Summary
Ca2+-ATPase activity was solubilized, partly purified, and separated from nonspecific alkaline phosphatase activity (APase) of dentinogenically active rat incisor odontoblasts. Attempts were made to extract the enzymes by various agents, such as Triton X-100, deoxycholate, butanol, EDTA, and buffers of decreasing ionic strength. Solubilization by butanol followed by extraction with low concentrations of EDTA proved to be most effective. Purification and separation were done by molecular sieve chromatography.
Ca2+-ATPase showed no activity againstp-nitrophenyl phosphate (p-NPP) or inorganic pyrophosphate (PPi) and was unaffected by R 8231 [(±)-6(m-bromophenyl)-5,6-dihydroimidazo(2,1-b)thiazole oxalate]. It was activated by Ca2+ and Mg2+ ions in equimolar concentrations with the substrate. The enzyme was rapidly inactivated in the solubilized state. An apparent molecular weight of about 18,000 was obtained from molecular sieve data. APase, showing activity against ATP, PPi, andp-NPP, was virtually totally inhibited by R 8231. It was activated by Mg2+ ions but slightly reduced in activity by Ca2+ ions. It had an apparent mol. wt. of 79,000. The results provide direct evidence for earlier suggestions of the existence in hard tissue forming cells of two phosphatases active at alkaline pH.
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Abbreviations
- APase:
-
nonspecific alkaline phosphatase (EC 3.1.3.1)
- Ca2+-ATPase:
-
adenosine triphosphatase (EC 3.6.1.3)
- PPiase:
-
inorganic pyrophosphatase (EC 3.6.1.1)
- p-NNP:
-
p-nitrophenyl phosphate
- PPi :
-
inorganic pyrophosphate
- R 8231:
-
(±)-6(m-bromophenyl)-5,6-dihydroimidazo(2,1-b)thiazole oxalate
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Granström, G., Jontell, M. & Linde, A. Separation of odontoblast Ca2+-ATPase and alkaline phosphatase. Calcif Tissue Int 27, 211–217 (1979). https://doi.org/10.1007/BF02441188
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DOI: https://doi.org/10.1007/BF02441188