Microassay of phosphate provides a general method for measuring the activity of phosphatases using physiological, nonchromogenic substrates such as lysophosphatidic acid

Since measurement of lysophosphatidate phosphatase activity is important in studies of tumorigenesis, we attempted to develop a simpler alternative to the more complex methods currently available. Measuring the phosphate rele ased would permit use of the same method for a variety of phosphatases with physiological substrates, many of which are nonchromogenic. The Malachite green method of K. Itaya and M. Ui (1966, Clin. Chim. Acta 14, 361) has ade quate sensitivity for quantitating phosphatase activity in biological sampl es. In samples with high endogenous phosphate concentrations pretreatment w ith 50 mg Dowex 1 x 10 (100-200 mesh, OH- form) usually permitted reliable determination of phosphatase activity. For 34 consecutive runs the mean rel ative difference [(phosphorus activity - vitamer activity)/phosphorus activ ity] obtained from the simultaneous measurement of both the phosphate relea sed and the corresponding organic product (pyridoxal and pyridoxine) was -0 .03 +/- 0.09. The within run and between run coefficients of variation (thr ee runs of four to five replicates) were 0.05 and 0.04, respectively. Pyrid oxine 5'-phosphate hydrolase activity (pH 10) in cultured skin cells (norma l and cancerous) ranged from 2 to 12 nmol phosphorus/min. mg protein. Lysop hosphatidate phosphatase activity (pH 7.4) ranged from 3 to 14 nmol phospho rus/min. mg protein. The current approach permits the measurement of phosph atase activity with a single method using a variety of substrates and incub ation conditions. (C) 2001 Academic Press.