Characterization of Ca2+-dependent endogenous phosphorylation of 160000- and 150000-dalton proteins of sarcoplasmic reticulum

I. Orr, Z. Gechtman, V. Shoshan-Barmatz

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Abstract

The 160 and 150 kDa proteins of sarcoplasmic reticulum (SR) are phosphorylated endogenously. The phosphorylation of both proteins has a marked requirement for Ca2+. Half-maximal and maximal phosphorylation was obtained at about 1 nM- and 1 μM-Ca2+ respectively, and a Hill coefficient of about 0.5 was calculated. The phosphorylation is also dependent on NaF as an inhibitor of the SR phosphoprotein phosphatase. The phosphorylation of these proteins is very rapid, and maximal phosphorylation is achieved in less than 15 s. The phosphorylation of the 160 kDa and 150 kDa polypeptides is completely inhibited by 5 mM-MgCl2 and by 75 μM-LaCl3, by very low concentrations of different detergents, and by preincubation of the SR for 2 min at 60 °C. The inhibition by Mg2+ is due to stimulation of ATP hydrolysis, thereby decreasing ATP concentration. Different phosphorylated peptides were obtained by digestion with protease V8 of the 160 kDa and 150 kDa protein bands, suggesting that the 160 kDa and 150 kDa proteins are distinct. The two phosphorylated proteins are present in different fractions and preparations of SR, with or without [3H]PN200-110 binding capacity. These and other results suggest that the phoshorylated SR proteins are distinct from the α1 and α2 subunits of the voltage-gated Ca2+ channel of the T-system membranes. Different inhibitors and activators of protein kinase C and calmodulin-dependent protein kinase have no effect on the endogenous phosphorylation of both polypeptides, suggesting that the phosphorylation is regulated solely by Ca2+. A possible regulatory function for this phosphorylation system is described in the accompanying paper [Gechtman, Orr and Shoshan-Barmatz (1991) Biochem. J. 276, 97-102].

Original languageEnglish
Pages (from-to)89-96
Number of pages8
JournalBiochemical Journal
Volume276
Issue number1
DOIs
StatePublished - 1 Jan 1991

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