Moriyama A, Sasaki M
J Biochem. 1983 Nov;94(5):1387-97. doi: 10.1093/oxfordjournals.jbchem.a134485.
Succinyltrialanine p-nitroanilide(STANA)-hydrolytic enzyme was purified 5,200-fold from porcine liver soluble fraction with a yield of 75% by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephadex G-150, and hydroxylapatite columns. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate (SDS). The pI of the enzyme was 4.9 by dis gel electrofocusing and the molecular weight was calculated to be 72,000 by gel filtration on a Sephadex G-150 column and 74,000 by SDS-polyacrylamide gel electrophoresis. Acidic amino acids amounted to 17.2% of the total amino acid residues, and the basic ones, 12.9%. No hexosamine was detected. The STANA-hydrolytic enzyme showed maximal activity at pH 7.4 against succinyltrialanine p-nitroanilide and at pH 6.5 against succinyl-Gly-Pro-4-methylcoumaryl 7-amide (MCA), and was stable between pH 6 and 7 in the presence of dithiothreitol. This enzyme hydrolyzed succinyl-Gly-Pro-Leu-Gly-Pro-MCA, succinyl-Gly-Pro-MCA, succinyl-Ala-Pro-Ala-MCA, and several proline-containing natural peptides in addition to succinyltrialanine p-nitroanilide, but was unable to hydrolyze the substrates of aminopeptidases, dipeptidylaminopeptidase IV, trypsin, and chymotrypsin. Elastatinal and chymostatin were effective inhibitors and their IC50 values were 8.7 micrograms/ml and 18.2 micrograms/ml, respectively. The enzyme was completely inhibited by 10(-7) M p-chloromercuribenzoic acid (pCMB), 10(-7) M p-chloromercuriphenylsulfonic acid (pCMPS), and 10(-4) M diisopropyl phosphofluoridate (DFP), but not by 1 mM E-64, which is known as an inhibitor specific to thiol proteinase. The enzyme was easily inactivated by agitation in a Vortex mixer, and its activity was recovered by the addition of thiol compounds such as dithiothreitol, 2-mercaptoethanol and cysteine. The effects of inhibitors and thiol compounds were substantially identical when the enzyme activity was measured with either succinyltrialanine p-nitroanilide or succinyl-Gly-Pro-MCA as a substrate. These results indicate that the STANA-hydrolytic enzyme in the liver soluble fraction is a post-proline cleaving enzyme [EC 3.4.21.26].
琥珀酰丙氨酰对硝基苯胺（STANA）水解酶从猪肝可溶性部分经硫酸铵分级分离以及在DEAE-葡聚糖凝胶、葡聚糖凝胶G-150和羟基磷灰石柱上的色谱法纯化了5200倍，产率为75%。通过在有无十二烷基硫酸钠（SDS）存在下的聚丙烯酰胺凝胶电泳判断，纯化后的酶是均一的。通过圆盘凝胶电聚焦测定该酶的pI为4.9，通过在葡聚糖凝胶G-150柱上的凝胶过滤计算分子量为72000，通过SDS-聚丙烯酰胺凝胶电泳计算为74000。酸性氨基酸占总氨基酸残基的17.2%，碱性氨基酸占12.9%。未检测到己糖胺。STANA水解酶对琥珀酰丙氨酰对硝基苯胺在pH 7.4时表现出最大活性，对琥珀酰甘氨酰-脯氨酰-4-甲基香豆素-7-酰胺（MCA）在pH 6.5时表现出最大活性，并且在二硫苏糖醇存在下于pH 6至7之间稳定。该酶除了水解琥珀酰丙氨酰对硝基苯胺外，还能水解琥珀酰甘氨酰-脯氨酰-亮氨酰-甘氨酰-脯氨酰-MCA、琥珀酰甘氨酰-脯氨酰-MCA、琥珀酰丙氨酰-脯氨酰-丙氨酰-MCA以及几种含脯氨酸的天然肽，但不能水解氨肽酶、二肽基氨基肽酶IV、胰蛋白酶和糜蛋白酶的底物。弹性蛋白酶抑制剂和糜蛋白酶抑制剂是有效的抑制剂，它们的IC50值分别为8.7微克/毫升和18.2微克/毫升。该酶被10⁻⁷M对氯汞苯甲酸（pCMB）、10⁻⁷M对氯汞苯磺酸（pCMPS）和10⁻⁴M二异丙基氟磷酸酯（DFP）完全抑制，但不被1mM E-64抑制，E-64是一种已知的巯基蛋白酶特异性抑制剂。该酶在涡旋混合器中搅拌时很容易失活，通过添加二硫苏糖醇、2-巯基乙醇和半胱氨酸等巯基化合物可恢复其活性。当以琥珀酰丙氨酰对硝基苯胺或琥珀酰甘氨酰-脯氨酰-MCA为底物测量酶活性时，抑制剂和巯基化合物的作用基本相同。这些结果表明，肝可溶性部分中的STANA水解酶是一种脯氨酸后切割酶[EC 3.4.21.2
