| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1985: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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
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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
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