(Na+ + K+)-activated adenosinetriphosphatase: fluorimetric determination of the associated K+ -dependent 3-O-methylfluorescein phosphatase and its use for the assay of enzyme samples with low activities.

Data are presented which prove that 3-O-methylfluorescein phosphate is a substrate for the K⁺-dependent phosphatase that is associated with Na⁺,K⁺-ATPase. Conditions for the continuous fluorimetric assay of 3-O-methylfluorescein phosphatase are described. Enzyme preparations from three different tissues with widely different specific activities exhibit similar K_m values for 3-O-methylfluorescein phosphate. Correlation between Na⁺,K⁺-ATPase activity and K⁺-dependent 3-O-methylfluorescein phosphatase activity is demonstrated in several partially purified enzyme preparations and crude tissue fractions. When the K⁺-dependent 3-O-methylfluorescein phosphatase of a crude rat-brain homogenate is assayed, the activity is a linear function of the amount of homogenate added to the assay mixture. The equivalent of 10 μg of brain tissue may be assayed under the conditions used. The potential value of this highly sensitive fluorimetric method for the assay of enzyme in small samples of various tissues is suggested.