Characterization of protein kinase C and its isoforms in human T lymphocytes.
Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function.
Beyers
A D
AD
Department of Medical Physiology, Stellenbosch Medical School, Tygerberg, Republic of South Africa.
Hanekom
C
C
Rheeder
A
A
Strachan
A F
AF
Wooten
M W
MW
Nel
A E
AE
D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D017830:Octoxynol; D011092:Polyethylene Glycols; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology*
D004075:Diglycerides; D007527:Isoenzymes; D011092:Polyethylene Glycols; D017830:Octoxynol; D011493:Protein Kinase C; C013965:diolein