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Placental alkaline phosphatase integrates via its carboxy-terminus into the microvillous membrane: its allotypes
Abstract:
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.
Insights
Human placental microvillous alkaline phosphatase (M-PLAP) is anchored to membranes by its carboxy-terminus. Subtilisin cleavage releases the enzyme, yielding a soluble, active form for further study.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Human placental microvillous alkaline phosphatase (M-PLAP) is an important membrane-bound enzyme.
- Understanding its membrane anchoring and structure is crucial for biochemical analysis.
Purpose of the Study:
- To investigate the membrane-binding domain and anchoring mechanism of M-PLAP.
- To characterize the effects of enzymatic cleavage on M-PLAP structure and activity.
Main Methods:
- Extraction of M-PLAP using butanol or subtilisin proteolysis.
- N-terminal sequencing of M-PLAP variants.
- Analysis of enzyme activity and molecular size using SDS-PAGE and starch gel electrophoresis.
Main Results:
- Subtilisin cleavage removes a ~2000 MW membrane-binding domain, leaving the catalytic site intact and soluble.
- M-PLAP is anchored via its carboxy-terminus, with the N-terminus exposed.
- Trypsin cleavage at the N-terminus also yields an active, membrane-associated fragment.
- Subtilisin-cleaved M-PLAP exhibits altered conformation and increased mobility on starch gels.
Conclusions:
- M-PLAP is anchored to the plasma membrane by its C-terminus.
- Enzymatic cleavage can yield soluble, active forms of M-PLAP, facilitating biochemical studies.
- Different cleavage sites and allelic variations influence M-PLAP's molecular size and properties.