Effects of various salts on the steady-state enzymatic activity of E. coli alkaline phosphatase.
Seventeen salts were tested at various concentrations for their effects on E. coli alkaline phosphatase steady-state activity. Three effects were distinguished: a general ionic strength effect, and weaker cation and anion effects. 1. All salts tested, including those with "noninteracting" cations and anions, stimulate alkaline phosphatase activity usually ca. 100% at moderate (0.05-0.3 M) concentrations. 2. Cations such as Na+ and Li+ produce further increases in activity at concentrations up to 1 M. 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" effectiveness such as thiocyanate also reduce activity at ca. 1 M concentrations. 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 the enzyme so there is no general effect of 1 M salts on the conformation of the protein. The Michaelis constant for the substrate, p-nitrophenylphosphate, and inhibition constant for inorganic phosphate are increased to some extent by salts, but the increase in activity is due to an increase in Vmax. Our working hypothesis is that increased ionic strength weakens electrostatic interactions, enabling noncovalently bound phosphate to dissociate more rapidly.
Poe
R W
RW
Department of Biochemistry, University of Georgia, Athens 30602.
Sangadala
V S
VS
Brewer
J M
JM
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000838:Anions; D002412:Cations; D002704:Chlorates / Q000494:pharmacology; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D008094:Lithium / Q000494:pharmacology; D009994:Osmolar Concentration; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012492:Salts / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013447:Sulfites / Q000494:pharmacology
D000838:Anions; D002412:Cations; D002704:Chlorates; D000644:Quaternary Ammonium Compounds; D012492:Salts; D013447:Sulfites; D012965:Sodium Chloride; D008094:Lithium; D012964:Sodium; D000469:Alkaline Phosphatase; C027917:tetramethylammonium; C032706:sodium chlorate; C025026:sodium sulfite