A highly conserved amino acid sequence 442GDASE446 in the ATP binding pocket of rat Na/K-ATPase was mutated, and the resulting proteins, G442A, G442P, D443A, S445A, and E446A, were expressed in HeLa cells to investigate the effect of individual ligands on Na/K-ATPase. The apparent Km for the high and low affinity ATP effects was estimated by ATP concentration dependence for the formation of the...

The use of protein phosphatase inhibitors has been instrumental in defining the intracellular roles of protein phosphatase 1 (PP1), PP2A and PP2B. Identification of the role of PP2C in vivo has been hampered, in part, by the unavailability of specific inhibitors. In order to facilitate the identification of novel and specific inhibitors of PP2C by random screening of compounds, and to further c...

The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The p...

PPM [metal-dependent protein phosphatase, formerly called PP2C (protein phosphatase 2C)] family members play essential roles in regulating a variety of signalling pathways. While searching for protein phosphatase(s) that act on AMPK (AMP-activated protein kinase), we found that PPM1A and PPM1B are N-myristoylated and that this modification is essential for their ability to dephosphorylate the α...

The interaction of Cu2+ with enzymatic activity of rabbit kidney Na+/K(+)-ATPase was studied in media with buffered, defined free Cu2+ levels. The IC50-values are 0.1 mumol/l for Na+/K(+)-ATPase and 1 mumol/l for K(+)-pNPPase. Dithiothreitol (DTT) reverses the inhibitory effect of Cu2+ in vitro. Cu2+ exerts non-competitive effects on the enzyme with respect to Na+, K+, ATP or pNPP, but has a mi...

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the aci...

An alkaline phosphatase (A LPase) from Saccharomyces cerevisiae strain 257 was purified 345-fold with specific activity of 54 533 nmol x min“ 1 x mg protein-1 . It was shown to be a dimeric protein (apparent mol. wt. approx. 130 kDa) with optimum activity at pH 8.6 8.8 and good stability at 50 °C. The A LPase was a non-specific enzyme hydrolyzing a wide vari­ ety of monophosphate esters. The en...