PURIFICATION AND CHARACTERIZATION OF AN ENZYME PRODUCED BY TREPONEMA-DENTICOLA CAPABLE OF HYDROLYZING SYNTHETIC TRYPSIN SUBSTRATES
PURIFICATION AND CHARACTERIZATION OF AN ENZYME PRODUCED BY TREPONEMA-DENTICOLA CAPABLE OF HYDROLYZING SYNTHETIC TRYPSIN SUBSTRATES
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DOI：
10.1128/iai.53.1.213-220.1986
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发表时间：
1986-07-01
影响因子：
3.1
通讯作者：
LOESCHE, WJ
中科院分区：
文献类型：
--
作者：
OHTA, K;MAKINEN, KK;LOESCHE, WJ
一种来自密螺旋体的酶，能水解合成胰蛋白酶底物N-。-苯甲酰- l-精氨酸-对硝基苯胺（BAPNA），经凝胶电泳鉴定，纯度接近均匀。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，酶的分子量约为69,000，经Sephadex G-100凝胶过滤，酶的分子量约为50,000。BAPNA水解的最适pH为8.5左右。该酶热不稳定，在低pH值下不可逆失活。Ca2+、Mg2+和Ba2+增强了酶活性，而Mn2+、Hg2+、Co2+和Zn2+抑制了酶活性。金属螯合剂和巯基试剂对该活性无影响。该酶被某些蛋白酶抑制剂如氟磷酸二异丙基、N- α抑制。-对甲酰基赖氨酸氯甲基酮，苯基甲基磺酰氟，l- 1-甲酰基酰胺-2-苯乙基氯甲基酮-1-抗胰蛋白酶和大豆胰蛋白酶抑制剂。BAPNA和N- α的Km值。-苯甲酰- l-精氨酸乙酯分别为0.05和0.12 mM，且Vmax值高于胰蛋白酶。虽然纯化后的酶能水解一些低分子量的合成胰蛋白酶底物，但不能水解酪蛋白、血红蛋白、偶氮酪蛋白、偶氮蛋白、牛血清白蛋白或明胶。因此，这种酶可能不是蛋白酶，但能够水解涉及精氨酸和赖氨酸羧基的酯、酰胺和肽键。
An enzyme from Treponema denticola that hydrolyzes a synthetic trypsin substrate, N-.alpha.-benzoyl-L-arginine-p-niroanilide (BAPNA), was purified to near homogeneity, as judged by gel electrophoresis. The molecular weight of the enzyme was estimated to be ca. 69,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ca. 50,000 by gel filtration on Sephadex G-100. The pH optimum for the hydrolysis of BAPNA was around 8.5. The enzyme was heat labile and irreversibility inactivated at low pH values. Enzyme activity was enhanced by Ca2+, Mg2+, and Ba2+ but inhibited by Mn2+, Hg2+, Co2+, and Zn2+. Metal chelators and sulfhydryl reagents had no effect on this activity. The enzyme was inhibited by certain protease inhibitors such as diisopropyl fluorophosphate, N-.alpha.-p-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethylchloromethyl ketone, .alpha.-1-antitrypsin, and soybean trypsin inhibitor. The Km values for BAPNA and N-.alpha.-benzoyl-L-arginine ethyl ester were 0.05 and 0.12 mM, respectively, and the Vmax values were higher than those observed with trypsin. Although the purified enzyme hydrolyzed some low-molecular-weight synthetic trypsin substrates, it did not hydrolyze casein, hemoglobin, azocasein, azocoll, bovine serum albumin, or gelatin. Thus, this enzyme is probably not a protease but is capable of hydrolyzing ester, amide, and peptide bonds involving the carboxyl group of arginine and lysine.