Piatigorsky J, Horwitz J
Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, MD 20892, USA.
Biochim Biophys Acta. 1996 Jul 18;1295(2):158-64. doi: 10.1016/0167-4838(96)00030-1.
Argininosuccinate lyase (ASL)/delta-crystallin, a major soluble protein of the transparent eye lens of birds and reptiles, is a mixture of tetramers comprising all possible combinations of two similar polypeptides (delta 1 and delta 2). Only the delta 2 polypeptide has ASL activity. In the present investigation we have purified each of the 5 major isoforms (delta A to delta E, pI 5.2 to 5.8) of delta-crystallin tetramers from the embryonic duck lens by isoelectric focussing and established by peptide sequencing that the delta 1 and delta 2 polypeptides are encoded in the previously identified, linked delta 1 and delta 2 genes, respectively. The relative amounts of the different tetramers in the 14-day-old embryonic lens were consistent with equal expression of the 2 delta-crystallin genes and no preference for assembly of the 2 delta polypeptides. The relative amount of ASL activity of the tetramers was a linear function of the relative amount of their delta 2 polypeptides, with delta A (only delta 1) lacking enzymatic activity altogether. delta B (3 delta 1:1 delta 2), delta C (2 delta 1:2 delta 2), delta D (1 delta 1:3 delta 2) and delta E (4 delta 2) all gave normal Michaelis-Menten kinetics for fumarate production from argininosuccinate at 40 degrees C and had a similar Km (average Km for mixture was 0.15 mM). delta E had a Km of 0.187 mM and a Vmax of 9 mumol/min per mg protein. Unlike bovine and like human ASL, both reported previously, embryonic duck ASL/delta-crystallin showed no evidence of cooperativity or activation by GTP. Each isoform had a similar far ultraviolet circular dichroism spectrum and thermal stability between 20 degrees C and 60 degrees C, with denaturation occurring at 65 degrees C. Our data suggest that gene duplication, structural modifications leading to greater thermal stability of the delta 1 and delta 2 polypeptides, and selective loss of ASL activity in the delta 1 polypeptide all occurred during the recruitment of ASL for a refractive role in the duck lens, resulting in the generation of ASL isoenzymes.
精氨琥珀酸裂解酶（ASL）/δ-晶体蛋白是鸟类和爬行动物透明晶状体中的一种主要可溶性蛋白质，它是由两种相似多肽（δ1和δ2）的所有可能组合构成的四聚体混合物。只有δ2多肽具有ASL活性。在本研究中，我们通过等电聚焦从鸭胚胎晶状体中纯化了δ-晶体蛋白四聚体的5种主要异构体（δA至δE，pI 5.2至5.8），并通过肽测序确定δ1和δ2多肽分别由先前鉴定的、连锁的δ1和δ2基因编码。14日龄胚胎晶状体中不同四聚体的相对含量与两个δ-晶体蛋白基因的等量表达一致，且对两种δ多肽的组装没有偏好。四聚体的ASL活性相对量是其δ2多肽相对量的线性函数，其中δA（仅含δ1）完全缺乏酶活性。δB（3个δ1:1个δ2）、δC（2个δ1:2个δ2）、δD（1个δ1:3个δ2）和δE（4个δ2）在40℃下从精氨琥珀酸生成富马酸时均呈现正常的米氏动力学，且具有相似的Km值（混合物的平均Km为0.15 mM）。δE的Km为0.187 mM，Vmax为每毫克蛋白质9 μmol/min。与先前报道的牛ASL不同，与人ASL相似，鸭胚胎ASL/δ-晶体蛋白没有协同作用或被GTP激活的证据。每种异构体在20℃至60℃之间具有相似的远紫外圆二色光谱和热稳定性，在65℃时发生变性。我们的数据表明，在鸭晶状体中，ASL被招募用于屈光作用的过程中，发生了基因复制、导致δ1和δ2多肽热稳定性增强的结构修饰以及δ1多肽中ASL活性的选择性丧失，从而产生了ASL同工酶。
