Phosphorylation of glycogen synthase in a homogenate of human polymorphonuclear leukocytes |
| |
Authors: | Henning Juhl Viggo Esmann |
| |
Institution: | 1. Dept. of Medicine, Marselisborg Hospital, DK-8000, ?rhus, C., Denmark
|
| |
Abstract: | Summary Glycogen synthase I in a homogenate of human polymorphonuclear leukocytes was phosphorylated under imitated physiological
conditions utilizing the endogenous protein kinases. At subsequent steps of phosphorylation the32P-labelled synthase was purified and characterized. Limited tryptic hydrolysis of the32P-labelled synthase released four phosphopeptides (t-A, t-B, t-C, t-D) and subsequent chymotrypsinization of the trypsin resistant
core released three phosphopeptides (c-A, c-B, c-C).
One Pi/subunit was incorporated within 8–10 min and 2.2 Pi/subunit within 60 min increasing the Kc for Gle-6-P to 4–6 mM. The initial phosphorylation up to 0.8 Pi/subunit occurred mainly in peptide c-A and a linear relation between ratio of independence (RI) of glycogen synthase in the
interval RI 0.85 to RI 0.05 and phosphorylation of this peptide to 0.5 Pi was observed. Phosphorylation of this peptide is responsible for the decrease in ratio of independence.
From experiments with inhibitors and activators, the initial phosphorylation was found predominantly catalysed by the endogenous
cAMP independent synthase kinase, however, the endogenous cAMP dependent protein kinase and phosphorylase kinase also phosphorylate
endogenous glycogen synthase I to a minor degree. Circumstantial evidence for a Ca-dependent synthase kinase different from
phosphorylase kinase is presented.
The endogenous Gle-6-P dependent glycogen synthase occurring in a homogenate of leukocytes disrupted in the presence of NaF incorporated 1.07 Pi/subunit and Kc for Glc-6-P was increased from 6–8 mM to 20 mM.
From the present and previous experiments 7] a total of 8 major phosphorylatable sites have been defined, one on each of
the peptides t-A, t-B, t-C, c-B and c-C and two on peptide c-A, which in addition may contain a third site for phosphorylase
kinase. Assuming identical subunits, only 13 out of 32 sites are thus covalently modified at maximum phosphorylation. The
operational defined synthase R (Kc for Glc-6-P 0.5 mM) and D (Kc for Glc-6-P 2–8 mM) activities correspond to synthase with about 0.8 Pi and 1.8–2.3 Pi/subunit, respectively. |
| |
Keywords: | |
本文献已被 PubMed SpringerLink 等数据库收录! |
|