Heme Regulation of Synthesis and Intracellular Localization of <delta> -Aminolevulinate Synthase Isozymes

1. 经木瓜蛋白酶有限水解，从大鼠网状细胞中纯化出< δ > -氨基乙酰丙酸（ALA）合成酶。细胞的裂解和酶的纯化在0.1% Triton X-100和0.1%脱氧胆酸钠存在下进行，以防止血红蛋白沉淀。通过硫酸铵分离、木瓜蛋白酶消化、凝胶过滤、羟基磷灰石柱层析、Q Sepharose离子交换层析和coa -琼脂糖亲和层析从溶血液中分离出该酶。经sds -聚丙烯酰胺凝胶电泳分析，最终产物基本均匀，分子量为49,000。纯化的红系酶表现出与木瓜酶消化的肝酶相似的动力学特性（分子量为51,000）。与肝酶相反，高浓度琥珀酰辅酶a对红系酶无抑制作用。用抗大鼠肝ALA合成酶IgG和抗鸡肝ALA合成酶IgG免疫化学方法分析了红细胞与肝ALA合成酶的关系。大鼠红系ALA合成酶与抗肝ALA合成酶抗体无交叉反应性，但大鼠、小鼠和鸡的肝ALA合成酶具有显著的交叉反应性。这些结果清楚地区分了红系和肝系ALA合成酶的同工酶关系，表明可能存在至少两种不同的ALA合成酶基因。Western blot分析显示，肾ALA合成酶和哈德氏腺ALA合成酶与肝酶具有相同的分子大小。通过免疫筛选<lambda> gtll cDNA文库，克隆了鸡红细胞和肝脏ALA合成酶的编码片段。以红系ALA合成酶cDNA和肝系ALA合成酶cDNA为探针进行Northern blot分析发现，肝系ALA合成酶mRNA仅能在肝脏的poly(A) <^+RNA>片段中检测到，且其分子大小（2.3kb）大于红系ALA合成酶mRNA(2.0kb)，而红系ALA合成酶mRNA仅能在网状红细胞的poly(A) <^+RNA>片段中检测到。将鸡红系ALA合成酶的部分cDNA序列及其推导的氨基酸序列与鸡肝ALA合成酶的部分cDNA序列进行了比较（Bothwick et al.（1985）报道）；两者在核苷酸和氨基酸序列上的同源性约为50%。少

1. <delta> -Aminolevulinate (ALA) synthase was purified from rat reticulocytes after limited proteolysis by papain. The lysis of the cells and the purification of the enzyme were carried out in the presence of 0.1 % Triton X-100 and 0.1 % sodium dexycholate to prevent hemoglobin precipitation. The enzyme was isolated from the hemolysate by a procedure involving ammonium sulfate fractionation, papain digestion, gel filtration, hydroxyapatite column chromatography, ion exchange chromatography on Q Sepharose, and affinity chromatography on CoA-Agarose. The final preparation was essentially homogeneous when analysed by SDS-polyacrylamide gel electrophoresis, showing a molecular weight of 49,000. The purified erythroid enzyme showed kinetic properties similar to those of the papain-digested hepatic enzyme (molecular weight, 51,000). In contrast with the hepatic enzyme, a high concentration of succinyl-CoA was not inhibitory to the erythroid enzyme.2. The relationship between erythroid and h … More epatic ALA synthases was analyzed immunochemically using anti-rat liver ALA synthase IgG and anti-chicken liver ALA synthase IgG. Rat erythroid ALA synthase showed no cross-reactivity with anti-liver ALA synthase antibodies, but hepatic ALA synthases from rat, mouse, and chicken share substantial cross-reactivity with one another. These result clearly distinguish the isozyme relationship between erythroid and hepatic ALA synthases and suggest that there may be at least two different ALA synthase genes. Western blot analysis showed that kidney ALA synthase and Harderian gland ALA synthase have the same molecular size with that of the hepatic enzyme.3. Fragments of the chicken cDNAs coding for erythroid and hepatic ALA synthases were cloned through the immunological screening of the <lambda> gtll cDNA library. Northern blot analysis using both the erythroid ALA synthase cDNA and the hepatic ALA synthase cDNA as the probe revealed that mRNA for hepatic ALA synthase could be detected only in poly(A) <^+RNA> fraction from the liver with the hepatic enzyme cDNA and its molecular size (2.3kb) was larger than that of erythroid ALA synthase mRNA(2.0kb), which could be detected only in poly(a) <^+RNA> from the reticulocytes by the erythroid enzyme cDNA.A partial cDNA sequence of chicken erythroid ALA synthase and its deduced amino acid sequence were compared with those of chicken liver ALA synthase (reported by Bothwick et al. (1985)); about 50% sequence homologies were observed between them for both the nucleotide and amino acid sequences. Less