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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.
Insights
Rat liver microsomal membranes exhibit unique biphasic protein phosphorylation. ATP fuels the first phase, while tripolyphosphate, an impurity, drives the second phase, revealing a novel endogenous phosphorylation system.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Signaling
Background:
- Rat liver microsomal membranes contain endogenous systems for protein phosphorylation.
- Previous studies indicated a biphasic phosphorylation pattern under specific conditions.
Purpose of the Study:
- To investigate the mechanism of endogenous protein phosphorylation in rat liver microsomes.
- To identify the specific phosphodonors involved in the observed biphasic phosphorylation.
- To characterize the properties of the solubilized and fractionated phosphorylation system.
Main Methods:
- Solubilization of microsomal membranes using Triton X-100.
- Chromatographic fractionation using DEAE-cellulose and Sepharose 4B.
- Phosphorylation assays using [gamma-32P]ATP and purified/synthesized tripolyphosphate.
Main Results:
- Two proteins (Mr = 145,000 and 130,000) were selectively phosphorylated.
- The first phase of phosphorylation utilized ATP, while the second phase used tripolyphosphate as the phosphodonor.
- Tripolyphosphate, present as an impurity in [gamma-32P]ATP, was confirmed as the second-phase phosphodonor.
- The system exhibited rapid dephosphorylation and turnover of phosphoryl groups.
- The tripolyphosphate-dependent phosphorylation was reversibly inhibited by ATP, ADP, and their analogues.
Conclusions:
- Rat liver microsomes possess a unique endogenous phosphorylation system involving both ATP and tripolyphosphate.
- Tripolyphosphate acts as a significant phosphodonor in this system, particularly after ATP depletion.
- The observed inhibition by adenine nucleotides suggests regulatory mechanisms for this phosphorylation process.