Effect of 1-p-bromotetramisole on phosphatases in neonatal hamster bone and tooth germs at different pH

This study was designed to compare the various phosphatases and pyrophosphatases in bone with those in developing teeth. Moreover the alkaline phosphatase inhibitor 1-p-bromotetramisole (1-pBTM) was assessed for its ability to discriminate between the several phosphatase activities. Enzyme activities were determined over the pH range 3.50 to 11.25 in homogenates of calcifying hamster tibiae, and whole tooth germs using p-nitrophenyl-phosphate (pNPP) and pyrophosphate (PPi) as substrates. Characterization of alkaline phosphatase inhibition with 1-pBTM showed that in tooth germs the pI50 values of 1-pBTM for whole tooth germs, ectodermal and mesenchymal phosphatases were identical (pI50 = 5.36), which was slightly but significantly lower than that for bone alkaline phosphatase (pI50 = 5.64). In bone, two pNPP-ase activities were found with optima at pH 5.5 and pH 10.3 respectively. The alkaline phosphatase activity was completely inhibited by 10(-4) M 1-pBTM. Inhibition of the acid phosphatase was incomplete. With PPi as substrate, three activities were found with optima at pH 4.8, 7 and 8.7 respectively. All these PPi-ase activities were strongly inhibited by 1-pBTM in developing teeth, only one phosphatase activity was found, which exhibited an alkaline pH (10.3) optimum with both substrates. This activity was inhibited by 1-pBTM with both substrates although the effect on pNPP-ase activity was more marked. From these results we conclude that in bone there are insufficient differences in the extent of inhibition by 1-pBTM to distinguish between the various phosphatase activities when more physiological substrates such as pyrophosphate (PPi) are used.(ABSTRACT TRUNCATED AT 250 WORDS)