Production of delta-aminolevulinate: subcellular localization and purification of murine hepatic L-alanine: 4,5-dioxovaleric acid aminotransferase.
1. L-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C57BL/6J and DBA/2J. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of delta-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on L-alanine-AH-Sepharose. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. The yield of the purification was ca 90 micrograms of protein per gram liver wet weight. 10. The purified enzyme had a subunit mol. wt of 146,000 +/- 5000 as determined by electrophoresis under denaturing conditions.
McKinney
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Department of Zoology, University of Maryland, College Park 20742.
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D000622:Aminolevulinic Acid / Q000378:metabolism*; D000818:Animals; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D007982:Levulinic Acids / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008970:Molecular Weight; D013154:Spleen / Q000378:metabolism; D014018:Tissue Distribution; D000637:Transaminases / Q000302:isolation & purification / Q000378:metabolism*
D007982:Levulinic Acids; D000622:Aminolevulinic Acid; D000637:Transaminases; C019687:aminolevulinate aminotransferase