title: Tripolyphosphate is an alternative phosphodonor of the selective protein phosphorylation of liver microsomal membrane.
abstract: Two proteins (Mr = 145,000 and Mr = 130,000) of rat liver microsomal membrane are selectively phosphorylated in a characteristic biphasic time course by incubating the membrane with [gamma-32P]ATP in the absence of exogenously added Mg2+ (Lam, K. S., and Kasper, C. B. (1980) J. Biol. Chem. 255, 259-266). This endogenous phosphorylation system was solubilized with Triton X-100 and fractionated by chromatography with DEAE-cellulose and Sepharose 4B. The resulting preparation lacked both ATPase and inorganic pyrophosphatase activity, but retained its original character: the first phase occurred in the presence of ATP but the second phase was initiated after its depletion, implying the presence of a phosphodonor other than ATP. The putative phosphoryl donors were demonstrated to be ATP in the first phase and in the second phase tripolyphosphate, which is present in [gamma-32P]ATP preparations as a radioactive impurity. The latter conclusion was corroborated by results showing that tripolyphosphate purified from a commercial [gamma-32P]ATP and chemically synthesized [32P] tripolyphosphate were both capable of phosphorylating the two proteins and that the unlabeled tripolyphosphate competed effectively against the phosphodonor. A rapid dephosphorylation was observed in both phases upon removal of substrates during the reaction, indicating that there is a continuous turnover of the phosphoryl groups being transferred to the proteins. The second phase of phosphorylation maintained by the tripolyphosphate was shown to be reversibly inhibited by micromolar levels of ATP, ADP, and nonhydrolyzable analogues of these compounds. The implications of this unique phosphorylation system are discussed.
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000648:ultrastructure*; D008970:Molecular Weight; D017830:Octoxynol; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011122:Polyphosphates / Q000378:metabolism*; D051381:Rats; D012969:Sodium Fluoride / Q000494:pharmacology
chemical_list: D011122:Polyphosphates; D011092:Polyethylene Glycols; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D017830:Octoxynol; C005692:triphosphoric acid