High-performance liquid chromatographic and mass spectrometric identification of phosphocitrate synthesized by human kidney homogenate.

Two high-performance liquid chromatographic techniques for the analysis of phosphocitrate, a metabolite synthesized by the cell that is thought to play a role in a number of physiological as well as pathological events, have been developed. The mass spectra of the isolated compound obtained under fast-atom bombardment and collisionally activated decomposition mass-analysed ion kinetic energy conditions are also reported.

Evidence in vitro for an enzymatic synthesis of phosphocitrate.

A biological synthesis of phosphocitrate is described from precursors, citrate and adenosinetriphosphate reacting in the presence of rat liver homogenate. Identity of the newly formed product was examined by enzymatic digestion of reactions mixtures, HPLC chromatography and 1H-NMR spectra. Authenticity of product was established by comparison to chemically synthesized phosphocitrate. Recognition of the existence of a biologically synthetic pathway adds credence to the known presence of phosphocitrate in mitochondria and a postulated role to control calcium phosphate deposition in that organelle.

Possible mechanism of inhibition of cartilage alkaline phosphatase by insulin.

The inhibition of alkaline phosphatase by insulin is a finding reported by many researchers but the mechanism of this inhibition has not been studied. Since alkaline phosphatase is an important factor in the mechanism of calcification and an impairment of mineralisation has been observed in diabetes mellitus, a study was carried out to assess the effect of the hormone on alkaline phosphatase measured in chondrocytes, in matrix vesicles and in a purified enzyme preparation. Enzyme activity was inhibited by insulin. The lowest active concentration was 10(-6) M and maximal inhibition was obtained at about 10(-4) M. The inhibition is of the uncompetitive type. Full recovery of the hormone-inhibited enzyme was obtained with 10(-4) M 2-mercaptoethanol. Data suggest direct interaction between the alkaline phosphatase and insulin molecules, involving either disulfide cross linkages or the metal chelating activity of insulin.

A possible role for polyamines in cartilage in the mechanism of calcification.

The role of polyamines in cartilage is not known: they may be somehow related to the mechanism of calcification. In epiphyseal cartilage from calf scapulas, they are more concentrated in the ossifying area, where calcification takes place, than in the resting region. Spermidine is present in greater amounts than spermine and putrescine. Since ornithine decarboxylase (EC 4.1.1.17) is measurable only in the resting region of the tissue, it is in this area that polyamine biosynthesis occurs, while they accumulate in the ossifying area. Immunohistochemical evidence is obtained that only in the ossifying zone is spermidine extracellular. It is at this level that the matrix is rearranged to become calcified, and proteoglycans are dissociated and partially removed. The effect of polyamines on solutions of proteoglycan subunits has been studied in vitro by following variations of turbidity and viscosity. While in the presence of putrescine the specific viscosity decreases to asymptotic values, in the presence of either 30 mM spermidine or 2.5-10 mM spermine, the decrement is more marked. At the same concentrations, increase of the turbidity of proteoglycan subunit solutions was observed. Only spermidine showed the capacity of displacing proteoglycan subunits from a column of Sepharose 4B-type II collagen: at 15 mM concentration, about 90% of proteoglycans were removed from the column. Alkaline phosphatase activity, which plays an important role in calcification, is enhanced by spermidine and spermine. These results obtained in vitro support the hypothesis that polyamines may be related to calcification of preosseous cartilage.

Alkaline phosphatase binds to collagen; a hypothesis on the mechanism of extravesicular mineralization in epiphyseal cartilage.

Affinity chromatography on Sepharose 4B-collagen gels was used to test the affinity of alkaline phosphatase for collagen. Results indicate that alkaline phosphatase of preosseous cartilage binds to collagen probably by electrostatic interactions, this interaction is inhibited by proteoglycan subunits. These results suggest that, in vivo, the formation of a collagen-alkaline phosphatase complex may be a step of the process leading to cartilage calcification.

Solubility properties of alkaline phosphatase from matrix vesicles.

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.

In vitro biosynthesis by articular chondrocytes of a specific low molecular size proteoglycan pool.

