guanosine-tetraphosphate and diguanosine-tetraphosphate

Commercial samples of GTP and guanosine 5'-tetraphosphate were analyzed, with or without previous treatment with alkaline phosphatase, by high-pressure liquid chromatography on a Hypersil ODS column. They showed the presence of diguanosine 5',5"'-Pl,Pn-tri, tetra-, and pentaphosphates in varying amounts depending on the sample, but usually in proportions of around 0.3%.

Topics: Alkaline Phosphatase; Dinucleoside Phosphates; Drug Contamination; Drug Industry; Guanine Nucleotides; Guanosine Tetraphosphate; Guanosine Triphosphate; Hydrolysis; Liver; Muscles

The activity of the endoribonuclease VI from Artemia is sensitive to several purine nucleotides. The enzyme is non-competitively inhibited by diguanosine tetraphosphate (Ki = 75 microM), a nucleotide abundant in Artemia encysted gastrulae and located in the same particulate fraction as the gastrular ribonuclease. Diguanosine triphosphate and diadenosine tetraphosphate are less efficient inhibitors (Ki congruent to 200 microM). The ribonuclease is non-competitively inhibited by 5'-AMP (Ki = 10 microM) and 5'-GMP (Ki = 50 microM) but is insensitive to the corresponding 5'-phosphates of cytosine and uridine. Other purine mononucleotides inhibit the enzyme activity less efficiently. The modulation of the enzyme activity by these nucleotides is discussed in relation with the changes in ribonuclease activity during early development of Artemia.

Topics: Animals; Artemia; Dinucleoside Phosphates; Endoribonucleases; Guanine Nucleotides; Guanosine Tetraphosphate; Kinetics; Purine Nucleotides; Structure-Activity Relationship

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

Compounds of general structure N(5')pn(5')N were used by the reovirus-associated RNA polymerase as primers for template-directed synthesis of virus-specific oligonucleotides and RNA. Structural requirements for activity included a guanosine residue and at least four phosphates--i.e., G(5')pppp(5')N. Gp4G incubated with viral cores in the presence of CTP yielded Gp4GpC and CpGp4GpC. In a complete transcription mixture, Gp4G was also incorporated into the 5' termini of full-length transcripts in the unmethylated forms Gp4GpC and CpGp4GpC, in contrast to viral mRNAs that contain 5'-terminal m7GpppGmpC formed de novo. Gp5G, Gp6G, and Gp4A but not Gp2G, Gp3G, and Ap4A also primed reovirus transcription and inhibited RNA methylation.

Topics: Dinucleoside Phosphates; Guanine Nucleotides; Guanosine Tetraphosphate; Mammalian orthoreovirus 3; Oligoribonucleotides; Reoviridae; RNA Caps; RNA, Messenger; RNA, Viral; Transcription, Genetic

A brain preparation, consisting of nuclei and perikarya, was able to bind tritium-labeled diguanosine tetraphosphate ([3H]Gp4G). The binding was linear with both time and amount of extract and apparently presented two dissociation constant values (KD) of 0.16 and 0.6 microM, respectively, as determined by equilibrium binding experiments. Inhibition of [3H]Gp4G specific binding by 50% at equilibrium was accomplished by cold Gp4G, guanosine 5'-tetraphosphate, diguanosine triphosphate, and GTP at 0.7, 2.8, 3.1, and 10 microM concentrations, respectively. Diadenosine tetraphosphate (Ap4A) at concentrations up to 100 microM did not affect the observed binding of [3H]Gp4G to brain. These results suggest that this binding is specific and requires the existence of 4 phosphates plus at least 1 guanosine residue in the molecule. The binding of Gp4G to brain increased with time of development reaching a plateau at about 20 days after birth. The data are discussed in relation to previous results on the binding of Ap4A to brain and to DNA polymerase-alpha (Grummt, F., Waltl, G., Jantzen, H-M., Hamprecht, K., Huebscher, U., and Kuenzle, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6081-6085; Rapaport, E., Zamecnik, P. C., and Baril, E. F. (1981) Proct. Natl. Acad. Sci. U. S. A. 78, 838-842).

Topics: Animals; Brain; Cell Nucleus; Dinucleoside Phosphates; Guanine Nucleotides; Guanosine Tetraphosphate; Kinetics; Mathematics; Rats; Time Factors