• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.420-426
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• 1998

An extracellular glutamyl aminopeptidase (EC 3.4.11.7) producing bacterium was isolated from soil and identified as Bacillus licheniformis based on its morphological and physiological characteristics. The aminopeptidase was purified to homogeneity by ammonium sulfate precipitation, Phenyl Sepharose, Resource Q, and Superose 12 column chromatographies. The specific activity of the purified aminopeptidase was 9.2 unit/mg for glutamyl p-nitroanilide with 17.6 purification folds. The purified aminopeptidase had an estimated molecular mass of 64 kDa consists of two different subunits (42 kDa and 22 kDa), and its isoeletric point was 5.2 measured by isoelectric focusing. The optimum pH and temperature of the aminopeptidase were 8.0 and 55$^{\circ}C$, respectively. The aminopeptidase was inhibited by EDTA and 1,10-phenanthroline, suggesting it be a metalloenzyme. Comparing with other aminopeptidase, the enzyme showed relatively high activity against peptide having glutamic acid as N-terminal.

• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.7-11
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• 1996

MR-387Al (ARPA-Val-Pro) and A2 (AHPA-Val-Hyp) were prepared as aminopeptidase N inhibitors through the synthesis of peptide MR-387A and B analogues which contained 3-amino-2-hydroxy-4-phenyl butanoic acid (ARPA) as a zinc-chelating moiety. They are competitive inhibitors of aminopeptidase N with inhibition constants(Ki) of 4.1 $\times 10^{-7}\;and 1.1 \times 10^{-6}$ M, respectively. MR-387Al also strongly inhibited aminopeptidase B of human myelogenous leukemia K-562 cell with $IC_50$ of 0.35 $\mu$ M. Inhibitions of aminopeptidase N activity by ARPA-bearing inhibitors of various peptide chain lengths also have been studied. $IC_ 50$ values of AHPA-Val (bestatin), ARPA-Val-Pro (MR-387Al) and ARPA-Val-Pro-Leu (MR-387C) compared against porcine kidney aminopeptidase N were 20.1, 0.60 and 0.08 $\mu$ M, respectively. These results support that a multiple interaction between the $S_1\to S'_3$ sites of aminopeptidase N and the $P_1\to P'_3$ of the inhibitor plays a crucial role in stabilizing strongly the enzyme-inhibitor complex.

• Applied Biological Chemistry
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• v.36 no.6
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• pp.539-546
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• 1993

This paper describes the purification and partial characteristics of aminopeptidase from Microccus sp. LL3 to utilize the microorganism as a potential agent for industrial application for the purpose of shortening ripening period of cheddar cheese. The optimal temperature and pH for enzyme activity were $35^{\circ}C$ and 7.0, respectively for L-leucine-p-nitroanilide as substrate. The enzyme remained stable for 10 minutes up to $50^{\circ}C$. The activity of aminopeptidase was stimulated by $Mg^{++}$ ion but strongly inhibited by $Hg^{++}$, metal complexing reagents, ethylenediaminetetraacetate (EDTA) and 1,10-phenanthroline. The enzyme was thought to be metallopeptidase. This enzyme had a broad substrate specificity, but was inactive on peptide with arginine as N-terminal amino acid. An intracellular aminopeptidase from Micrococcu sp. LL3 was purified by chromatography on DEAE-Sephacel and filtration on Sepacryl S-300. The enzyme has a molecular weight of 43,500.

• Journal of the Korean Chemical Society
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• v.35 no.1
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• pp.78-84
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• 1991

In an effort to increase effective action of enkephalins, several peptide inhibitors of enkephalin aminopeptidase have been synthesized. The peptides contain 3-amino-2-hydroxy amino acid as a zinc binding site and side chains of substrate pattern. The peptides were synthesized in solution by chain elongation from C-terminal end using DCC/HOBt as coupling reagent. The peptides are shown to have very strong inhibitory activity against enkephalin aminopeptidase.

• Microbiology and Biotechnology Letters
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• v.23 no.1
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• pp.47-52
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• 1995

Since the original productivity of new aminopeptidase M inhibitors MR-387A and B by Streptomyces sp. SL-387 (KCTC 0102BP) was not enough for further chemical and biological evaluation, mutation of parent strain by the treatment of N-methyl-N'-nitro-N-nitrosoguanidine was performed in order to obtain a clone with greater inhibitory activity. Mutant N-3 was selected due to a 6-fold greater productivity (40 $\mu$g/ml) than that of the wild type(6.7 $\mu$g/ml). This mutant was resistant to 3,4-dehydro-DL-proline, an antimetabolite of proline, with 25 $\mu$g/ml of minimum inhibitory concentration. Furthermore, the characteristic morphological change from spiral spore chain in wild type to straight in mutant was observed. An aminopeptidase M nhibitor different from MR-387A and B was isolated from the culture broth of the mutant. This inhibitor was composed of 2 proline, 1 valine, and an unknown amino acid which is presumably 3-amino-4-phenylbutanoic acid. IC$_{50}$ value (89.1 $\MU$g/ml) of the purified inhibitor was lower than that of other inhibitors, which may be due to the absence of 2(S)-hydroxyl group within the structure of 3-amino-4-phenyl- butanoic acid.

