Streptozotocin induced diabetes: Significant changes in the kinetic properties of the soluble form of rat bone alkaline phosphatase

A soluble form of an alkaline phosphatase, obtained from the osseous plate of streptozotocin-induced diabetic rats, was purified 90-fold with a yield of 26%. The calculated M-r of the purified enzyme was 80,000 by denaturing polyacrylamide gel electrophoresis and 160,000 by gel filtration on Sephacr yl S-300, suggesting a dimeric structure for its native form. In the absenc e of metal ions, the p-nitrophenylphosphatase activity of the purified enzy me was 4223.1 U/mg. Magnesium or calcium ion concentrations up to 2 mM incr eased the specific activity of the enzyme to 9896.5 and 10,796.2 U/mg, resp ectively. The enzyme was stimulated to a lesser extent by MnCl2 (5390.1 U/m g) and CoCl2 (5088.2 U/mg). The purified soluble alkaline phosphatase showe d a broad substrate specificity, and among the less hydrolyzed substrates w ere pyrophosphate (1517.6 U/mg) and bis-p-nitrophenylphosphate (499.6 U/mg) . The enzyme was relatively stable at 45 degrees for periods as long as 180 min, but was denatured rapidly above 50 degrees, following first order kin etics with T-1/2 values ranging from 3.5 to 57.7 min. The results reported herein suggested that the soluble form of alkaline phosphatase from strepto zotocin-induced diabetic rats had its kinetic properties altered, apparentl y as a consequence of changes in metal-binding properties. (C) 1999 Elsevie r Science Inc.