Studies on alkaline phosphatases. 1. Kinetics of plasma phosphatase of normal and rachitic chicks

Abstract

The optimum pH for the hydrolysis of [beta]-glycerophosphate and phenyl phosphate by fowl plasma phosphatase was found to change to higher values with increases in the initial concentration of substrate. The pH optima for various concentrations of phenyl phosphate were higher than the optima for the corresponding concentrations of [beta]-glycerophosphate. On the acid side of pH optima the rate of hydrolysis with dilute substrate was greater than with substrate present in high concentrations. At relatively high pH values, hydroxyl ions inhibited the hydrolysis of dilute glycerophosphate in a manner which suggested competitive inhibition. When velocities were measured at pH 9. 88 for all dilutions of glycerophosphate, Km decreased with increases in the amount of enzyme in the digest, i. e. Km varied with variations in the enzyme: inhibitor ratio. Under similar conditions of pH, Km for plasma of rachitic chicks, having a high phosphatase activity, was lower than K for the plasma of chicks fed with vitamin D, possessing low phosphatase activity. When velocities were measured at the optimum pH for each concentration of substrate, variations in Km due to differences in enzyme concentration were greatly reduced. The 1/v: 1/[S] plots of velocities obtained in this manner also resolved the data into two parts from which 2. 2 values for Km were calculated for each preparation of enzyme, namely Km1 for data with dilute substrate and Km2 for data with high concentrations of substrate. The presence or absence of vitamin D in the diet of chicks had no effect on Km1 and Km2 for plasma phosphatase when the velocities were measured at the optimum pH for each concentration of substrate. When velocities were measured at the same pH for all concentrations of phenyl phosphate, Km values for fowl plasma phosphatase increased with increasing alkalinity from 0. 15 m[image] at pH 8. 9 to 8. 3 m[image] at pH 10. 3. Km values, plotted as pKm versus pH, fell along a straight line with a slope of -1. 3 when the velocities were measured in the presence of optimum amounts of magnesium chloride in the digest. The effect of activators on the relationship between pH and Km is discussed.