guanosine-triphosphate and diguanosine-tetraphosphate

Topics: Amides; Amino Acids; Animals; Artemia; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dinucleoside Phosphates; Enzyme Inhibitors; Enzyme Stability; Guanosine Triphosphate; Molecular Structure; Nucleotidyltransferases; Phosphoric Acids; Phosphorus Radioisotopes

The chemical nature of the enzyme-nucleotide phosphoramidate reaction intermediate employed by the unique GTP:GTP guanylyltransferase from yolk platelets of Artemia franciscana cysts to synthesize diguanosine tetraphosphate (Gp4G) has been investigated. Labeling of the enzyme with [alpha-32P]GTP followed by isolation of the labeled phosphoamino acid by periodate treatment and alkaline hydrolysis and comparison of the product with phosphoamino acid standards by thin-layer and ion-exchange chromatography showed that the linkage involves the Nepsilon2 ring nitrogen of an enzyme histidyl residue. Thus, this enzyme is distinct from the mRNA capping enzymes which can also synthesize Gp4G but which employ a lysyl-nucleotide intermediate. Based on its reaction mechanism and substrate specificity, GTP:GTP guanylyltransferase may belong to the GAFH superfamily which includes the histidine triad proteins, Ap4A phosphorylases, and galactose-1-phosphate uridylyltransferase.

Topics: Animals; Artemia; Dinucleoside Phosphates; Guanosine Triphosphate; Histidine; Multienzyme Complexes; Nucleotidyltransferases; Phosphoric Monoester Hydrolases

The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze Gp4G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.

Topics: Animals; Artemia; Binding, Competitive; Dinucleoside Phosphates; GTP Phosphohydrolases; Guanosine Triphosphate; Precipitin Tests; Proto-Oncogene Proteins p21(ras); Substrate Specificity

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 aquatic fungi Achlya ambisexualis and Blastocladiella emersonii were grown in the presence of 32Pi and the 32P-labeled acid-soluble nucleotide fractions were analyzed by ion-exchange chromatography on DEAE-cellulose. Selected column fractions containing diguanosine tri- and tetra-phosphates (Gp3G and Gp4G) added as chromatographic markers were analyzed further for 32P by chromatography and (or) enzyme hydrolysis. The results of these experiments clearly indicate that neither Gp4G nor Gp3G is synthesized during vegetative growth of these organisms and cast doubt on the hypothesis that diguanosine nucleotides are important metabolic regulators in fungi.

Topics: Blastocladiella; Chromatography, DEAE-Cellulose; Dinucleoside Phosphates; Fungi; Guanine Nucleotides; Guanosine Triphosphate; Oligonucleotides; Oligoribonucleotides