Purification et propriétés de la phosphatase alcaline du cerveau de bœuf

Abstract Purification and properties of alkaline phosphatase from beef brain 1. 1.|Alkaline phosphatase from beef brain (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) has been purified over 1840-fold by butanol extraction, acetone and (NH 4 ) 2 SO 4 precipitation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. Two active peaks were separated; one of these peaks contains 2–3% of the total activity, the other fraction is homogeneous on cellulose acetate and polyacrylamide gel electrophoresis. 2. 2.|The molecular weight based on elution volume is estimated to be about 190 000. 3. 3.|This enzyme is probably a Zn 2+ metalloenzyme and is activated by Mg 2+ . The following monoesters are hydrolysed: p -nitrophenylphosphate, phenylphosphate, β-glycerophosphate, dl -phosphoserine, O -phosphorylethanolamine. The K m values have been determined. Pyrixodal 5-phosphate is very slightly hydrolysed. The optimum pH is 10.2, using p -nitrophenyl phosphate as substrate in carbonate buffer. 4. 4.|The beef-brain alkaline phosphatase has also an inorganic pyrophosphatase activity, which is optimum in Tris-HCl buffer (pH 8.8) and inhibited by excess Mg 2+ and substrate. 5. 5.|A study of inhibition of these two activities has established some differences. The pyrophosphatase activity is more sensitive to the action of EDTA and is recovered more slightly by metal ions. However, our data are consistent with previous results on alkaline phosphatases from different sources in which the phosphatase and pyrophosphatase activities has been shown to be located on the same site. The role of active groups is discussed. 6. 6.|Phosphotransferase activity has been found in a medium containing p -nitro-phenylphosphate (donor) and Tris or ethanolamine (acceptor). The percentage of phosphate transfered is 50%.