Inhibitors of in vitro mineralization from rabbit aorta and their role in biomineralization

Abstract

Mineralization of aorta is known to occur late in life and appears to be a pathological phenomenon. In vitro studies revealed that the matrix prepared from the thoracic aorta pieces after their extraction with 3% Na2HPO4 and 0.1 mM CaCl2 were mineralized under physiological conditions of temperature, pH, and ionic strength of the media to form matrix-bound mineral phase resembling hydroxyapatite in nature. However, the matrix identically prepared from the unextracted rabbits aortae failed to mineralize under identical assay conditions. The addition of the aorta extract in the assay system inhibited the above mineralization process. Standard biochemical techniques, e.g., dialysis, ion exchange, and molecular sieve chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis by high-performance liquid chromatography were employed to isolate, purify, and characterize the potent inhibitory biomolecules from the aorta extract. The inhibitory activity of the aorta extract was found to be primarily due to the presence of three biomolecules having molecular weights of 66, 45, and 27-29 kDa. The above inhibitory biomolecules loosely associated with aorta may be involved in the control of calcification associated with arteriosclerosis.

Peeping into Human Renal Calcium Oxalate Stone Matrix: Characterization of Novel Proteins Involved in the Intricate Mechanism of Urolithiasis

“…Proteins present in the organic matrix of renal stones have been proposed to play an important role in kidney stone disease for several decades [36], [37]. Identification of proteins was hampered in the past by limitations in protein identification methods thereby making the identification of novel proteins quite difficult without prior knowledge of their involvement in this process.…”

Tendon gradient mineralization for tendon to bone interface integration

“…11,12,23 Adding the tendon extracts (obtained during preparation of extracted tendons) in the calcium and phosphate homogenous system can also inhibit precipitation of calcium and phosphate ions as a mineral phase. 12 Based on previous studies, two hypotheses could explain calcification produced by Na 2 HPO 4 extraction: 1) Na 2 HPO 4 specifically promotes dissociation from the tissue of a substance that inhibits calcification in situ; and 2) phosphate residuals alone, or in combination with endogenous Ca 2þ , bind at appropriate matrix sites, thus providing centers for nucleation and growth of calcium phosphate crystals. 10,11,13 The indentation test was used to evaluate mechanical properties of tendon in previous studies.…”

“…[10][11][12] This theory is supported by data showing that tendon extraction with 3% Na 2 HPO 4 leads to a quick mineralization and re-adding the extract significantly inhibits the rates of calcium and phosphate uptake from the soluble phase. [11][12][13] However, a recent study showed that when the Na 2 HPO 4 -extracted tendon was incubated in a solution containing both calcium and phosphate, the mineralization process only lasted for several hours. 12 A decline in free calcium occurred quickly because of the spontaneous formation of apatite crystals in solution containing increased levels of calcium and phosphate.…”

“…[11][12][13] However, a recent study showed that when the Na 2 HPO 4 -extracted tendon was incubated in a solution containing both calcium and phosphate, the mineralization process only lasted for several hours. 12 A decline in free calcium occurred quickly because of the spontaneous formation of apatite crystals in solution containing increased levels of calcium and phosphate. 14 Studies by Price et al 15,16 indicated that fetuin, which is synthesized in the liver and is found at high concentrations in mammalian serum and bone, can sustain elevated calcium and phosphate levels in solution and favor mineralization within the collagen fibrils by selectively preventing apatite crystal growth in the solution outside the fibril, thereby enhancing intrafibrillar mineralization.…”

Mode of interaction of calcium oxalate crystal with human phosphate cytidylyltransferase 1: a novel inhibitor purified from human renal stone matrix

“…The interaction between calcium and acidic Asp, Glu and Gla is certainly plausible, but it is equally conceivable that basic residues that are normally protonated at urinary pH and positively charged might experience an attraction toward negatively charged oxalate groups [27]. This is corroborated by the presence of basic amino acids too in the inhibitory proteins [28]. In either case steric constraints from 3D conformation of the molecule might limit the number of these simple interactions [27].…”