Microassay of phosphate provides a general method for measuring the activity of phosphatases using physiological, nonchromogenic substrates such as lysophosphatidic acid
Jd. Mahuren et al., Microassay of phosphate provides a general method for measuring the activity of phosphatases using physiological, nonchromogenic substrates such as lysophosphatidic acid, ANALYT BIOC, 298(2), 2001, pp. 241-245
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.