title: In vitro phosphorylation of phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum.
abstract: Previously, we described two distinct classes of phosphoenolpyruvate carboxylase (PEPC) isoforms in the green alga Selenastrum minutum. Class 1 PEPC (PEPC1) is a homotetramer composed of 102 kDa subunits (p102), whereas Class 2 PEPCs exist as three large protein complexes (PEPC2-PEPC4) containing varying proportions of structurally dissimilar p102 and 130 kDa (p130) PEPC catalytic subunits. In the current study, a p102 calcium-independent protein kinase was shown to co-purify with PEPC1, but not PEPC2. However, the p130 subunit of PEPC2 was phosphorylated in vitro during its incubation in the presence of [gamma-(32)P]ATP and a clarified algal extract. Treatment of purified PEPC2 with protein phosphatase 2A(2) increased its apparent M(r) as judged by Superose 6 gel filtration chromatography. The presence of the protein phosphatase inhibitors NaF and microcystin-LR throughout PEPC purification significantly influenced the activity and structural organization of Class 2, but not Class 1, PEPC isoforms. The results are consistent with the notion that under the culture conditions employed: (i) Class 1 and Class 2 PEPC isoforms exist in vivo mainly in their dephosphorylated and phosphorylated forms, respectively, and (ii) phosphorylation of Class 2 PEPCs leads to a significant reduction in their activity and native M(r). We propose that protein kinase-mediated phosphorylation is involved in the control and structural organization of green algal PEPC.
authors.affiliation: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada. rivoalj@ms.umanitoba.ca
authors.forename: Jean
authors.initials: J
authors.lastname: Rivoal
authors.forename: David H
authors.initials: DH
authors.lastname: Turpin
authors.forename: William C
authors.initials: WC
authors.lastname: Plaxton
mesh_terms: D000460:Chlorophyta / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic / Q000494:pharmacology; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology
chemical_list: D007527:Isoenzymes; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D012969:Sodium Fluoride; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases; D010730:Phosphoenolpyruvate Carboxylase; C057862:cyanoginosin LR