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 Mr of the purified enzyme was 80,000 by denaturing polyacrylamide gel electrophoresis and 160,000 by gel filtration on Sephacryl S-300, suggesting a dimeric structure for its native form. In the absence of metal ions, the p-nitrophenylphosphatase activity of the purified enzyme was 4223.1 U/mg. Magnesium or calcium ion concentrations up to 2 mM increased the specific activity of the enzyme to 9896.5 and 10,796.2 U/mg, respectively. The enzyme was stimulated to a lesser extent by MnCl2 (5390.1 U/mg) and CoCl2 (5088.2 U/mg). The purified soluble alkaline phosphatase showed a broad substrate specificity, and among the less hydrolyzed substrates were 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 kinetics with T1/2 values ranging from 3.5 to 57.7 min. The results reported herein suggested that the soluble form of alkaline phosphatase from streptozotocin-induced diabetic rats had its kinetic properties altered, apparently as a consequence of changes in metal-binding properties.
Fernandes
S S
SS
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto/Universidade São Paulo, Brasil.
Furriel
R P
RP
Petenusci
S O
SO
Leone
F A
FA
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001842:Bone and Bones / Q000201:enzymology* / Q000502:physiology; D002113:Calcification, Physiologic; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D013311:Streptozocin
D013311:Streptozocin; D000469:Alkaline Phosphatase