RNA is required for enzymatic conversion of glutamate to delta-aminolevulinate by extracts of Chlorella vulgaris.
Formation of delta-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNAglu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs with glutamate.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000378:metabolism*; D002384:Catalysis; D002708:Chlorella / Q000201:enzymology / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D006868:Hydrolysis; D007982:Levulinic Acids / Q000378:metabolism*; D009249:NADP; D012343:RNA, Transfer / Q000378:metabolism*; D012260:Ribonucleases / Q000494:pharmacology; D013379:Substrate Specificity
D005971:Glutamates; D007982:Levulinic Acids; D009249:NADP; D000622:Aminolevulinic Acid; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D012260:Ribonucleases