• Journal of Plant Biology
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• v.13 no.2
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• pp.11-14
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• 1970

Identification and observations of leucine aminopeptidase (LAP) and multiple molecular forms of the enzyme, isozymes, were made with a technique of starch-gel electrophoresis for various legume pollen. Plants tested other than Leguminosae demonstrated either no indication of the presence or at least tract of enzymes and the isozymes, although all legume pollen tested showed strong LAP patterns. The electrophoretic patterns of LAP failed to be shown if the extracts were heated or otherwise denatured. Extent of zymogrammatic appearance of LAP and the isozymes were characteristic of a species.

• Applied Biological Chemistry
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• v.41 no.8
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• pp.608-610
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• 1998

To investigate the inhibitory activity of 2-hydroxy-3-amino-4-phenylbutyrate-harboring aminopeptidase N inhibitors, p-nitro-AHPA-peptide derivatives (1 and 2) and an AHPA-peptide derivative (3) were synthesized by chain elongation from C-terminal end using DCC/HOBt as a coupling reagent. The peptides $1{\sim}3$ exerted strong inhibitory activities against aminopeptidase N with $IC_{50}$ values of 1.8, 7.3 and $24.0\;{\mu}g/ml$, respectively, and cytotoxicity on cancer cell lines in vitro.

• BMB Reports
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• v.32 no.3
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• pp.317-319
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• 1999

A leucine aminopeptidase has been isolated from the culture medium of the soil fungus, Aspergillus flavus. The enzyme was found to be a glycoprotein, as judged by electrophoresis analysis and the subsequent staining by the periodic acid-Schiff's reagent. Carbohydrate moieties could be cleaved by N-glycosidase, but not by O-glycosidase, indicating that the glucans are linked to the asparagine residue in the protein. Removal of N-glucans was observed without prior denaturation of the protein, implying that the N-glycosidic linkage is exposed and accessible to glycosidase. When the activity of native or deglycosylated enzyme was measured in the presence of various metal ions, removal of carbohydrates increased the aminopeptidase activity of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.5 no.6
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• pp.319-323
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• 1995

A chromogenic mimic of phenlyalanyl-dipeptide, L-phenylalanyl-L-2-sulfanilylglycine (PSG), was synthesized and examined for its usability in leucine aminopeptidase (LAP) assay. The enzyme activity was easily determined by measuring the amount of diazotized adduct of sulfanilic acid released upon hydrolysis of PSG ($\varepsilon^{420}$=18,000/M/cm). Under the experimental conditions employed, PSG showed a Km of 0.063 mM and a Kcat of 1683/min, assessable less than 0.1 $\mu$ g of LAP per milliliter. And the presence of aminopeptidase M (APM) was suggested to be negligible in LAP assay. This novel assay can circumvent the occasional yellow background in biological systems, i.e., serums, etc..

• Journal of Microbiology and Biotechnology
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• v.6 no.4
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• pp.250-254
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• 1996

MR-387A [(2S, 3R)-2-hydroxy-3-amino-4-phenylbutanoyl-L-valyl-L-prolyl-(2, 4-trans)- L-4-hydroxy-proline] reversibly inhibits aminopeptidase N (BC 3.4.11.2) in a process that is remarkable for its unusual degree of time dependence. The time required to inactivate the enzyme by 50$%$ ($t_{1/2}$) for establishing steady-state levels of $EI^*$complex was approximately 5 minutes. This indicates that the inhibition is a slow-binding process. In dissociation experiments of $EI^*$ complex, enzymic activity was regained slowly in a quadratic equation, indicating that the inhibition of aminopeptidase N by MR-387A is tight-binding and reversible. Thus, the binding of MR-387A by aminopeptidase N is slow and tight, with $K_{i}$ (for initial collision complex, EI) and $K_i{^*}$ (for final tightened complex, $EI^*$) of $2.2\times10^{-8}$ M (from Lineweaver-Burk plot) and $4.4\times10^{-10}$ M (from rate constants), respectively. These data indicate that MR-387A and aminopeptidase N are bound approximately 200-fold more tightly in the final $EI^*$complex than in the initial collision EI complex.