Reet Toomik

Characterization of protein kinase C-mediated phosphorylation of the short cytoplasmic domain isoform of C-CAM

C‐CAM is a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family. Two co‐expressed isoforms, C‐CAM‐L and C‐CAM‐S, are known, having different cytoplasmic domains both of which can be phosphorylated in vivo. Here we have characterized the PKC‐mediated phosphorylation of the short cytoplasmic domain isoform, C‐CAM‐S. Phorbol myristyl acetate induced phosphorylation of C‐CAM‐S in transfected CHO cells. Using synthetic peptides and Edman degradation we identified Ser449 as the PKC‐phosphorylated amino acid residue. Binding experiments with modified peptides indicated that this phosphorylation decreases the ability of the cytoplasmic domain of C‐CAM‐S to bind calmodulin.

A potential pitfall in protein kinase assay: phosphocellulose paper as an unreliable adsorbent of produced phosphopeptides.

The ability of phosphocellulose paper to retain phosphorylated peptides containing basic amino acid residues was investigated. Some peptide substrates that are commonly used for three different protein kinases were tested. The adsorption onto phosphocellulose paper was strongly dependent on the amino acid composition of the peptides. None of the phosphopeptides studied was adsorbed completely, the amount bound varied from 7 to 93%. Phosphopeptides containing two basic amino acids each differed remarkably in the degree of binding to the phosphocellulose paper (40% RRASVA, 60% FRRLSI, and 80% HRASV was bound). The results presented here indicate that data from phosphorylation experiments obtained so far for different peptides using the phosphocellulose paper method should be judged with caution.

Preparation of ferric adsorbent paper and its interaction with phosphate-containing biomolecules

A procedure for the synthesis of a selective adsorbent for phosphate-containing biomolecules is described. The sorbent is based on Whatman chromatography paper, which is activated with epichlorohydrine, followed by the coupling of iminodiacetic acid to the active surface of the sorbent. The immobilized complex-forming chelating groups are saturated with ferric ions. The synthesized adsorbent is a counterpart to Chelating Sepharose and makes it possible to extend the use of immobilized ferric chelating groups for analytical purposes. It displays a high affinity towards compounds containing free terminal phosphate groups (phosphopeptides, nucleotides). The results of the binding experiments are compared to the corresponding data obtained with Chelating Sepharose gels.

Erythrocytic protein kinase C activity in primary hypertension

Ek P, Toomik R, Eriksson S, Frithz G, Ronquist G, Engström L (University of Uppsala and University Hospital, Uppsala; and Central Hospital, Eskilstuna, Sweden). Erythrocytic protein kinase C activity in primary hypertension. J Intern Med 1998; 243: 299–305.