guanosine-tetraphosphate and diadenosine-tetraphosphate

LDHk is a cancer-associated lactate dehydrogenase which is also found at high levels in normal mammalian retina. Such retinas share with most cancer tissues a dependence on aerobic glycolysis, leading to high production of lactate. However, retinas of lower vertebrate species are significantly less dependent on aerobic glycolysis. We find that retinas of species less dependent on aerobic glycolysis express significantly lower levels of an LDHk-like activity, less than or equal to the low levels seen in brains. The enzymes from lower species differ from the mammalian retinal enzyme in their pH optima and responsiveness to oxygen; but share a similar degree of inhibition by 5'-5'-dinucleoside tetraphosphates. Therefore, the expression pattern of LDHk in brain and retina of diverse vertebrate species suggests a link with the Warburg effect.

Topics: Adenine Nucleotides; Aerobiosis; Anaerobiosis; Animals; Brain; Chickens; Dinucleoside Phosphates; Glycolysis; Goldfish; Guanosine Tetraphosphate; Hydrogen-Ion Concentration; Isoenzymes; L-Lactate Dehydrogenase; Rana pipiens; Rats; Retina; Turtles; Xenopus laevis

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.

Topics: Acid Anhydride Hydrolases; Adenine Nucleotides; Animals; Dinucleoside Phosphates; Guanine Nucleotides; Guanosine Tetraphosphate; Kinetics; Liver; Molecular Weight; Phosphodiesterase I; Phosphoric Diester Hydrolases; Phosphoric Monoester Hydrolases; Rats; Substrate Specificity

The occurrence and distribution of bis-(5'-guanosyl) tetraphosphatase activity towards dinucleoside tetraphosphates between the 27 000 g supernatant and sedimented fraction were studied in liver, kidney, brain, muscle and intestinal mucosa from rat. The p1p4-bis-(5'-guanosyl) tetraphosphate-hydrolysing activities found in total homogenates were 0.77, 1.44, 0.39, 0.36 and 2.14 units (mumol/min)/g respectively. The activities found in the 27000 g-sedimented fractions were 74, 49, 11, 4 and 96% of those present in the homogenates respectively. The properties of the soluble enzymes were investigated. All of them have low Km values for p1p4-bis-(5'-guanosyl) tetraphosphate (from 2 to 50 microM), are competitively inhibited by guanosine 5'-tetraphosphate with K1 values from 10 to 160 nM, have molecular weights of about 21 000, require Mg2+ or Mn2+ and are inhibited by Ca2+. These properties show that bis-(5'-guanosyl) tetraphosphatase (EC 3.6.1.17), an enzyme previously characterized in Artemia salina and rat liver [Warner & Finamore (1965) Biochemistry 4, 1568-1575; Vallejo, Sillero & Sillero (1974) Biochim, Biophys. Acta 358, 117-125; Lobatón, Vallejo, Sillero & Sillero (1975) Eur. J. Biochem. 50, 495-501], is present in all the rat tissues examined. The inhibition of the enzyme by Ca2+ could be related to the effect of p1p4-bis-(5'-adenosyl) tetraphosphate as a trigger of DNA synthesis [Grummt, Waltl, Jantzen, Hamprecht, Huebscher & Kuenzle (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6081-6085].

Topics: Acid Anhydride Hydrolases; Adenine Nucleotides; Animals; Calcium; Dinucleoside Phosphates; Female; Guanosine Tetraphosphate; Hydrolysis; Kinetics; Phosphoric Monoester Hydrolases; Rats; Subcellular Fractions; Tissue Distribution