Srivastava O P, Srivastava K, Chaves J M
Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Mol Vis. 2008;14:1872-85. Epub 2008 Oct 20.
The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses.
An inactive, Arg-bond hydrolyzing proteinase in the alpha-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclusion Agarose A1.5 m chromatography, non-denaturing preparative gel-electrophoresis, and size-exclusion HPLC. The purified proteinase was characterized for the proteinase type, proteolysis of bovine recombinant gammaB-, gammaC-, and gammaD-crystallins, and its presence in three different protein fractions of human lenses (i.e., alpha-crystallin, beta(H)-crystallin, and membrane fractions).
An inactive, Arg-bond hydrolyzing proteinase present in the alpha-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl beta-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the alpha-crystallin fraction since it eluted at a lower molecular weight species than alpha-crystallin during size-exclusion Agarose A1.5 m chromatography. Following a three-step purification procedure, the enzyme showed three species between 22 kDa and 25 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The three protein bands were identified as betaA3-, betaB1-, and betaB2-crystallin by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitor studies revealed that the enzyme was a serine-type proteinase. Among the recombinant betaA3-, betaB1-, or betaB2-crystallins, only the betaA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of gammaC- and gammaD- crystallins, and the cleavage of gammaD-crystallin at M(1)-G(2), Q(54)-Y(55), M(70)-G(71), and Q(103)-M(104) bonds. Further, the enzyme was also present in three fractions of human lenses (alpha-crystallin, beta(H)-crystallin, and membrane fractions).
A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents. The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.
本研究旨在表征与βA3-晶状体蛋白相关的蛋白酶活性特性，该蛋白是从人晶状体的α-晶状体蛋白组分中分离得到的。
从60至70岁人晶状体的水溶性（WS）蛋白组分中分离出的α-晶状体蛋白组分中的一种无活性的、能水解精氨酸键的蛋白酶，经脱氧胆酸钠处理后被激活。通过三步法对激活后的酶进行纯化，包括尺寸排阻琼脂糖A1.5m色谱法、非变性制备凝胶电泳和尺寸排阻高效液相色谱法。对纯化后的蛋白酶进行蛋白酶类型、对牛重组γB-、γC-和γD-晶状体蛋白的蛋白水解作用以及其在人晶状体三种不同蛋白组分（即α-晶状体蛋白、β（H）-晶状体蛋白和膜组分）中的存在情况进行表征。
α-晶状体蛋白组分中存在的一种无活性的、能水解精氨酸键的蛋白酶在用去污剂如脱氧胆酸钠、Triton X-100、辛基β-D-吡喃葡萄糖苷和CHAPS（3-[(3-胆酰胺丙基)二甲基铵基]-1-丙烷磺酸盐）处理后表现出活性。脱氧胆酸钠激活的酶从α-晶状体蛋白组分中释放出来，因为在尺寸排阻琼脂糖A1.5m色谱分析中，它以比α-晶状体蛋白分子量更低的物种形式洗脱。经过三步纯化程序后，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析中，该酶显示出22kDa至25kDa之间的三种物种。通过基质辅助激光解吸/电离飞行时间（MALDI-TOF）和串联质谱（ES-MS/MS）方法，这三条蛋白带被鉴定为βA3-、βB1-和βB2-晶状体蛋白。抑制剂研究表明该酶是一种丝氨酸型蛋白酶。在重组βA3-、βB1-或βB2-晶状体蛋白中，只有βA3-晶状体蛋白在去污剂处理和尺寸排阻色谱后表现出蛋白酶活性。该蛋白酶还表现出对γC-和γD-晶状体蛋白的蛋白水解作用，以及γD-晶状体蛋白在M(1)-G(2)、Q(54)-Y(55)、M(70)-G(71)和Q(103)-M(104)键处的切割。此外，该酶也存在于人晶状体的三种组分（α-晶状体蛋白、β（H）-晶状体蛋白和膜组分）中。
一种丝氨酸型βA3-晶状体蛋白酶以无活性状态存在于α-晶状体蛋白组分中，并被去污剂激活。该酶能蛋白水解αA-、αB-、γC-和γD-晶状体蛋白，且存在于60至70岁人晶状体的三种组分（α-晶状体蛋白、β（H）-晶状体蛋白和膜组分）中。
