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Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase.

ABSTRACT: Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.

SUBMITTER: Cox RP

PROVIDER: S-EPMC1198286 | biostudies-other | 1967 Oct

REPOSITORIES: biostudies-other

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Project description:IntroductionThe physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PP(i)) and generates phosphate (P(i)). Humans and mice deficient in the PP(i)-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPP(i)ase), which generates P(i) via PP(i) hydrolysis with neutral pH optimum, remains unknown. We assessed iPP(i)ase in Enpp1(-/-) and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPP(i)ase regulates collagen I expression.MethodsWe treated mouse calvarial osteoblasts with ascorbate and beta-glycerol phosphate to promote calcification, and we assessed cytosolic P(i) and PP(i) levels, sodium-dependent P(i) uptake, Pit-1 P(i) co-transporter expression, and iPP(i)ase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts.ResultsInorganic pyrophosphatase but not TNAP was elevated in Enpp1(-/-) calvariae in situ. Cultured primary Enpp1(-/-) calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PP(i) in early culture, Enpp1(-/-) osteoblasts maintained cytosolic P(i) levels comparable to WT osteoblasts, in association with increased iPP(i)ase, enhanced sodium-dependent P(i) uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1(-/-) osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic P(i) and decreased cytosolic but not extracellular PP(i), and induced both collagen I synthesis and calcification.ConclusionsIncreased iPP(i)ase is associated with "P(i) hunger" and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPP(i)ase induces collagen I at the levels of mRNA expression and synthesis and, unlike TNAP, stimulates calcification by osteoblasts without reducing the extracellular concentration of the hydroxyapatite crystal inhibitor PP(i).

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Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase.

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