Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum.
Enzymically active intestinal alkaline phosphatase exists in both soluble and membrane-bound forms in the suckling rat. Antiserum prepared against purified soluble alkaline phosphatase (anti-AlP) was shown to be monospecific when assessed by Ouchterlony double-diffusion analysis and immunoelectrophoresis. The two forms of alkaline phosphatase were antigenically identical and possessed similar affinities for anti-AlP. To study the biosynthesis of the two forms, 14-day-old rats were injected intraperitoneally with [(3)H]leucine. The labelling kinetics of alkaline phosphatase, extracted from supernatant and brush-border membrane fractions with anti-AlP, was followed over 20h. Incorporation of [(3)H]leucine into membrane-bound alkaline phosphatase was rapid, reaching a plateau at 6h. The soluble enzyme showed slower incorporation of label and maximal radioactivity was not reached until 12h after labelling, a lag of 6h 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 labelling brush-border membrane proteins in vivo by intraluminal injection of diazotized [(125)I]iodosulphanilic acid was used. The appearance of (125)I in soluble and membrane alkaline phosphatase was then monitored over a 7h period, encompassing the lag between maximal leucine labelling of the two forms. The results failed to show either 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. Therefore there does not appear to be a serial precursor/product relationship between the soluble and membrane-bound forms of suckling-rat intestinal alkaline phosphatase.
D000469:Alkaline Phosphatase / Q000096:biosynthesis*; D000828:Animal Population Groups / Q000378:metabolism*; D000818:Animals; D000833:Animals, Suckling / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D003979:Diazonium Compounds / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007082:Ileum / Q000201:enzymology*; D007527:Isoenzymes / Q000096:biosynthesis*; D007930:Leucine / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D003902:Detergents; D003979:Diazonium Compounds; D007527:Isoenzymes; D011092:Polyethylene Glycols; C018909:diazoiodosulfanilic acid; D017830:Octoxynol; C010615:Nonidet P-40; D000469:Alkaline Phosphatase; D007930:Leucine