Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum

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Abstract
Enzymically active intestinal alkaline phosphatase exists in soluble and membrane-bound forms in the suckling rat. Antiserum prepared against purified soluble alkaline phosphatase (anti-A1P) was monospecific when assessed by Ouchterlony double-diffusion analysis and immunoelectrophoresis. The 2 forms of alkaline phosphatase were antigenically identical and possessed similar affinities for anti-A1P. To study the biosynthesis of the 2 forms, 14-day-old rats were injected i.p. with [3H]leucine. The labeling kinetics of alkaline phosphatase, extracted from supernatant and brush-border membrane fractions with anti-A1P, was followed over 20 h. Incorporation of [3H]leucine into membrane-bound alkaline phosphatase was rapid, reaching a plateau at 6 h. The soluble enzyme showed slower incorporation of label and maximal radioactivity was not reached until 12 h labeling, a lag of 6 h behind the membrane-bound enzyme. Soluble alkaline phosphatase could not have been a precursor of the membrane form, as there was no early peak of radioactivity in the soluble form. To determine if the soluble enzyme was irreversibly derived from the membrane enzyme, a newly developed technique of labeling brush-border membrane proteins in vivo by intraluminal injection of diazotized [125I]iodosulfanilic acid was used. The appearance of 125I in soluble and membrane alkaline phosphatase was then monitored over a 7 h period, encompassing the lag between maximal leucine labeling of the 2 forms. The results failed to show a proportional transfer of radioactivity from membrane to soluble alkaline phosphatase or an absolute increase in radioactivity of the soluble form during degradation of brush-border alkaline phosphatase. Thus there does not appear to be a serial precursor/product relationship between the soluble and membrane-bound forms of suckling-rat intestinal alkaline phosphatase.

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