title: A novel assay method for calcium calmodulin dependent phosphatase from bovine brain extract.
abstract: Calcium calmodulin dependent protein ser/thr phosphatase, also referred to as protein phosphatase 2B (PP2B), is rich in neural tissue, and plays an important role in the overall function of the nervous system. Routinely phosphatase assay employs, para-Nitrophenlylphosphate (p-NPP), as a substrate, is also extended to assay PP2B. However, in the present study, the differential spectral characterstic property of tyrosine and phopshotyrosine has been exploited to employ the latter as a candidate substrate for the PP2B assay. The specific activity of PP2B using phosphortyrosine in bovine Bos Taurus indicus brain extract (Bos Taurus indicus), was measured in presence of different metal ions like Ca(2+), Mn(2+) and Mg(2+). Further modulators like dithiothreitol (DTT), calmodulin (CaM) and metal chelators such as EGTA and EDTA were applied to confirm the role of divalent cations and to determine calcium calmodulin dependent phoshphatase activity. PP2B activity was higher with phosphotyrosine in presence of Ca(2+) than with p-NPP. Further experiments, involving calmodulin as a modulator, confirmed phosphotyrosine as a better substrate over p-NPP. Calmodulin further enhanced the effect of phosphotyrosine as a potential substrate confirming calcium calmodulin dependent phosphatase activity. Phosphotyrosine is proposed as a better substrate in assaying calcium dependent phosphatase activity when compared to para-nitrophenylphosphate.
authors.affiliation: Department of Neurochemistry, National Institute of Mental Health and Neurosciences, Bangalore 560 029, India. ksdevaraju@bub.ernet.in
authors.forename: K S
authors.initials: KS
authors.lastname: Devaraju
authors.affiliation: Department of Zoology, Maharani's Science College for Women, Bangalore 560 056, India.
authors.forename: B S Mohan
authors.initials: BS
authors.lastname: Kumar
authors.affiliation: Department of Clinical Research, Padmashree Group of Institutions, Bangalore 560 072, India.
authors.forename: S V
authors.initials: SV
authors.lastname: Sureshbabu
authors.affiliation: Department of Biotechnology, Bangalore University, Bangalore 560 056, India.
authors.forename: A
authors.initials: A
authors.lastname: Gopi
authors.affiliation: Department of Biotechnology, Bangalore University, Bangalore 560 056, India.
authors.forename: R
authors.initials: R
authors.lastname: Saraswathi
authors.affiliation: Department of Biotechnology, Bangalore University, Bangalore 560 056, India.
authors.forename: B M
authors.initials: BM
authors.lastname: Harish
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D019703:Calcineurin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D019000:Phosphotyrosine / Q000737:chemistry; D014020:Tissue Extracts / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D002147:Calmodulin; D014020:Tissue Extracts; D019000:Phosphotyrosine; D014443:Tyrosine; D019703:Calcineurin; D002118:Calcium