Two Low K , Hydrolytic Activities on Dinucleoside 5 ’ , 5 ” ’ - P 1 , P 4 - tetraphosphates in Rat Liver

Ninety per cent of total rat liver hydrolytic activity (1.4 unitslg of fresh tissue) on diadenosine or diguanosine 5’,5”’-P’,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; Lobaton, C. D., Vallejo, C. G., Sillero, A., and Sillero, M. A. G. (1975) Eur. J . Biochem. 50, 495501). Particulate activity, as extracted with Triton X100, is composed of two enzymes separable by gel filtration. One of them was a low K , (1 p~ Gp4G, 5 p~ Ap4A) 22,000-dalton enzyme, strongly inhibited by guanosine 5‘-tetraphosphate ( IC i = 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. K , values were 12 MM (Gp4G) and 8 p~ (Ap4A). Guanosine 5’-tetraphosphate was a competitive inhibitor (Ki = 2 pM). 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 Zn”. 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.