Hare D F, Jarroll E L, Lindmark D G
Department of Biology, Cleveland State University, Ohio 44115.
Exp Parasitol. 1989 Feb;68(2):168-75. doi: 10.1016/0014-4894(89)90094-5.
The proteinase activity of Giardia lamblia trophozoites, Portland 1 strain, was characterized with respect to substrate specificities and inhibitor sensitivities. Proteinase activity with urea-denatured hemoglobin (UDH), alpha-N-benzoyl-DL-arginine-2-naphthylamide (BANA), and alpha-N-benzoyl-argininamide (BAA) as substrates exhibited pH optima of 5.8, 3.8, and 5.0, respectively. For BANA, the apparent Km was 0.20 mM and the Vmax was 2.56 microM. For BAA, the apparent Km was 4.0 mM and the Vmax was 8.69 microM. Dithiothreitol (DTT, 5 mM) enhanced proteinase activity threefold for UDH, fourfold for BAA, and fivefold for BANA. Iodoacetamide, L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), each at 1 mM, inhibited proteinase activity by greater than 90% with BANA and BAA. Iodoacetamide inhibited proteinase activity by 35% with UDH; TPCK and TLCK inhibited activity greater than 70% with UDH. Activity on BAA was inhibited by 91% with Zn2+ and activity on UDH was inhibited by 30% with Cu2+. Virtually complete inhibition of proteinase activity on BANA and BAA was obtained with leupeptin and chymostatin at 1 microgram/ml. Pepstatin A, chelators, and other heavy metals had no apparent effect on proteinase activity. Two polypeptide bands (ca. 105 and 40 kDa) indicative of proteinase activity were visualized by sodium dodecyl sulfate-gelatin polyacrylamide gel electrophoresis. The 105 kDa band was visible over the pH range of 4 to 7, but with greater intensity from pH 5 to 7. The 40 kDa band, while present at pH 5, was most intense at pH 6 and 7.(ABSTRACT TRUNCATED AT 250 WORDS)
对蓝氏贾第鞭毛虫波特兰1株滋养体的蛋白酶活性进行了底物特异性和抑制剂敏感性方面的表征。以尿素变性血红蛋白（UDH）、α-N-苯甲酰-DL-精氨酸-2-萘酰胺（BANA）和α-N-苯甲酰精氨酸酰胺（BAA）为底物时，蛋白酶活性的最适pH分别为5.8、3.8和5.0。对于BANA，表观Km为0.20 mM，Vmax为2.56 μM。对于BAA，表观Km为4.0 mM，Vmax为8.69 μM。二硫苏糖醇（DTT，5 mM）使UDH的蛋白酶活性提高了三倍，使BAA提高了四倍，使BANA提高了五倍。碘乙酰胺、L-对甲苯磺酰胺-2-苯乙基氯甲基酮（TPCK）和N-α-对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK），浓度均为1 mM，对以BANA和BAA为底物的蛋白酶活性抑制率均大于90%。碘乙酰胺对以UDH为底物的蛋白酶活性抑制率为35%；TPCK和TLCK对以UDH为底物的活性抑制率大于70%。Zn2+对以BAA为底物的活性抑制率为91%，Cu2+对以UDH为底物的活性抑制率为30%。用1 μg/ml的亮抑酶肽和抑糜酶素对以BANA和BAA为底物的蛋白酶活性几乎完全抑制。胃蛋白酶抑制剂A、螯合剂和其他重金属对蛋白酶活性无明显影响。通过十二烷基硫酸钠-明胶聚丙烯酰胺凝胶电泳可观察到两条指示蛋白酶活性的多肽带（约105和40 kDa）。105 kDa的条带在pH 4至7范围内可见，但在pH 5至7时强度更大。40 kDa的条带在pH 5时出现，在pH 6和7时最明显。（摘要截短至250字）
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