Alkaline phosphatase from rat osseous plates: purification and biochemical characterization of a soluble form

• Published in: Biochimica et biophysica acta Vol. 1074; no. 2; pp. 256 - 262
• Main Authors: Say, JoséC., Ciuffi, Katia, Furriel, Rosa P.M., Ciancaglini, Pietro, Leone, Francisco A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 08.07.1991; Elsevier
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - chemistry; Alkaline Phosphatase - isolation & purification; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Bone Matrix; Chromatography, Gel; Divalent ion; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Growth Plate - enzymology; Hydrolases; Hydrophobic chromatography; Kinetics; Molecular Weight; Nitrophenols - pharmacology; Organophosphorus Compounds - pharmacology; Osseous matrix; Osteogenesis; p-Nitrophenylphosphate; Rat; Rats; Solubility; Substrate Specificity; Alkaline phosphatase; Rat; Osseous matrix; Hydrophobic chromatography; Divalent ion; AMPOL; PNPP; PNPPase; BIS; p-Nitrophenylphosphate; Specific activity; Vertebrata; Mammalia; Purification; Molecular weight determination; Enzyme; Rodentia; Soluble form; Bone matrix; Ossification
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(91)90161-9

A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of M r 80 000 on SDS-PAGE and an apparent molecular weight of 163 000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl 2 was 19 027 U/mg and the hydrolysis of p-nitrophenyl phosphate ( K 0.5 = 92 μM) showed positive cooperativity ( n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis( p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20–25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.