• 한국미생물·생명공학회지
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• 제26권5호
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• pp.420-426
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• 1998

Glutamic acid의 분해능이 뛰어난 aminopeptidase를 생산하는 세균을 토양으로부터 분리하였다. 이 균은 형태적 생리적 특성으로부터 Bacillus licheniformis로 동정되었다. 이 균을 최적 배지에 접종하고 37$^{\circ}C$에서 진탕배양한 후 균이 생산한 aminopeptidase를 ammonium sulfate 침전, Phenyl Sepharose CL-4B, Resource Q, Superose 12HR column을 통해 분리하여서 20.6%의 수율로 17.6배 정제된 glutamyl p-nitroanilide을 기질로 했을 때 9.2unit/mg의 순수효소를 얻었다. SDS-PAGE와 Native-PAGE로 부터 이 효소의 분자량이 42,000Da와 22,000Da으로 구성된 헤테로다이커로 약 64,000Da임이 밝혀졌고 효소의 등전점은 5.2로 나타났다. 이 효소의 반응 최적 온도는 55$^{\circ}C$, 반응최적 pH는 8.0이었고, EDTA와 1,10-phenanthroline에 의해서 효소활성이 저해되는 metalloenzyme으로 판명되었다.

• Journal of Microbiology and Biotechnology
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• 제6권1호
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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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• 제36권6호
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• pp.539-546
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• 1993

본 연구에서는 Cheddar 치즈의 숙성기간을 단축시키고 flavor를 증진시키기 위하여 Micrococcus sp. LL3를 치즈 제조시에 첨가할 목적으로 본 균주가 생성하는 aminopeptidase의 특성을 조사하였으며, 또한 본 효소를 정제하였다. L-Leucine-p-nitroanilide를 기질로 사용 하였을 때 본 효소의 최적온도와 pH는 각각 $30^{\circ}C$ 및 7.0이었다. 본 효소는 $50^{\circ}C$까지의 온도에서 10분간 방치하였을 경우에도 안정하였다. $Mg^{++}$ ion에 의해 본 효소의 활성이 촉진되었으나 $Hg^{++}$ ion과 EDTA나 1,10-phenanthroline에 의해서 효소활성이 거의 실활되어 metallopeptidase인 것으로 추정되었다. 본 효소의 기실 특이성은 광범위 하였으나, N-terminal amino acid로서 arginine을 함유한 peptide는 분해하지 못했다. 본 균주가 생성하는 aminopeptidase의 정제를 위하여 DEAE-Sephacel ion chromatography및 gel filtration을 실시하였으며, 이때 분자량 46,500의 효소를 얻을 수 있었다.

• 대한화학회지
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• 제35권1호
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• pp.78-84
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• 1991

Enkephalin의 작용을 연장하기 위한 연구이 하나로서 수개의 enkephalin aminopeptidase의 저해물을 합성하였다. 이 펩티드성 저해물은 zinc 결합자리로서 3-amino-2-hydroxy amino acid와 기질양상의 곁사슬을 가진다. 펩티드들은 용액 중에서 DCC/HOBt를 축합시약으로 사용하여 C-말단으로부터 사슬 연장법으로 합성하였다. 합성한 펩티드 저해물들은 모두 enkephalin aminopeptidase에 대하여 강한 저해 작용을 나타내었다.

• 한국미생물·생명공학회지
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• 제23권1호
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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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• 제13권2호
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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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• 제41권8호
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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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• 제32권3호
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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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• 제5권6호
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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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• 제6권4호
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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.