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

血红素对δ-氨基乙酰丙酸合酶同工酶的合成和细胞内定位的调节

基本信息

批准号：
60570105
负责人：
HAYASHI Norio
金额：
$ 1.02万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60570105/
关键词：
<delta> -Aminolevulinate Synthase Purification Isozyme cDNA

项目摘要

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

1.以大鼠网织红细胞为原料，经木瓜酶有限酶解后，纯化了β-氨基酮戊酸合成酶。细胞裂解和酶的纯化是在0.1%Triton X-100和0.1%脱氧胆酸钠的存在下进行的，以防止血红蛋白沉淀。该酶经硫酸铵分级沉淀、木瓜酶消化、凝胶过滤、羟基磷灰石柱层析、Q琼脂糖离子交换层析、CoA-琼脂糖亲和层析等步骤分离得到。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析，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(分子量为51,000)相似的动力学性质。与肝酶相比，高浓度的琥珀酰辅酶A对红系酶无抑制作用。红系与h-…的关系用抗大鼠肝ALA合酶抗体和抗鸡肝ALA合酶抗体对更多的表型ALA合成酶进行了免疫化学分析。大鼠红系ALA合酶与抗肝ALA合酶抗体无交叉反应，而大鼠、小鼠和鸡的肝ALA合酶具有显著的交叉反应。这些结果清楚地区分了红系和肝脏ALA合酶之间的同工酶关系，提示可能至少存在两个不同的ALA合酶基因。Western印迹分析表明，肾脏ALA合成酶和哈德氏腺ALA合成酶与肝脏ALA合成酶的分子大小相同。通过对鸡红细胞和肝脏丙氨酸合成酶基因文库的免疫学筛选，克隆了鸡红细胞和肝脏丙氨酸合成酶的基因片段。以红细胞型丙氨酸合成酶和肝型丙氨酸合成酶基因为探针进行Northern印迹分析表明，只有肝酶的聚(A)+RNA&gt；组分才能检测到肝丙氨酸合成酶的mRNA，且其分子大小(2.3kb)大于红细胞型丙氨酸合成酶(2.0kb)，只有聚(A)+RNA&gt；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Bothwick等报道的鸡肝脏ALA合成酶的部分序列进行了比较。(1985))；它们之间在核苷酸和氨基酸序列上都观察到大约50%的序列同源性。较少