PHOSPHODIESTERASE ACTIVITY IS A NOVEL PROPERTY OF ALKALINE-PHOSPHATASE FROM OSSEOUS PLATE

Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K (0.5)) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics with a K-0.5 of 500 mu M. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 mu M, and K-0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K-0.5 = 300 mu M for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K-0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and K-1 values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K-0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pK(a) values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in understanding the mineralization process.