Placental alkaline phosphatase integrates via its carboxy-terminus into the microvillous membrane: its allotypes differ in conformation.

Human placental microvillous alkaline phosphatase (M-PLAP) was extracted from microvilli either by butanol extraction or subtilisin proteolysis. The data indicate that subtilisin cleavage of PLAP removes a membrane-binding domain of approximately 2000 molecular weight, leaving the catalytic site intact and the protein in solution. Sequencing studies on the N-terminal 13 amino acids of both the subtilisin-cleaved and uncleaved forms of M-PLAP indicate that the enzyme is anchored to the plasma membranes by its carboxy-terminus. The N-terminal 13 amino acids of A-PLAP were the same as those of M-PLAP. Trypsin solubilization failed to release M-PLAP from these membranes and it appears to cleave a portion of molecular weight of about 9K from the amino terminus, leaving an enzymatically active portion of PLAP associated with the membrane. On SDS gels, subtilisin-cleaved M-PLAP showed an apparent dimeric molecular size larger than that of the original uncleaved enzyme, presumably due to the generation of a less compact conformational state. On starch gels, cleaved M-PLAP showed a single zone of enzyme activity with a mobility sightly greater than that of A-PLAP, which did not require the presence of Triton X-100 to enter the gel. Variations in the apparent molecular sizes of the different allelic forms of PLAP were also observed.