Proteoglycans synthesized by articular and epiphyseal chondrocytes in culture were compared. Proteoglycans extruded by the two types of cells into the culture medium are of identical Mr. On the other hand, the proteoglycans of cells or pericellular matrix synthesized by the articular chondrocytes are characterized by an heterogeneous fraction of low-Mr which is not present in the material derived from epiphyseal chondrocytes. There are at least two components in this fraction: the first seems to be a precursor of aggregated proteoglycans, the other may represent a component of cell coat. Stimulation of the cell cultures with vitamin D metabolites and somatomedin enhances proteoglycan biosynthesis but no modification is observed in the proteoglycan Mr.

Influence of calcium depletion on medullary bone of laying hens.

Medullary bone of birds maintained on a low-calcium diet represents a good model to study modifications of matrix composition in calcified tissue undergoing intense formation and resorption. The composition of the bone matrix during the low-calcium diet has been analyzed by both chemical and histological techniques. Sixty White Leghorn pullets 1 year old were used for the experiment. Fifteen birds served as controls and were killed on day zero; the remaining birds were placed on a calcium-deficient diet (0.13% calcium) and sacrificed after 4, 7, and 12 days of treatment in groups of 15. Serum levels of calcium, PTH, and estrogens were also measured. Chemical analysis of the samples were made for total nitrogen, hydroxyproline, hexosamine, hexoses, calcium, and phosphorus. Collagen and proteoglycans of the matrix of medullary bone of the egg-laying hens were found to be affected by the low-calcium diet. They either increased or decreased during the experiment but never in parallel. The increment of serum PTH is considered responsible for the variations in the amount of collagen. The effects of this hormone are magnified by the fall of serum estrogens as shown also by variations in the amounts of noncollagenous protein. In the late phase of the diet the matrix is represented by poorly calcified osteoid tissue rich in noncollagenous protein, i.e., proteoglycans and glycoproteins.

Ca2+-binding glycoprotein in avian bone induced by estrogen.

The discovery in calcifying cartilage of a glycoprotein, endowed with high calcium affinity and alkaline phosphatase activity, has prompted the investigation of the presence of this compound in other calcified tissues. From medullary bone, a tissue which is highly mineralized under estrogen stimulus, a glycoprotein has been extracted which had the properties described. Besides the high calcium affinity (KD = 10(-7)M), this protein shows phosphatase activity and rate of hydrolysis of ATP, GTP and pyrophosphate was measured. Analysis of the chemical composition of the matrix of the medullary bone indicates that proteoglycans are present in large amounts. The calcium binding glycoprotein appears to be a compound present in different calcified tissues.

Enzymatic properties of the Ca2+-binding glycoprotein isolated from preosseous cartilage.

The Ca2+-binding glycoprotein isolated from preosseous cartilage shows also alkaline phosphatase activity. The purification procedure indicates that the enzyme is inhibited in crude extract and conceivably in the intact tissue; the activity may be controlled by the proteoglycans present in the matrix. Other substrates are hydrolyzed by the purified enzyme in addition to p-nitrophenylphosphate; the highest specific activity was measured with ATP and pyrophosphate (PPi) at pH 7.5 and 9.0 Mg2+ induces an activation of ATP and PPi hydrolysis; Ca2+ activates hydrolysis of ATP but inhibits that of PPi. The glycoprotein shows also transphosphorylase activity, L-serine being the best phosphate acceptor. The release or transfer of Pi catalyzed by the glycoprotein can be an important step in calcium phosphate precipitation.

Glycosaminoglycans and endochondral calcification.

Present controversy about endochondral calcification and ossification is concerned with changes in glycosaminoglycans. Some authors report a rise in amounts of glycosaminoglycans while others a fall. Cartilage was carefully sliced under microscopic control to provide samples of material from different functional zones of the developing tissues. The following zones were studied histochemically and analyzed for their content of total nitrogen, hydroxyproline, total hexosamines, uronic acid and phosphorus: the resting zone; the zone of proliferating and maturing cells; the calcifying zone, characterized by degenerating hypertrophic cells and early mineral deposition; the ossifying region, where early bone formation takes place. Serial analyses provided evidence that glycosaminoglycans increases before calcification starts. Afterwards, part of the glycosaminoglycan content is removed. This biphasic process appears to occur during the calcification of other tissues too, such as secondary bone and dentine.