Partial purification and some properties of human liver alkaline phosphatase.
1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.
D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D006801:Humans; D007422:Intestines / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D009439:Neuraminidase / Q000494:pharmacology; D009596:Nitrophenols / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011092:Polyethylene Glycols; D012794:Sialic Acids / Q000032:analysis; D015032:Zinc / Q000494:pharmacology
D000596:Amino Acids; D009596:Nitrophenols; D010710:Phosphates; D012794:Sialic Acids; D011092:Polyethylene Glycols; D000469:Alkaline Phosphatase; D009439:Neuraminidase; D008274:Magnesium; D015032:Zinc