the midgut cells from Rhynchosciara americana larvae, and are recovered in soluble form after papain treatment. The major papain-released aminopeptidase (Mr 207,000 and pI 7.8) was shown to be a true aminopeptidase with a broad specificity toward aminoacyl-beta-naphthylamides and to be more active on tetra and tripeptides than on dipeptides. The purified aminopeptidase is inactivated by EDTA according to a kinetics which is half order in relation to EDTA. Leucine hydroxamate (Ki 27 microM) and hydroxylamine (Ki 5.4 mM) completely protect the enzyme from inactivation by EDTA, whereas isoamyl alcohol (Ki 62 mM) increases the inactivation rate. There are 2.3 binding sites in the enzyme for phenanthroline, which makes the binding of the substrate in the enzyme difficult, changes the enzyme-substrate into a more productive complex, and increases the inactivation rate of the enzyme by EDTA by 87-fold. The data support the proposal that the enzyme has a metal ion which is catalytically active and that the enzyme displays two subsites in its active center: a hydrophobic subsite, to which isoamyl alcohol binds exposing the metal ion, and a polar subsite, to which hydroxylamine binds.

Aminopeptidases↗

PubMed1975

Topological studies on the hydrolases bound to the intestinal brush border membrane. II. Interactions of free and bound aminopeptidase with a specific antibody.

The position of the intestinal brush border aminopeptidase with respect to the lipid bilayer has been investigated with the aid of right side out vesicles prepared from the brush border and an immunological technique using an unlabelled or peroxidase-labelled antibody specific for aminopeptidase. The finding that the bound form of the enzyme was almost as readily inhibited and agglutinated as the free form during incubation with the antibody was consistent with the view that the majority of the aminopeptidase surface emerged from the bilayer. This finding was entirely corroborated by the observation that only a few antigenic determinants were not free to react with the antibody in bound aminopeptidase. This immunological technique may be applied to other membrane proteins provided that preparations of the pure proteins and of specific antibodies are available.

Aminopeptidases↗

PubMed1993

The oligomeric structure of renal aminopeptidase N from bovine brush-border membrane vesicles.

Bovine renal brush-border membrane vesicle aminopeptidase N at various stages of purity was treated with two bifunctional cross-linking agents. A pattern of emergence of higher molecular weight forms was observed. By using a cleavable cross-linker, aminopeptidase N was shown to cross-link both to itself and to its breakdown products as well as to dipeptidyl peptidase IV. Using this technique it was possible to identify three of the breakdown products as 45 kDa, 66 kDa and 95 kDa peptides. N-terminal amino acid sequence analysis was used to define the precise cleavage points for the bovine renal aminopeptidase N breakdown products. The short amino acid sequences obtained show strong sequence similarity with the human intestinal and rat kidney aminopeptidase N.

Amino Acid Sequence↗

PubMed1989

Molecular cloning and amino acid sequence of rat kidney aminopeptidase M: a member of a super family of zinc-metallohydrolases.

Using a polyclonal antibody, a partial cDNA clone for rat aminopeptidase M was identified in a lambda gt11 library from rat kidney. A synthetic oligonucleotide probe derived from the sequence of the insert was used to screen a randomly primed lambda gt10 library. This allowed the identification of several overlapping clones encoding the full sequence of the enzyme. The reading frame, 2898 base pairs in length, encodes a 966 amino acid polypeptide. A highly hydrophobic segment, 24 amino acids in length, located close to the aminoterminus, is proposed to serve as the membrane-spanning domain for this membrane-bound enzyme. The sequence includes nine potential N-linked glycosylation sites and one potential sulfation site. In addition, the rat aminopeptidase M sequence contains an eight amino acid consensus sequence believed to serve as the zinc binding domain in a family of zinc-metallohydrolases. Rat aminopeptidase M shows 77% similarity with the recently cloned human enzyme, as well as weaker but significant similarity with aminopeptidase N from E. coli (18%) and with human leukotriene A4 hydrolase (21%).

Amino Acid Sequence↗

PubMed1985

Vascular, plasma membrane aminopeptidase M. Metabolism of vasoactive peptides.

Aminopeptidase M (EC 3.4.11.2), an enzyme present on the cell surface of vascular endothelium and/or smooth muscle, rapidly hydrolyzes leucyl- and arginyl-2-naphthylamides and a number of vasoactive peptides at physiologic pH. Utilizing both thin-layer chromatography and high pressure liquid chromatography, it was found that vascular aminopeptidase M converted kallidin to bradykinin and inactivated des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P and hexa(6-11)substance P. Aminopeptidase M did not, however, hydrolyze bradykinin, angiotensin I, angiotensin II, saralasin, vasopressin, oxytocin or any form of substance P containing a component of the Arg-Pro-Lys-Pro sequence. Both the naphthylamidase and peptidase activities were inhibited similarly by known amino-peptidase M inhibitors including o-phenanthroline, amastatin, bestatin and puromycin. However, inhibitors of angiotensin I converting enzyme (captopril), carboxypeptidase N (MERGETPA), neutral endopeptidase (phosphoramidon), post proline cleaving enzyme and dipeptidyl(amino)peptidase IV (diisopropylphosphofluoridate, DFP) were without effect. These results demonstrate that vascular, cell surface aminopeptidase M can selectively metabolize vasoactive peptides and may play a role in modulating their levels in the circulation and/or within the vessel wall.

