EFFECTS OF VARIOUS SALTS ON THE STEADY-STATE ENZYMATIC-ACTIVITY OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE

Seventeen salts were tested at various concentrations for their effect s on E. coli alkaline phosphatase steady-state activity. Three effects were distinguished: a general ionic strength effect, and weaker catio n and anion effects. 1. All salts tested, including those with ''nonin teracting'' cations and anions, stimulate alkaline phosphatase activit y usually ca. 100% at moderate (0.05-0.3 M) concentrations. 2. Cations such as Na+ and Li+ produce further increases in activity at concentr ations up to 1 N4. The noninteracting cations tetramethylammonium and tetrapropylammonium produce lower activities at these concentrations. These do not provide the secondary stimulatory effect of cations such as Na+ or Li+. 3. Anions associated with greater ''salting in'' effect iveness such as thiocyanate also reduce activity at ca. 1 M concentrat ions. These latter effects are not dependent on protein concentration so they probably do not involve subunit dissociation. There is little effect on the fluorescence or fluorescence-polarization spectrum of th e enzyme so there is no general effect of 1 M salts on the conformatio n of the protein. The Michaelis constant for the substrate, p-nitrophe nylphosphate, and inhibition constant for inorganic phosphate are incr eased to some extent by salts, but the increase in activity is due to an increase in V(max). Our working hypothesis is that increased ionic strength weakens electrostatic interactions, enabling noncovalently bo und phosphate to dissociate more rapidly.