ALKALINE-PHOSPHATASE (EC 3.1.3.1) IN SERUM IS INHIBITED BY PHYSIOLOGICAL CONCENTRATIONS OF INORGANIC-PHOSPHATE

Natural and artificial manipulation of tissue nonspecific alkaline pho sphatase activity indicates that pyrophosphate, phosphoethanolamine, a nd pyridoxal 5'-phosphate are among the natural substrates for this en zyme. Although inorganic phosphate has been recognized as a competitiv e inhibitor of this enzyme for many years, the influence of phosphate on alkaline phosphatase activity in serum under physiological conditio ns has not been previously reported. We examined the kinetics of tissu e nonspecific alkaline phosphatase fi-om bovine kidney and sera from 4 9 patients with a wide range of endogenous phosphate concentrations us ing pyridoxine 5'-phosphate as a substrate at pH 7.4. For the bovine k idney enzyme, the K-m was 0.42 +/- 0.04 mu mol/L, and the K-i for phos phate was 2.4 +/- 0.2 mu mol/L. Analysis of the kinetics using pyridox ine 5'-phosphate in undiluted serum from 10 subjects with phosphorus r anging from 0.5-2.1 mmol/L and alkaline phosphatase activity ranging f rom 41-165 nmol/min.mL gave estimates for the K-m of 56 +/- 11 mu mol/ L and for the K-i of 540 +/- 82 mu mol/L for phosphate. This indicates that under physiological conditions alkaline phosphatase activity tow ard pyridoxine 5'-phosphate is reduced approximately 50% by the normal phosphate concentration and that it will increase or decrease signifi cantly in response to changes in phosphate concentration within the ra nges observed clinically.