Purification And Properties Of Alkaline Phosphatase From Bovine Mammary Tissue PDF Download

A general procedure for the purification of alkaline phosphatase from human tissues using affinity chromatography on a phosphonic acid- Sepharose derivative was developed. The enzymes from human liver, kidney, intestine, placenta and the serum of a patient with Paget's disease of bone were purified to apparent homogeneity as judged by several criteria. Some catalytic properties of alkaline phosphatase purified from human liver were studied. Under in vitro conditions the enzyme catalysed the hydrolysis of a number of phosphomonoesters which have been suggested to be possible physiological substrates. The V values for the hydrolysis of most substrates were very similar and the lowest K values were obtained at near-neutral pH. A calcium-sensitive ATPase activity could not be demonstrated, even in the presence of a calcium-dependent regulator protein. Ca and Mg ions abolished the ATPase and pyrophosphatase activities of liver alkaline phosphatase by formation of metal-substrate complexes. Phosphodiesters and phosphonic acids were not substrates of the enzyme although the latter compounds were inhibitors. The inhibition of liver alkaline phosphatase by phosphate (K^ = 35 yM) suggested that the enzyme activity in vivo may be largely inhibited. Vanadate was a potent competitive inhibitor of alkaline phosphatases from several sources (K_^ values were less than 1 yM) . The inhibition at physiological concentrations of vanadate, and the reversal of this inhibition by compounds such as L-epinephrine indicated that alkaline phosphatase may be regulated by vanadate. The removal of sialic acid residues from liver alkaline phosphatase by neuraminidase treatment caused the isoelectric point to change from 4.0 to 6.5, but had no influence on the specific activity of the enzyme, the values for six substrates, or the inhibition by L-homoarginine. The neuraminidase-treated enzyme was inactivated by heating at 56°C at the same rate as the native enzyme, but was inactivated by SDS more rapidly than the native enzyme. The amino acid compositions from 13 reports of alkaline phosphatase from mammalian and bacterial sources were compared. With the exception of the enzyme from human intestine, the results suggested that a high degree of compositional similarity was present. Apparent subunit molecular weight values, determined by SDS-PAGE for human alkaline phosphatases from liver, kidney, intestine, and Paget's serum were similar (92000-96000). The corresponding value for the enzyme from placenta w T as 74000. Maps of the radioiodinated peptides of alkaline phosphatase purified from human liver, kidney and Paget's serum were highly similar whereas the corresponding maps of the enzyme purified from intestine or placenta were different from each other and from the maps for the enzymes from the other tissues. These results and the data from heat inactivation and differential inhibition studies strongly suggest that three different structural genes code for the protein moiety of the alkaline phosphatase present in these tissues. The general purification protocol and the procedure for mapping the tryptic peptides from alkaline phosphatase radioiodinated within polyacrylamide gels appear to be useful for the characterization and structural identification of tumor-associated alkaline phosphatases.