Twentyfold increase in alkaline phosphatase activity by sequential reversible activation of the enzyme followed by coupling with a copolymer of ethylene and maleic anhydride.
Alkaline phosphatase, APase, (EC 3.1.31) from calf intestine, after shifting the equilibrium by effector molecules towards the dimeric form of the enzyme, was coupled (ratio 1:2, protein: copolymer) to a copolymer of ethylene and maleic anhydride, EMA. The water-soluble APase-EMA was separated from APase and the unbound EMA by DEAE-cellulose ion exchange chromatography. The specific activity of the APase-EMA, compared to APase, increased 26-fold at pH 7.1 and 10-fold at pH 8.6. The pH optimum of APase-EMA was shifted down from pH 9.5 (native APase) to 8.6. This change could be interpreted in terms of polyelectrolyte theory. APase-EMA retained 50-70% of its optimum activity in the pH range 7-8, while APase retained only 5-15% of its optimum activity within the same pH range. Its isoelectric point, pI, was 4.2 (APase 6.0) and it migrated on polyacrylamide gel electrophoresis in a single band, anodic movement twice as fast as APase. Parallel with the kinetic measurements, the reactive-enzyme sedimentation method was used to measure S20,w values. S20,w values obtained for APase-EMA, activated APase, and APase dialyzed against water were 6.56S, 6.46S, and 5.17S, respectively. Molecular weights, Mr, were determined by equilibrium sedimentation: the values obtained were 180,000, 160,000, and 84,500. Mr values of APase-EMA and APase (native) estimated by Sepharose-4B gel filtrations were essentially the same. The above-mentioned values remained unchanged for APase-EMA after intensive dialysis against water, whereas for the activated APase, separation from the effector molecules caused the equilibrium to shift back to the monomeric, very slightly active enzyme with concomitant changes of S20,w to 5.15 and Mr to 82,000.
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006358:Hot Temperature; D007422:Intestines / Q000201:enzymology; D007525:Isoelectric Focusing; D008298:Maleates; D008970:Molecular Weight; D010649:Phenylalanine / Q000494:pharmacology; D011095:Polyethylenes; D014461:Ultracentrifugation
D008298:Maleates; D011095:Polyethylenes; D010649:Phenylalanine; C007668:ethylene-maleic anhydride copolymer; D000469:Alkaline Phosphatase