Abstract
The roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated mineralization have been studied by selectively releasing the enzyme from a wide variety of matrix vesicle preparations using treatment with a bacterial phosphatidylinositol-specific phospholipase C and by demineralization of the vesicles using isosmotic pH 6 buffer. Following depletion of 50-90% of the alkaline phosphatase activity or treatment with citrate buffer, the vesicles were tested for their ability to accumulate 45Ca2+ and 32Pi from a synthetic cartilage lymph. Removal of alkaline phosphatase by phospholipase C treatment caused two principal effects, depending on the matrix vesicle preparation. In rapidly mineralizing vesicle fractions which did not require organic phosphate esters (Po) to accumulate mineral ions, release of alkaline phosphatase had only a minor effect. In slowly mineralizing vesicles preparations or those dependent on Po substrates for mineral ion uptake, release of alkaline phosphatase caused significant loss of mineralizing activity. The activity of rapidly calcifying vesicles was shown to be dependent on the presence of labile internal mineral, as demonstrated by major loss in activity when the vesicles were decalcified by various treatments. Ion uptake by demineralized vesicles or those fractionated on sucrose step gradients required Po and was significantly decreased by alkaline phosphatase depletion. Uptake of Pi, however, was not coupled with hydrolysis of the Po substrate. These findings argue against a direct role for alkaline phosphatase as a porter in matrix vesicle Pi uptake, contrary to previous postulates. The results emphasize the importance of internal labile mineral in rapid uptake of mineral ions by matrix vesicles.
Highlights
The activitoyf rapidly calcifying ves- may play a role in the transport of phosphate during mineral ion uptake by matrix labile internal mineral, as demonstrated by major loss vesicles[33,34,35,36]
This study represents the first time that the three most commonlyused types of matrix vesicle preparations have been directly compared with respect to their abilities and requirements for accumulation of 45Ca2+and 32Pions from metastable synthetic lymphs in vitro
We examined conventional collagenase-released matrix vesicles [7], matrix vesicle-enriched microsomes (MVEM) prepared by homogenization of fresh cartilage tissue [35], and MVEM further purified by sucrose step gradient fractionation [37]
Summary
Buffer (50 mM TES, 1.4 mMMgC12, 10%w/v sucrose, pH 7.5) and differential centrifugation,the MVEM were resuspended in TMS "Ca2+ and 3zPiUptake-Assays of &Ca2+and 32Piuptake were alizing activity of these fractions was significantly reduced; slight increases above the control in ion uptake of Ca2+(Fig. 2 A ) and Pi (Fig. 2B) were again observed in the alkaline phosphatase-depleted MVEM. In less active preparations (Fig. 2, C and D)uptake of mineral ion was slower, performed in SCL [21, 41] as previously described [34] except that with a longer lag phase In these MVEM, phospholipase C filters were presoaked in SCL (instead of 20 mM CaCl, or Pi buffer) treatment resulted in a significant reduction in the accumuto reduce nonspecific absorption of radiolabel to the filters. In the absence of AMP, release of alkaline phosphatase by phospholipase C caused significant reduction in the ratoef Ca2+uptake, citrate treatof Ca2+in these MVEM preparations. Alkaline phosphatase activity of the phospholipase C-treated fractions was 45.7 and 28.8%of the TMS-and citrate-treated controls, respectively
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