Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa |
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Authors: | Rita Maria Ulloa Hector Norberto Torres Claudia M. Ochatt Maria Teresa Téllez-Iñón |
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Affiliation: | (1) Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Argentina;(2) Facultad de Ciencias Exactas y Naturales Obligado 2490, 1428 Buenos Aires, Argentina |
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Abstract: | DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca2+ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca2+ and 1 µg/ml of the modulator. The stimulatory effect of the Ca2+-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca2+-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme. |
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Keywords: | Ca2+ calmodulin kinase Neurospora crassa autophosphorylation |
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