Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.
Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.
Gómez
M L
ML
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI) and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Ochatt
C M
CM
Kazanietz
M G
MG
Torres
H N
HN
Téllez-Iñón
M T
MT
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D017886:Durapatite; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D010703:Phorbol Esters / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000254:growth & development
D007527:Isoenzymes; D010703:Phorbol Esters; D017886:Durapatite; D011493:Protein Kinase C