Characterization of the multiple forms of hydroxymethylbilane synthase from rat spleen.
Phenylhydrazine treatment induced hydroxymethylbilane synthase activity (EC 4.3.1.8) in rat spleen, erythrocytes and liver by 40-fold, 7.5-fold and 6-fold respectively. Five multiple forms of the enzyme were resolved by DEAE-cellulose chromatography. In the presence of phenylmethanesulphonyl fluoride only three forms, two major and one minor, were resolved by the fractionation, suggesting that two of the original forms arose by proteolytic modification. Heat treatment (70 degrees C) in the presence of proteinase inhibitor converted one of the major forms into the other major form. Product isomer analysis suggested that this heat-labile form represented an enzyme-substrate covalent intermediate and not a hydroxymethylbilane synthase-uroporphyrinogen III synthase complex. Identical elution profiles and kinetic properties of the enzymes from rat spleen and erythrocytes suggested that the enzyme isolated from spleen was possibly from stored erythrocytes. Sephadex G-75 chromatography of the heat-stable DEAE-cellulose-purified form yielded pure enzyme as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr was found to be 43000 +/- 1500. Initial-velocity studies on all enzyme forms showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. For the heat-stable form Vmax, varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 7.4 and 8.8 are important for catalysis. Km decreased with decreasing pH on the acid side of the pH optimum, suggesting the absence of ionization of a group with pK 7.4 in free enzyme or substrate.
D000642:Ammonia-Lyases / Q000378:metabolism*; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004790:Enzyme Induction / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology; D006358:Hot Temperature; D011163:Hydroxymethylbilane Synthase / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D010659:Phenylhydrazines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen / Q000201:enzymology*
D007527:Isoenzymes; D010659:Phenylhydrazines; C030299:phenylhydrazine; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases