Comparative Studies on the Enzymatic Properties of two Trypsin-like Enzymes from Menhaden, Brevoortia tyranus

혈합육어 멘헤이든의 장기조직분포Trypsin-유사효소에 관한 비교효소학적 연구

Two trypsin-like enzymes, designated trypsin A and 3, purified from the intestine of menhaden by $(NH_4)_2SO_4$ fractionation, Benzamidine-Sepharose 6B affinity chromatography, DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The two trypsins were subjected to compare the enzymatic properties of the trypsin-like enzymes from the other dark fleshed fishes. Both trypsins catalysed the hydrolysis of N$\alpha$-benzoyl-DL-arginine-p-nitroanilide and they were remarkably inhibited by several well known trypsin-inhibitors, tosyllysyl chloromethyl ketone, soybean trypsin inhibitor, be-nzamidine, leupeptin and antipain, etc. Therefore, it was ascertained that the two enzymes are serine-type trypsins. The molecular weights of these enzymes were about 25,000 and 26,200, respectively, ;Is determined by SDS-PAG electrophoresis and by Sephadex G-100 gel filtration, and the molecular weights of these two enzymes are somewhat fewer than those from the other dark fleshed fishes. Both enzymes had less basic amino acids such as arginine and Iysine, whereas they had slightly high contents of neutral amino acids, glycine, alanine and tryptophane. The enzymes showed a pH optimum of $8\~11$ at $60^{\circ}C$ against the $N\alpha$-benzoyl-DL-argi-nine-p-nitroanilide substrate and they were quite unstable above $40^{\circ}C$ and under the atidic pH region. The Km constant of the two enzymes against the $N\alpha$-benzoyl-DL-arginine-p-nitroanilide was $1.4\times10^{-4}M$ for trypsin A and $4.3\times10^{-5}M$ for trypsin B, respectively.

멘헤이든의 장기에서 황산암모늄염석, 친화성크 로마토그라피(Benzamidine-Sepharose 6B), 이온교환크로마토그라피 (DEAE-Sephacel), 겔여과크로마토그라피(Sephadex G-75)등의 정제과정을 거쳐 2종의 trypsin-유사효소를 정제하고 다른 혈압육어 trypsin의 성질과 비교 검토할 수 있는 효소학적 성질에 관하여 분석하였다. 이들 두 효소는 trypsin에 대한 선택성 합성기질인 $N\alpha$-benzoyl-DL-arginine-p-nitroanilide ( BA-p-NA)를 분해하고, 이미 알려져 있는 trypsin 저해제 tosyl Iysyl chloromethyl ketone(TLCK), soybean trypsin inhibitor(SBTI), benzamidine, leupetin, antipain 등에 의하여 현저히 저해를 받으므로서 serine계의 trypsin임이 확증되었다. 이들 두 효소의 분자량은 겔여과법과 SDS-polya-crylamide 전기영동법에 의하여 trypsin A가 약 25,000, trypsin B가 약 26,200이었으므로 이미 밝혀진 혈압육어의 trypsin 중에서는 비교적 작았다. 이들 효소는 다른 혈압육어들에 비하여 염기성아미노산에 속하는 arginine과 Iysine이 다소 적었든 반면, 중성아미노산인 glycine과 alanine, 그밖에 tryptophan이 조금 많았다. 한편, 이들 효소는 BA-p-NA 기질에 대하여 $60^{\circ}C$전후, pH $8\~11$에서 최대활성을 보였으며, 산성 pH의 조건과 $40^{\circ}C$ 이상의 온도에서는 극히 불안정하였다. 이들 두 효소의 BA-p-NA에 대한 Km 정수는 trypsin A가 $1.4\times10^{-4}M$, trypsin B가 $4.3\times10^{-5}M$였다.