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Comparative kinetic studies on the two interconvertible forms of Streptococcus faecalis inorganic pyrophosphatase.

ABSTRACT: In this work the two interconvertible forms of inorganic pyrophosphatase (EC 3.6.1.1) of Streptococcus faecalis were shown to differ in kinetics. The highly active form of the enzyme was more sensitive to the changes in the Mg2+ concentration, and thus also more sensitive to the inhibition caused by ATP, which competes with PPi for the chelation of Mg2+ ions. We have previously described a kinetic model for the less-active form of S. faecalis inorganic pyrophosphatase [Lahti & Jokinen (1985) Biochemistry 24, 3526-3530]. The kinetic model of the highly active enzyme form is proposed to be a modification of the model of the less-active form in which enzyme activation by free Mg2+ is necessary for the reaction to occur. In this model the enzyme exists in two states, referred to as R- and T-states. In the absence of ligands the enzyme is in the T-state. R-state, i.e. the catalytically active state, exists only in the presence of free Mg2+. Mg1PPi2- is the primary substrate, and free pyrophosphate is a weak inhibitor that cannot serve as a substrate for the highly active form of S. faecalis inorganic pyrophosphatase. This model closely resembles that previously presented for yeast inorganic pyrophosphatase.

SUBMITTER: Lahti R

PROVIDER: S-EPMC1152772 | biostudies-other | 1985 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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Comparative kinetic studies on the two interconvertible forms of Streptococcus faecalis inorganic pyrophosphatase.

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