Two low Km hydrolytic activities on dinucleoside 5',5"'-P1,P4-tetraphosphates in rat liver. Characterization as the specific dinucleoside tetraphosphatase and a phosphodiesterase I-like enzyme.
Ninety per cent of total rat liver hydrolytic activity (1.4 units/g of fresh tissue) on diadenosine or diguanosine 5',5"'-P1,P4-tetraphosphate (Ap4A and Gp4G) present in isotonic homogenates sedimented at 37,000 X g. Supernatant activity corresponded to the earlier described, cytosolic and specific, bis(5'-guanosyl) tetraphosphatase or dinucleoside tetraphosphatase (EC 3.6.1.17; Lobatón, C. D., Vallejo, C. G., Sillero, A., and Sillero, M. A. G. (1975) Eur. J. Biochem. 50, 495-501). Particulate activity, as extracted with Triton X-100, is composed of two enzymes separable by gel filtration. One of them was a low Km (1 microM Gp4G, 5 microM Ap4A) 22,000-dalton enzyme, strongly inhibited by guanosine 5'-tetraphosphate (Ki = 9 nM), and likely identical to the cytosolic specific enzyme. The other Triton-extracted form was unspecific, with an estimated molecular weight of 150,000 (sucrose gradient) or 450,000 (gel filtration), both in the presence of detergent. Substrate specificity was broad, requiring a nucleoside 5'-phosphoryl residue with a free 3'-hydroxyl group, and acting on 5'-5' and 5'-3' compounds. Km values were 12 microM (Gp4G) and 8 microM (Ap4A). Guanosine 5'-tetraphosphate was a competitive inhibitor (Ki = 2 microM). It required bivalent cations since a residual activity after dialysis was abolished by EDTA and enhanced by Mg2+, Mn2+, or Ca2+. In the absence of other added cations, the enzyme, inhibited by 1 mM EDTA, is fully reactivated by an equimolar amount of Zn2+. The possible identity of this activity with phosphodiesterase I (EC 3.1.4.1; Razzell, W.E. (1963) Methods Enzymol. 6, 236-258) is discussed, and its potential role in the metabolism of dinucleoside tetraphosphates is indicated.
D017766:Acid Anhydride Hydrolases; D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D015226:Dinucleoside Phosphates; D006150:Guanine Nucleotides / Q000378:metabolism*; D006159:Guanosine Tetraphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D015226:Dinucleoside Phosphates; D006150:Guanine Nucleotides; D006159:Guanosine Tetraphosphate; C015138:diguanosine tetraphosphate; C020733:diadenosine tetraphosphate; D010744:Phosphoric Monoester Hydrolases; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)