Characterization and comparison of soluble and membranous forms of intestinal alkaline phosphatase from the suckling rat.

In the suckling rat intestine, alkaline phosphatase is present in boths oluble and membranous forms. These forms were purified from the 105,000 X g ileal supernatant and jejunal particulate fractions, respectively. Protease inhibitorsa dded to theh omogenate buffer did not significantly alter the yield of soluble and membranous enzyme. Moreover, cytoplasmic fractions from suckling rat ileum containing active lysosomal enzymes degraded rather than solubilized the membranous form. The two forms were found to be kinetically similar and immunologically identical. The soluble form had M, = 108,000 in nondenaturing sodium dodecyl sulfate gels and a single subunit in denaturing sodium dodecyl sulfate gels with M, = 47,000. Comparable values for the membranous forms were M, = 130,000 and 66,000. With Triton polyacrylamide gel electrophoresis,
the RF of the soluble form was 0.48, faster than the membranous form (RF 0.20). These values were identical for crude and purified enzymes. Analysis of carbohydrate content revealed that the soluble form contained more fucose (20 versus 8 mol of sugar/g of protein X lo-’) and N-acetylgalactosamine (32 versus 14 mol of sugar/g of protein X lo-‘) than did the membranous form. The membranous enzyme displayed strong lipid affinity by ready incorporation into liposomes, while the soluble form failed to be included. The two enzyme forms had different patterns of molecular heterogeneity by isoelectric focusing, with bands of activity at pH 4.97 and 5.06 for the soluble form and 4.33, 4.43, and 4.60 for the membranous enzyme. The papainsolubilized membranous enzyme migrated similarly to the soluble form on Triton- and sodium dodecyl sulfate
gel-electrophoresis but reproduced the pattern of the membranous form on isoelectric focusing. From these studies, we conclude that: (a) the soluble and membranous forms of intestinal alkaline phosphatase are closely related butd istinct isozymes; and (b)t he soluble form is not simply a proteolytically solubilized product of the membranous enzyme.