ISOLATION AND PROPERTIES OF CYTOCHROME-C OXIDASE FROM RAT-LIVER AND QUANTIFICATION OF IMMUNOLOGICAL DIFFERENCES BETWEEN ISOZYMES FROM VARIOUS RAT-TISSUES WITH SUBUNIT-SPECIFIC ANTISERA
ISOLATION AND PROPERTIES OF CYTOCHROME-C OXIDASE FROM RAT-LIVER AND QUANTIFICATION OF IMMUNOLOGICAL DIFFERENCES BETWEEN ISOZYMES FROM VARIOUS RAT-TISSUES WITH SUBUNIT-SPECIFIC ANTISERA
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DOI：
10.1111/j.1432-1033.1985.tb08905.x
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发表时间：
1985-01-01
期刊：
影响因子：
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通讯作者：
KADENBACH, B
中科院分区：
文献类型：
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作者：
KUHNNENTWIG, L;KADENBACH, B
用细胞色素-c-Sepharose4B亲和层析和DEAE-Sepharose4B亲和层析从大鼠肝脏中分离得到细胞色素c氧化酶。两种制剂的十二烷基硫酸酯凝胶电泳图显示相同的亚基图谱，由13个不同的多肽组成。两种制剂的动力学分析表明，DEAE-Sephacel柱层析分离的酶具有较高的Vmax。针对10个核编码亚基中的9个，在兔体内制备了特异性抗血清。通过Western印迹分析，每个抗血清与其对应的亚基发生单特异性反应，从而排除了由于亚基的蛋白分解、聚集或构象修饰而导致的分离酶凝胶电泳谱中的人工条带。用大鼠肝细胞色素c氧化酶抗血清进行Western印迹分析，发现猪肝、肾细胞色素c氧化酶与心肌、骨骼肌细胞色素c氧化酶亚基Via和Vii亚基的反应性不同。为了定量分析免疫学差异，建立了一种硝酸纤维素酶联免疫吸附试验。抗大鼠肝细胞色素C氧化酶13个亚基中的12个亚基的单特异性抗血清，随着不同组织线粒体总蛋白的增加而滴定，这些蛋白溶解在十二烷基硫酸盐中，并点缀在硝酸纤维素上。测定了由第二种过氧化物酶标记的抗体形成的可溶性染料的吸光度。线粒体编码的细胞色素C氧化酶催化亚基I-III在所有大鼠组织中可能是相同的。所有9个被研究的细胞色素c氧化酶核编码亚基在2个或2个以上组织之间显示出免疫差异。肝脏、肾脏或脑与心脏或骨骼肌之间的免疫学差异较大。心脏和骨骼肌之间以及肝、肾和脑之间的某些亚单位存在微小但显著的差异。胎儿和成人肝脏、心脏和骨骼肌组织中相应的细胞色素c氧化酶核编码亚基之间存在明显的免疫学差异。这可能解释了由于细胞色素C氧化酶缺乏而导致的致命性婴儿肌病。
Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c-Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the 2 preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against 9 of the 10 nuclear encoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoreoic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the 2nd peroxidase-conjugated antibody was measured. The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. All 9 investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between 2 or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeltal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. This may explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.