Hapak R C, Zhao H, Boschi J M, Henzl M T
Biochemistry Department, University of Missouri, Columbia 65211.
J Biol Chem. 1994 Feb 18;269(7):5288-96.
CPV3 is a novel avian parvalbumin. It displays an isoelectric point of 4.6, intermediate between that of avian thymic hormone (pI = 4.3) and the muscle parvalbumin isoform (pI = 5.2). Expression of CPV3, like that of avian thymic hormone (ATH), is restricted to the thymic stroma. However, the CPV3 content of chicken thymus tissue (120 micrograms/g tissue) is 4 times lower than that of ATH (500 micrograms/g tissue). The polymerase chain reaction (PCR) was used to gain access to the nucleotide sequence of CPV3. A 147-base pair fragment of the coding sequence, corresponding to residues 48-97, was amplified from total chicken cDNA using degenerate PCR primers. A RACE-PCR strategy was then used to extend the known sequence in both the 5' and 3' directions. The cDNA sequence thus obtained includes 671 base pairs. Primer extension analysis suggests that the cloned cDNA corresponds to a full-length transcript. Northern analysis of chicken mRNA indicates that the average CPV3 transcript is approximately 800 nucleotides in length, significantly smaller than the ATH message (approximately 1000 nucleotides). Southern analysis suggests the presence of a single CPV3 gene in the chicken genome. The translated nucleotide sequence, displaying 108 residues between the initiator and termination codons, is that of a beta-parvalbumin. The CPV3 sequence exhibits 58% identity with ATH and 52% identity with the chicken muscle isoform. Interestingly, CPV3 and the mammalian oncodevelopmental parvalbumin called oncomodulin are identical at 73 of 108 residues (68% identity). Correspondingly, flow-dialysis measurements with 45Ca2+ indicate that the Ca(2+)-binding domains are inequivalent, as in oncomodulin. The apparent dissociation constants, at pH 7.4 in 150 mM NaCl, are approximately 10 nM and 80 nM.
CPV3是一种新型禽类小清蛋白。它的等电点为4.6，介于禽胸腺激素（pI = 4.3）和肌肉小清蛋白亚型（pI = 5.2）之间。与禽胸腺激素（ATH）一样，CPV3的表达仅限于胸腺基质。然而，鸡胸腺组织中CPV3的含量（120微克/克组织）比ATH（500微克/克组织）低4倍。采用聚合酶链反应（PCR）来获取CPV3的核苷酸序列。使用简并PCR引物从鸡总cDNA中扩增出编码序列的一个147个碱基对的片段，对应于第48 - 97位残基。然后采用RACE - PCR策略在5'和3'方向上延伸已知序列。由此获得的cDNA序列包含671个碱基对。引物延伸分析表明克隆的cDNA对应于一个全长转录本。对鸡mRNA的Northern分析表明，CPV3转录本的平均长度约为800个核苷酸，明显小于ATH的信息（约1000个核苷酸）。Southern分析表明鸡基因组中存在单个CPV3基因。翻译后的核苷酸序列在起始密码子和终止密码子之间显示有108个残基，是β - 小清蛋白的序列。CPV3序列与ATH的同一性为58%，与鸡肌肉亚型的同一性为52%。有趣的是，CPV3与被称为癌调蛋白的哺乳动物肿瘤发生相关小清蛋白在108个残基中的73个残基处相同（同一性为68%）。相应地，用45Ca2+进行的流动透析测量表明，钙结合结构域不等价，与癌调蛋白情况相同。在pH 7.4、150 mM NaCl条件下，表观解离常数约为10 nM和80 nM。
