A microfluorometric method for alkaline phosphatase: Application to the various segments of the nephron

A microtechnique has been developed for the measurement of alkaline phosphatase in minute amounts of renal tissue. This microtechnique utilizes the known fluorescent property of 4-methylumbelliferyl phosphate following enzymatic hydrolysis. The reaction is sensitive and reproducible and is inhibited by l-bromotetramisole, a specific alkaline phosphatase inhibitor. The microdetermination of alkaline phosphatase activity in the various segments of the mouse nephron allowed the localization of the enzyme in the glomeruli, and in the proximal convoluted tubule where the activity progressively decreases from the capsule of Bowman to the more distal segments. The enzyme was absent from the pars recta or S3 and from the rest of the nephron. This technique is applicable to very small amounts (0.1 μg of protein) of any tissue containing alkaline phosphatase.