Fujikawa-Adachi K, Nishimori I, Taguchi T, Onishi S
First Department of Internal Medicine, Kochi Medical School, Nankoku, Kochi 783-8505, Japan.
J Biol Chem. 1999 Jul 23;274(30):21228-33. doi: 10.1074/jbc.274.30.21228.
A cDNA clone for a novel carbonic anhydrase (CA) isozyme was isolated from human pancreas and salivary glands. The cDNA sequence of 1182 base pairs encoded a 317-amino acid protein with a predicted mass of 36.4 kDa. The highest similarity of this cDNA and the deduced amino acid sequence is to human CA V (mitochondrial CA), hereafter referred to as CA VA. Recombinant protein expressed in COS-7 cells transfected with this cDNA clone was enriched in a mitochondrial fraction. Confocal fluorescence microscopy showed cytoplasmic granular signals in COS-7 cells expressing a fusion protein of the novel CA and green fluorescent protein. Several lines of evidence suggest that the cDNA clone presented herein encodes a novel human mitochondrial CA isozyme, designated CA VB. CA VB has a hydrophobic N-terminal mitochondrial signal sequence (33 amino acid residues). Western blot analysis showed a 36-kDa protein precursor and a 32-kDa mature protein for CA VB. Similar to CA VA, CA VB is a "low activity" enzyme with a sensitivity to acetazolamide. The CA VB gene is located on Xp22.1. Northern blot analysis in normal human tissues demonstrated expression of a 1.3-kilobase transcript in heart and skeletal muscle, and reverse transcription-polymerase chain reaction analysis showed expression of CA VB in pancreas, kidney, salivary glands, and spinal cord but not in liver. CA VA mRNA expression was observed only in liver. These findings indicate these are two genetically distinct isoforms of human CA V, designated CA VA and CA VB, which have different patterns of tissue-specific distribution, suggest different physiological roles for the two mitochondrial isozymes.
从人胰腺和唾液腺中分离出一种新型碳酸酐酶（CA）同工酶的cDNA克隆。1182个碱基对的cDNA序列编码一个317个氨基酸的蛋白质，预测分子量为36.4 kDa。该cDNA及其推导的氨基酸序列与人类CA V（线粒体CA）的相似性最高，以下简称CA VA。用该cDNA克隆转染的COS-7细胞中表达的重组蛋白富集在线粒体部分。共聚焦荧光显微镜显示，在表达新型CA与绿色荧光蛋白融合蛋白的COS-7细胞中存在细胞质颗粒信号。多项证据表明，本文呈现的cDNA克隆编码一种新型人类线粒体CA同工酶，命名为CA VB。CA VB具有一个疏水的N端线粒体信号序列（33个氨基酸残基）。蛋白质印迹分析显示CA VB有一个36 kDa的蛋白质前体和一个32 kDa的成熟蛋白。与CA VA相似，CA VB是一种对乙酰唑胺敏感的“低活性”酶。CA VB基因位于Xp22.1。正常人组织的Northern印迹分析表明，在心脏和骨骼肌中有一个1.3千碱基转录本的表达，逆转录-聚合酶链反应分析显示CA VB在胰腺、肾脏、唾液腺和脊髓中表达，但在肝脏中不表达。仅在肝脏中观察到CA VA mRNA的表达。这些发现表明，人类CA V有两种基因上不同的同工型，即CA VA和CA VB，它们具有不同的组织特异性分布模式，提示这两种线粒体同工酶具有不同的生理作用。
