In vitro phosphorylation and inactivation of spinach leaf sucrose-phosphate synthase by an endogenous protein kinase.
(1) Partially purified preparations of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) contain an endogenous protein kinase that phosphorylates and inactivates the enzyme with [gamma-32P]ATP. (2) The kinetic effect of phosphorylation is to alter affinities for substrates and the effector inorganic phosphate without affecting maximum velocity. (3) Two-dimensional peptide mapping of tryptic digests of in vitro labeled SPS yielded two phosphopeptides (designated sites 5 and 7). Labeling of the two sites occurred equally with time, and both correlated with inactivation. Maximum inactivation was associated with incorporation of 1.5 to 2.0 mol P/mol SPS tetramer, and about 70% of the phosphoryl groups were incorporated into one of the sites (phosphopeptide 7). (4) Phosphorylation and inactivation were strongly inhibited by NaCl, and the presence of salt alters some characteristics of the kinase reaction. In the absence of salt, the apparent Km for Mg.ATP was estimated to be 5 microM. (5) The dependence of the rate of phosphorylation on SPS concentration suggested that SPS and the protein kinase are distinct enzymes, but have some tendency to associate especially in the presence of ethylene glycol. (6) Ca2+/EGTA and polyamines have no effect on the rate of phosphorylation, whereas polycations (polylysine, polybrene and protamine) are inhibitory. (7) Of the metabolic intermediates tested, Glc 6-P inhibited phosphorylation and inactivation of the enzyme. The inhibition was not antagonized by inorganic phosphate, which suggests that Glc 6-P may be an effector of the kinase, rather than the target protein. Regulation by Glc 6-P may be of physiological significance.
Huber
S C
SC
U.S. Department of Agriculture, North Carolina State University, Raleigh 27607.
Huber
J L
JL
D000255:Adenosine Triphosphate / Q000378:metabolism; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010944:Plants / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism*
D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase; D011494:Protein Kinases