Mineralization Alkaline phosphatase

Cell differentiation studies are used to follow the development of cell phenotype by analyzing the concentration of two proteins directly related to bone extracellular matrix mineralization alkaline phosphatase and the osteocalcin. Cell differentiation studies performed on the G5 glass have shown that it induces an earher differentiation of the osteoblastic cells than the polysterene plate controls (unpublished data) (see Fig. 2). Consequently, a faster bone formation could be obtained. 

Acid- and alkaline phosphatases act on a variety of mono- and multiple phosphate carrying low molecular mass molecules. In addition, they hydrolyze many, but not all, phosphoproteins. They are in use for decades to easily screen for diseases, however, somewhat unspe-cifially. For instance, acid phosphatase is used as biomarker for prostate cancer, and alkaline phosphatase to monitor bone (de-) mineralization and liver tumors. 

Phosphates of pharmaceutical interest are often monoesters (Sect. 9.3), and the enzymes that are able to hydrolyze them include alkaline and acid phosphatases. Alkaline phosphatase (alkaline phosphomonoesterase, EC 3.1.3.1) is a nonspecific esterase of phosphoric monoesters with an optimal pH for catalysis of ca. 8 [140], In the presence of a phosphate acceptor such as 2-aminoethanol, the enzyme also catalyzes a transphosphorylation reaction involving transfer of the phosphoryl group to the alcohol. Alkaline phosphatase is bound extracellularly to membranes and is widely distributed, in particular in the pancreas, liver, bile, placenta, and osteoplasts. Its specific functions in mammals remain poorly understood, but it seems to play an important role in modulation by osteoplasts of bone mineralization. 

Alkaline phosphatase Hypophosphatasia due to accumulation of inhibitors of mineralization such as pyrophosphate, and inability to raise phosphorus levels at the site of calcium-phosphorus deposition into hydroxyapatite... 

The acoustic micrograph in Fig. 1.5(a) came from a 5-week-old preparation. It was fixed in alcohol, and stained for alkaline phosphatase and, with von Kossa stain, for biomineral material. The biomineral material of interest here is hydroxyapatite, the principal crystalline mineral constituent of bone. The ordered structure visible within the matrix is not seen with either the light or electron microscopes. But the acoustic microscope can also work perfectly well with unfixed, unstained specimens. Figure 1.5(b) is an acoustic micrograph of matrix and cells from a 17-year-old male. In addition to the standard ingredients of culture medium, these cells were specifically stimulated with beta-glycerolphosphate and a vitamin C preparation. Because the acoustic... 

The non-specific alkaline phosphatases present in bone and calcifying cartilage have several properties in common. The ATPases concerned in the formation of different hard tissues seem to be isozymes. It could be shown that two enzymes capable of degrading ATP exist. One of them can be inhibited by levamisole and R 8231 and is probably identical with non-specific alkaline phosphates. The activity of the other enzyme, tentatively named Ca-ATPase , is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The Ca-ATPase is unaffected by ouabain and ruthenium red. It may be speculated that the Ca-ATPase is concerned with the transmembranous transport of Ca2+-ions to the mineralization front229. ... 

FIGURE 2-21 The pH optima of some enzymes. Pepsin is a digestive enzyme secreted into gastric juice trypsin, a digestive enzyme that acts in the small intestine alkaline phosphatase of bone tissue, a hydrolytic enzyme thought to aid in bone mineralization. 

Alkaline phosphatase is well known to be involved in mineralization, possibly through hydrolysis of organic phosphates to raise the product [Ca2+][Pi] to a level where precipitation occurs. 

Acid and alkaline phosphatases with phosphorylated intermediates are inhibited by vanadate. This has been exploited in the study of the role of alkaline phosphatase in mineralization.1069 Vanadate also inhibits the ATP-dependent degradation of proteins in reticulocytes.1070... 

Glucocorticoids can even cause osteoporosis when they are used for long-term replacement therapy in the Addison s disease, as has been shown by a study of 91 patients who had taken glucocorticoids for a mean of 10.6 years, in whom bone mineral density was reduced by 32% compared with age-matched controls (SEDA-19, 377 198). However, these results contrasted with the results of a Spanish study in patients with Addison s disease, in which no direct relation was found between replacement therapy and either bone density or biochemical markers of bone turnover of calcium metabolism (alkaline phosphatase, osteocalcin, procollagen I type, parathormone, and 1,25-dihydroxycolecalciferol) (SEDA-19, 377 199). 

In 70 postmenopausal women with completely resected breast cancers who were disease-free after taking tamoxifen for 2—3 years, a switch to exemestane resulted in increases in serum bone alkaline phosphatase and the carboxy-terminal telopeptide of type I collagen and a fall in parathormone bone mineral density worsened (28). 

The two enzyme systems most frequently mentioned in connection with biomineralization are (1) carbonic anhydrase and (2) alkaline phosphatase. Little information, however, has been presented on their specific role in the deposition of minerals. Recent advances in the field of biochemistry may shed new light on this intriguing problem. 

The only essential components of the mineral deposition mechanism that are fairly certain at this time relate to phosphate. Even for phosphate, alternative mechanisms are proposed, which are not mutually exclusive but probably function in parallel, in the regulation of different aspects of skeletal calcium transport, and to some extent provide redundancy that allows many mineral transport disorders to be survivable. Alkaline phosphatase activity is essential to produce phosphate. Its major substrate is pyrophosphate. In the absence of the alkaline phosphatase, normally highly expressed as an ectoenzyme by osteoblasts, there is little matrix mineralization... 

The deposition of limited quantities of hydroxyapatite in extracellular matrix has been observed without bounding cells. Cartilage calcification is such a case where local pH control and Ca2+ are dependent upon diffusion and the rate of mineral deposition is driven by phosphate presentation. Chondrocytes produce alkaline phosphatase that generates the required phosphate, but cartilage is not delimited by any cellular structures and transfer of Ca2+ and H+ is by diffusion from extracellular fluid. 

Hessle L, Johnson KA, Anderson HC, Narisawa S, Sali A, Coding JW, Terkeltaub R, Millan JL. 2002. Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization. Proc Natl Acad Sci USA 99 9445—9. 

A less common circumstance leading to hypercalcemia is development of a form of osteomalacia characterized by a profound decrease in bone cell activity and loss of the calcium buffering action of bone. In the absence of kidney function, any calcium absorbed from the intestine accumulates in the blood. Therefore, such patients are very sensitive to the hypercalcemic action of l,25(OH)2D. These individuals generally have a high serum calcium but nearly normal alkaline phosphatase and PTH levels. The bone in such patients generally has a high aluminum content, especially in the mineralization front, which may block normal bone mineralization. These patients do not respond favorably to parathyroidectomy. Deferoxamine, an agent used to chelate iron (see Chapter 58 ... 

When distances of micrometers are involved, and the ions are in solution, diffusion becomes very important. There are several indications that one biological strategy in diffusion control is to separate temporally, and maybe even spatially, the introduction of the cation and anion components into the microenvironment of mineralization. Several observations point to the fact that many of the charged matrix macromolecules that are components of the preformed matrix framework, do not adopt a regular conformation unless calcium is present as a counterion [39, 95]. This implies that the framework loads up first with the cation, and then only when the anion is introduced does mineralization begin. This may well be the reason why the enzymes that control the concentrations of the relevant forms of calcium carbonate and calcium phosphate (carbonic anhydrase and alkaline phosphatase respectively) are so intimately associated with the mineralization site [27, 96]. This is clearly an excellent strategy for maintaining control. 

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