Aminopeptidases↗

PubMed1991

Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P.

In addition to angiotensin I converting enzyme (ACE; EC 3.4.15.1) and carboxypeptidase N (CPN; EC 3.4.17.3), other peptidases contribute to bradykinin (BK) degradation in plasma. Rat plasma degraded BK by hydrolysis of the N-terminal Arg1-Pro2 bond, and the characteristics of hydrolysis are consistent with identification of aminopeptidase P (APP; EC 3.4.11.9) as the responsible enzyme. BK and BK[1-5] N-terminal hydrolysis was optimal at neutral pH, was inhibited by 2-mercaptoethanol, dithiothreitol, o-phenanthroline and EDTA, but was unaffected by the aminopeptidase inhibitors amastatin, puromycin and diprotin A, the endopeptidase-24.11 inhibitors phosphoramidon and ZINCOV, and the ACE and CPN inhibitors captopril and D,L-mercapto-methyl-3-guanidinoethylthiopropanoic acid (MERGETPA), respectively. Although kallidin (Lys-BK) was not metabolized directly by APP, conversion to BK by plasma aminopeptidase M (EC 3.4.11.2) resulted in subsequent degradation by APP. BK analogs containing N-terminal Arg1-Pro2 bonds, including [Tyr8-(OMe)] BK and [Phe8 psi(CH2NH)Arg9]BK (B2 agonists), des-Arg9-BK and [D-Phe8]des-Arg9-BK (B1 agonists), and [Leu8]des-Arg9-BK (B1 antagonist), were degraded by APP with Km and Vmax values comparable to those found for BK (Km = 19.7 +/- 2.6 microM; Vmax = 12.1 +/- 1.2 nmol/min/mL). In contrast, B2 antagonists containing D-Arg0 N-termini, including D-Arg[Hyp3,Thi5.8,D-Phe7]BK and D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, were resistant to APP-mediated hydrolysis. These data support a role for plasma aminopeptidase P in the degradation of circulating kinins, and a variety of B2 and B1 kinin agonists and antagonists. However, APP does not participate in the degradation of D-Arg0-containing antagonists.

3-Mercaptopropionic Acid↗

PubMed1986

Metabolism of thyrotropin-releasing hormone in human cerebrospinal fluid. Isolation and characterization of pyroglutamate aminopeptidase activity.

Pyroglutamate aminopeptidase, which catalyzes metabolism of thyrotropin-releasing hormone (TRH) to cyclo(His-Pro), is the major enzyme of TRH metabolism in human CSF. The partially purified CSF pyroglutamate aminopeptidase has a pH optimum between 6.0 and 7.4, and a Km of 15.9 +/- 3.1 microM. A number of potential competitive inhibitors of the enzymatic activity were examined, of which luteinizing hormone-releasing hormone and bombesin were the most effective. An examination of the structure of various peptides that inhibit pyroglutamate aminopeptidase activity indicated that the enzyme generally prefers a substrate having amino-terminal pyroglutamic acid (pGlu) and a COOH-terminal that is either blocked or distant from amino-terminal pGlu. Heavy metals, EDTA and reducing agents inactivated the enyzme, whereas benzamidine, phenylmethylsulfonylfluoride, trypsin inhibitor and alkylating agents had little or no effect on the enzymatic activity. Thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid), however, considerally inhibited the enzymatic activity. We hypothesize that CSF pyroglutamate aminopeptidase may play a role in the biologic actions of TRH.

Aminopeptidases↗

PubMed1988

Differential effects of aminopeptidase inhibitors on angiotensin-induced pressor responses.

Recent iontophoretic data suggest that conversion of angiotensin II (AII) to angiotensin III (AIII) may be necessary before the peptide can activate central angiotensin-sensitive neurons. Furthermore, this conversion may be inhibited by the aminopeptidase A inhibitor, amastatin. In the present study we investigated the importance of aminopeptidase activity on central angiotensin-induced pressor responses. Intracerebroventricular (i.c.v.) pretreatment with amastatin, suppressed i.c.v. AII-induced pressor responses. Pretreatment with the aminopeptidase B inhibitor, bestatin, increased pressor responses to AIII. Pressor responses induced by the aminopeptidase-resistant analogue, [Sar1]angiotensin II, were not affected by pretreatment with angiotensin inhibitors. These results support the hypothesis that AII must be converted to AIII to be active in the brain.

