Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates
Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates
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DOI：
10.1046/j.0014-2956.2001.02667.x
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发表时间：
2002-01-01
期刊：
影响因子：
--
通讯作者：
Gauthier, F
中科院分区：
文献类型：
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作者：
Brillard-Bourdet, M;Réhault, S;Gauthier, F
除了klikrein hK3外，一种丝氨酸蛋白酶通常被报道为PSA（前列腺特异性抗原）。人精浆中至少有另外两种酶也可切割源自人精胶蛋白序列的合成肽基底物。我们已经确定其中一个是前列腺酸性磷酸酶（PAP），它是前列腺液的主要成分，其生理功能尚不清楚。另一种是在精浆中以低浓度存在的高M-r碱性蛋白，其特征尚待确定。从刚射精的精浆中纯化PAP至均匀性。测定了其n端序列和磷酸酶性质（在低pH下水解对硝基苯基磷酸），并测定了氟化钠对其的抑制作用。纯化的和商业化的PAP在中性和略碱性的ph下，对来自于semenogelin序列的肽底物也具有酶解活性。k(cat)/ k -m值在10(2)-10(3)M-1之间。s（-1）范围内，使用荧光性semenogin衍生的底物，其肽基片段包含的裂解位点与hK3的裂解位点不同，并且主要位于P1=Gln残基或带有羟基的残基之间，PAP的酶解活性被所有目前使用的Aide谱蛋白酶抑制剂抑制得很差。只有3-4二氯异香豆素和苯甲胺抑制纯化的PAP，纯化的人和商业的PAP在中性PH下清洗纯化的人卵磷脂：两个主要的裂解位点在Tyr292和Ser170（卵磷脂I序列）。只有前者在体外通过精浆分析被鉴定出来。
In addition to kallikrein hK3, a serine protease generafly reported as PSA (prostate-specific antigen). at least two other enzymes in human seminal plasma also cleave synthetic peptidyl substrates derived from the sequence of human semenogelins. We have identified one of these as prostatic acid phosphatase (PAP), a major component of prostatic fluid whose physiological function is unclear. The other is a high M-r basic protein present at low concentrations in seminal plasma and that remains to be characterized. PAP was purified to homogeneity from freshly ejaculated seminal plasma. Its N-terminal sequence and its phosphatase properties (hydrolysis of para-nitrophenylphosphate at low pH) were determined, and its inhibition by sodium fluoride measured. Both purified and commercial PAP also had amidolytic activity on peptide substrates derived from the semenogelin sequence at neutral and slightly basic pH. The k(cat)/K-m values were in the 10(2)-10(3) M-1.s(-1) range using fluorogenic semenogelin-derived substrates whose peptidyl moiety included cleavage sites that had been identified ex PAP cleavage sites differed from those of hK3 and were mainly at P1=Gln residues or between residues bearing hydroxyl groups, PAP amidolytic activity was poorly inhibited by all currently used Aide spectrum proteinase inhibitors. Only 3-4 dichloroisocoumarin and benzamidine inhibited purified PAP, Purified human semenogelin was cleaned by purified and commercial PAP at neutral PH: the two main cleavage sites were at Tyr292 and Ser170 (semenogelin I sequence). only the former has been identified ex vivo by analysis of seminal plasma.