Rat intestinal phosphodiesterase II. Properties of the highly purified enzyme and its inactivation by iodoacetic acid.
A highly purifed preparation of rat intestinal phosphodiesterase II (oligonucleate 3'-nucleotidohydrolase, EC 3.1.4.18) has been studied using a synthetic substrate, thymidine 3'(2,4-dinitrophenyl) phosphate. The enzyme was most active between pH 6.1 and pH 6.7 and was inhibited by Cu2+ and Zn2+ but unaffected by EDTA, Mg2+, Co2+, and Ni2+. The reaction rate decreased at high levels of enzyme because of competitive inhibition by deoxythymidine 3'-phosphate, a reaction product, which showed a Ki of 2-10(-5) M. The molecular weight of the enzyme by gel-filtration was 150 000-170 000. In electrofocusing experiments multiple peaks of activity were found at pH 3.4, 4.2-4.5and 7.2. Polyacrylamide gel electrophoresis of freshly purified phosphodiesterase II showed up to 10 protein bands in the gels. If the preparations were stored at 4 degrees C for some time only one or two bands appeared. Investigation of the reaction of rat intestinal phosphodiesterase II with a number of possible phosphodiesterase substrates indicated that the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis. Thus compounds such as NAD, ATP, bis-(p-nitrophenyl)phosphate, thymidine 5'-(p-nitrophenyl)phosphate, glycerylphosphorylcholine, guanylyl-(2' leads to 5')-adenosine and 3',5'-cyclic AMP which contain phosphodiester bonds, nevertheless were not substrates for the enzyme. The enzyme was inhibited reverisbly by p-chloromercuribenzoate and p-chloromercuriphenylsulfonate and inactivated irreversibly by iodoacetic acid. Activity of the phosphodiesterase II was reduced to 50% by incubation with 2.0-10(-3)--5.0-10(-3) M iodoacetate for 20--30 min at 24 degrees C at pH 5.0--6.1. Iodoacetamide had no effect. The degree of inactivation by iodoacetate was reduced by the presence of a substrate for the enzyme or, more effectively by deoxythymidine 3'-phosphate, a competitive inhibitor. It is concluded that iodoacetic acid alkylates an essential residue at the active centre of the enzyme.
D000818:Animals; D002413:Cations, Divalent; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007421:Intestine, Small / Q000201:enzymology*; D007461:Iodoacetates / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D009841:Oligonucleotides; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification; D051381:Rats
D002413:Cations, Divalent; D007461:Iodoacetates; D009841:Oligonucleotides; D010726:Phosphodiesterase Inhibitors; D004492:Edetic Acid; D010727:Phosphoric Diester Hydrolases