Aminopeptidases↗

PubMed1989

Increased blood pressure induced by central application of aminopeptidase inhibitors is angiotensinergic-dependent in normotensive and hypertensive rat strains.

Two aminopeptidase inhibitors, amastatin (AM) and bestatin (BE), were employed in 3 strains of rats, spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD), to investigate the central angiotensinergic system. The results indicate that intracerebroventricular (i.c.v.) injections of AM and BE induced pressor elevations in all 3 strains of rats. In order to test for the possibility of spillage into peripheral vasculature, members from all 3 strains were peripherally infused with AM, BE, or 0.15 NaCl via jugular vein catheters. The SHRs were significantly more responsive to the aminopeptidases than the normotensive strains, however their overall pressor responses were only 33% of those to i.c.v. infusion. Next, in order to test the notion that these aminopeptidase inhibitors are having their effect via the central angiotensinergic system, and not some other peptidergic system, the specific angiotensin receptor antagonist, Sar1, Thr8-AII (sarthran) was employed. Intracerebroventricular pretreatment with sarthran prevented subsequent pressor responses to i.c.v. AM and BE in members of all 3 strains, thereby suggesting that these aminopeptidase inhibitors are having their effect via the central angiotensinergic system.

Aminopeptidases↗

PubMed1992

Comparison of soluble aminopeptidases in human cerebral cortex, skeletal muscle and kidney tissues.

In an attempt to elucidate the cellular function of the soluble aminopeptidases (with which the majority of tissue aminopeptidase activity is usually associated) we have determined the distribution and characteristics of these enzymes in three functionally dissimilar human tissues (cerebral cortex, skeletal muscle and kidney cortex), using a systematic experimental approach. Following fractionation of brain, muscle or kidney soluble extracts via anion exchange chromatography, four aminopeptidase enzymes types (alanyl-, arginyl-, leucyl- and pyroglutamyl-) were identified; the absolute and relative activities for corresponding enzymes were similar in each tissue. Following further purification of each enzyme type from each tissue (via liquid chromatography/preparative electrophoresis), corresponding enzyme types were found to have similar characteristics (pH optimum of activity, action of enzyme effectors, substrate specificity and molecular mass). Since the same enzymes, with correspondingly similar distribution and characteristics are present in such functionally dissimilar tissues, it is suggested that the principal role for the soluble aminopeptidases (in contrast to the membrane-associated enzymes, which may function in the catabolism of neuropeptides) is in the final stages of the general intracellular protein catabolism cascade, via hydrolysis of oligopeptide intermediates to free amino acids.

Aminopeptidases↗

PubMed1985

Biosynthesis of intestinal microvillar proteins. The intracellular transport of aminopeptidase N and sucrase-isomaltase occurs at different rates pre-Golgi but at the same rate post-Golgi.

The kinetics of processing and microvillar expression of aminopeptidase N (EC 3.4.11.2) and sucrose alpha-D-glucohydrolase-oligo-1,6-glucosidase (sucrase-isomaltase, EC 3.2.1.48 and EC 3.2.1.10) were compared by labelling of pig small intestinal mucosal explants with [35S]methionine. The conversion from transient (high mannose glycosylated) to mature (complex glycosylated) form was 1.7-times slower for sucrase-isomaltase than for aminopeptidase N, indicating a slower rate of migration from the rough endoplasmic reticulum to the Golgi complex. Likewise, sucrase-isomaltase appeared in the microvillar fraction at a slower rate than aminopeptidase N. The relative pool sizes of mature and transient forms of both enzymes in intracellular membranes (Mg2+-precipitated fraction) were determined to obtain information on the relative time, spent pre- and post-Golgi, respectively, prior to microvillar expression. This ratio was 0.24 +/- 0.06 (mean +/- SD) for sucrase-isomaltase as compared to 0.40 +/- 0.04 (mean +/- SD) for aminopeptidase N. Considering the slower rate of pre-Golgi transport for sucrase-isomaltase, this indicates that the two microvillar enzymes have rather similar if not identical rates of post-Golgi transport.

Aminopeptidases↗

PubMed1993

Cholesterol crystallization-promoting activity of aminopeptidase-N isolated from the vesicular carrier of biliary lipids.

Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle-associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase-N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase-N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 micrograms/ml. Aminopeptidase-N showed a concentration-dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase-N may play a critical role in the pathogenesis of cholesterol gallstone disease.

Amino Acid Sequence↗

PubMed1993

Purification and characterization of the cystinyl bond cleaving yeast aminopeptidase yscXVI.

Aminopeptidase yscXVI was purified from the yeast Saccharomyces cerevisiae. By SDS-PAGE the enzyme has a molecular weight of 45,000 Da, and in chromatofocusing, elution was observed at pH 6.2. The synthetic substrate cystinyl-4-nitroanilide (Km 22.5 microM, Vmax 12.9 mU/mg) is cleaved most efficiently in the pH range 7-8. Besides cleaving this standard substrate, aminopeptidase yscXVI acts on several other 4-nitroanilide substrates with unsubstituted N-terminal L-amino acids. Highest hydrolysis rate was measured with Lys-4-nitroanilide and Leu-4-nitroanilide. The activity of aminopeptidase yscXVI is abolished by chelating agents and restored by Zn2+, Mn2+ and Co2+ ions. Bestatin and amastatin are both strong inhibitors of the enzyme, with Ki values of 0.53 microM and 0.93 microM, respectively. Aminopeptidase yscXVI is detectable in the logarithmic growth phase, stationary phase, and in starved cultures of yeast.

Amino Acid Sequence↗

PubMed1994

Identification of a 130-kilodalton human biliary concanavalin A binding protein as aminopeptidase N.

BACKGROUND/AIMS: Human gallbladder bile contains a group of nonmucin glycoproteins that binds to the lectin concanavalin A (con A) and has been reported to promote cholesterol monohydrate crystal nucleation, an event preceding the formation of gallstones. Several of these proteins, including a 130-kilodalton protein, have been isolated and shown to promote nucleation in vitro. The aim of this study was to identify this and other major biliary con A binding glycoproteins. METHODS: Gallbladder bile was chromatographed on con A agarose, and the eluted proteins were electrophoresed, blotted, and subjected to amino-terminal sequence analysis. RESULTS: The major con A binding proteins were identified as aminopeptidase N (a 130-kilodalton protein), alpha 2 macroglobulin, hemopexin, immunoglobulin heavy chains, and the beta chain of haptoglobin. After further purification, aminopeptidase N was found to be enzymatically active and to promote cholesterol crystallization at its approximate physiological concentration in bile. CONCLUSIONS: It is likely that aminopeptidase N is the previously characterized 130-kilodalton biliary crystallization promoting protein. Aminopeptidase N is probably released from the biliary canalicular membrane by the detergent activity of bile salts and may be one factor that promotes cholesterol crystallization in the gallbladder.

Amino Acid Sequence↗

PubMed1984

Kinin-converting aminopeptidase from human urine. Further purification and characterization through kinetic and inhibitory studies.

An aminopeptidase from human urine (HUA) able to hydrolyze L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and to convert both MLBK and LBK to BK has been further purified and characterized. The preparation now obtained showed a 3-fold higher specific activity than the previously described one and a single active protein band in 7% polyacrylamide gel electrophoresis accounting for 86% of total protein. Kinetic constants for this kinin-converting enzyme were determined using L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and LBK. The Km values for different naphthylamides were in the 10(-5) M range while that for L-Leu-p-nitroanilide was 3.6 X 10(-4) M. With LBK as substrate the aminopeptidase activity showed the highest catalytic efficiency in spite of a Km in the mM range. The enzyme was poorly inhibited by -SH and -S-S- group reagents. Some L-aminoacids, as well as mono- and diamines, indomethacin, puromycin and bestatin were equipotent competitive inhibitors of both arylamidase and aminopeptidase activities. Results obtained in this paper are compatible with our conclusion that human urine, unlike other enzyme sources, contains only one aminopeptidase, and that this enzyme displays both arylamidase and kinin-converting activities. The enzyme's action may be important in the metabolism of kinins, yielding peptides which could interact with both B-1 and B-2 kinin receptors in the kidney.

Amines↗

PubMed1993

Molecular properties of aminopeptidase Ey as a zinc-metalloenzyme.

1. Aminopeptidase Ey from hen's egg yolk contains 1.0 g atom of zinc/mol of a subunit having molecular weight of 150 kDa. The inactive, Zn(2+)-free apoenzyme was reactivated by Co2+, Mn2+, Ca2+, Cd2+, Cu2+ and Ni2+ in addition to Zn2+, whereas Mg2+ and Fe2+ were ineffective. 2. The enzymatical properties of reconstituted enzymes, except for Zn(2+)-reconstituted enzyme, differed from native enzyme. The values for the activation energy were calculated by aminopeptidase Ey and Co(2+)-reconstituted enzyme. 3. The isoelectric point of the enzyme was about 2.8 as determined by isoelectric focusing. An asialo form of the enzyme, obtained by treatment with Arthrobacter sialidase, had an isoelectric point of 4.4. 4. The amino terminal sequence of aminopeptidase Ey was determined to be acyl-Xaa-Xaa-Pro-Glu-Ala-Ala-Ser-Leu-Pro-Gly. There was no identity with any known sequences of aminopeptidase.

Amino Acid Sequence↗