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

1. <delta>用木瓜蛋白酶对大鼠网织红细胞进行限制性酶解，纯化出β-氨基乙酰丙酸（ALA）合成酶。在0.1%Triton X-100和0.1%右旋胆酸钠存在下进行细胞裂解和酶纯化以防止血红蛋白沉淀。通过硫酸铵分级分离、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-琼脂糖亲和层析等步骤从溶血液中分离该酶。当通过SDS-聚丙烯酰胺凝胶电泳分析时，最终制剂基本上是均匀的，显示分子量为49，000。纯化的红细胞酶显示出与木瓜蛋白酶消化的肝酶（分子量，51，000）相似的动力学性质。与肝酶相比，高浓度琥珀酰辅酶A对红系酶无抑制作用.红系与h的关系 ...更多信息 使用抗大鼠肝ALA合酶IgG和抗鸡肝ALA合酶IgG免疫化学分析Escherichia ALA合酶。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合成酶相互之间具有显著的交叉反应性。这些结果清楚地区分了红细胞和肝细胞ALA合成酶的同工酶关系，并提示可能存在至少两种不同的ALA合成酶基因。Western blot分析表明，肾脏和哈氏腺ALA合成酶的分子大小与肝脏酶相同.通过免疫学筛选，从鸡gt 11 cDNA文库中克隆了编码鸡红系和肝系ALA脱氢酶的cDNA片段<lambda>。用红系ALA合成酶cDNA和肝ALA合成酶cDNA作为探针的北方印迹分析表明，肝ALA合成酶的mRNA仅能在肝酶cDNA及其分子大小的肝poly（A）&lt;^+RNA&gt;组分中检测到（2.3kb）大于红系ALA合成酶mRNA（2.0kb），这只能在poly（a）&lt;^+RNA&gt;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cDNA序列进行了比较（由Bothwick等人（1985）报道）;在它们之间观察到约50%的核苷酸和氨基酸序列同源性。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