한국산 뱀(유혈목이 : Natrix tigrina lateralis) 에서 스파르가눔(sparganum) 충체를 수집하고, 그들의 분비 배설물을 ion-exchange chromatography와 affinity chromatogrphy를 실시하여 cysteine proteinase를 순수 정제하였다. 정제된 효소의 최적 pH는 5.5이었고, 최적 mol 농도는 50 mM(50 mM sodium acetate, pH 5.5)이었다. 정제된 효소는 thiol-dependent이고, $4^{circ}C$와 $37^{circ}C$에서 24시간 동안 95%의 안전성올 보였다. 효소의 활성도는 저분자 합성기질인 CBZ-phe-arg-AFC에 대하여 활성이 높았다. 정제된 효소는 cysteine proteinase 특이 억제인자인 E-64, NEM, iodoacetic acid에 의해 활성이 완전히 억제되었고, serine proteinase 특히 억제인자, aspartic proteinase 특이 억제인자 그리고 metallo proteinase 억제인자에 의해 활성이 억제되지 않았다. Ultrogel AcA 54 column chromatography로 정제된 cysteine proteinase의 분자량을 측정한 결과 28 kDa이었다. 정제된 효소는 collagen type I과 hemoglobin을 분해하였다. 이상의 결과에서 스파르가눔은 cysteine proteinase를 이용하여 숙주 체내이동, 조직 침투성 및 영양소 섭취에 관여하는 것으로 생각된다.
Adult worms of Spirometra plerocercoids (spargana) were dissected out from the tissues of naturally infected snakes (natrix trigria lateralia). Fresh spargana were incubated in distilled water and their excretory/secretory (ES) products were collected. Proteinse activity of the ES products was assayed with a synthetic fluorescent substrate, carbobenzoxy-phenylalanyl-arginyl-7-amino-4-trifluoromethylcoumarin. By means of DEAE-Trisacryl M ion-exchange chromatography and thiolpropyl-sepharose affinity chromatography, the proteinase in the ESP resulted in a 15.6-fold purification. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified materials revealed a single 28 kDa band, consistent with the apparent native molecular weight (gel filtration chromatography) of approximately 28 kDa. The proteinase purified from the ES products were examined for various biochemical characteristics : 1) its inhibitor profile indicated it belonged to cysteine endopeptidases; 2) it exhibited identical pH curves with optimal at pH 5.5 and a 50% activity range from pH 3.7~7.1; 3) it completely degraded collagen chains to three identical products; and 4) it showed only minor activity toward hemoglobin. These results indicate that the proteinase may be involved in the parasite nutrition acquisition and/or penetration and lysis of host tissues.
