Purification and analysis of the microvillous (M) and A forms of placental alkaline phosphatase.
Microvillous M-PAP and A-PAP were purified to homogeneity from butanol extracts of syncytiotrophoblastic microvillous membranes using DEAE Sepharose CL-6B chromatography. The two isoenzymes had indistinguishable pH optima and Km values. Under reducing conditions in SDS-polyacrylamide gels, both forms showed a similar subunit molecular weight of 69 000. In the absence of reduction M-PAP was present in a dimeric form similar to A-PAP. However, the mobility of the dimer of M-PAP corresponded to a molecular weight of 116 000 compared with 130 000 for A-PAP. It was concluded that this difference is due to the A and M dimers being in different conformations. In SDS-polyacrylamide gels the M form also showed a higher molecular weight species corresponding to a mobility of 180 000, but species corresponding to the native molecular weight of 725 000 wer not found. Under different alkylating conditions the polypeptide subunits of both forms showed similar isoelectric banding patterns. The M-PAP showed some extra bands with a relatively high pI value. The hydrophobicity of the different forms of PAP was investigated by testing their affinities for columns of various hydrocarbon-coated agaroses. Unalkylated agarose did not bind the enzyme; C6 alkyl chains bound A, B and M-PAP. C2 alkyl chains bound M-PAP, but not A or B-PAP. This provided a potentially new and simple method of purifying M-PAP, and supports the view that M-PAP is hydrophobic relative to the A and B forms. The structural implications of this hydrophobicity are discussed.
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D010920:Placenta / Q000648:ultrastructure*; D011247:Pregnancy
D007527:Isoenzymes; D000469:Alkaline Phosphatase