Purification and properties of molecular-weight variants of human placental alkaline phosphatase.
1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8.6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4.2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined.
D000418:Albumins / Q000032:analysis; D000469:Alkaline Phosphatase / Q000032:analysis / Q000037:antagonists & inhibitors; D000596:Amino Acids / Q000032:analysis; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D003956:Dialysis; D004586:Electrophoresis; D005260:Female; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D014461:Ultracentrifugation
D000418:Albumins; D000596:Amino Acids; D000469:Alkaline Phosphatase