Purification and analysis of the microvillous (M) and A forms of placental alkaline phosphatase
Related Experiment Videos
Abstract:
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.
Insights
This study purified microvillous alkaline phosphatases (M-PAP and A-PAP), revealing distinct structural differences and hydrophobicity. M-PAP exhibits unique hydrophobic properties, offering a novel purification method and insights into enzyme structure.
Area of Science:
- Biochemistry
- Enzymology
- Membrane Protein Purification
Background:
- Alkaline phosphatases (AP) exist in various forms, including microvillous alkaline phosphatase (M-PAP) and placental alkaline phosphatase (A-PAP).
- Understanding the structural and biochemical differences between these isoenzymes is crucial for their characterization and potential applications.
Purpose of the Study:
- To purify and characterize microvillous M-PAP and compare its properties with A-PAP.
- To investigate the structural differences, including molecular weight, conformation, and hydrophobicity, between M-PAP and A-PAP.
Main Methods:
- Purification of M-PAP and A-PAP from syncytiotrophoblastic microvillous membranes using DEAE Sepharose CL-6B chromatography.
- Analysis of enzyme properties including pH optima, Km values, subunit molecular weight (SDS-PAGE), and oligomeric states.
- Investigation of hydrophobicity using affinity chromatography on hydrocarbon-coated agaroses.
Main Results:
- Both M-PAP and A-PAP showed similar pH optima and Km values, with subunit molecular weights of 69,000 under reducing conditions.
- Dimeric forms differed in molecular weight (M-PAP: 116,000; A-PAP: 130,000), suggesting conformational variations.
- M-PAP demonstrated unique binding to C2 alkyl chains, indicating higher hydrophobicity compared to A-PAP and B-PAP.
Conclusions:
- M-PAP and A-PAP are distinct isoenzymes with differing dimeric conformations and hydrophobicity.
- The differential hydrophobicity of M-PAP provides a novel and simple purification strategy.
- These findings contribute to understanding the structural diversity and properties of microvillous alkaline phosphatases.