Effect of vanadate, a potent alkaline phosphatase inhibitor, on 45Ca and 32Pi uptake by matrix vesicle-enriched fractions from chicken epiphyseal cartilage

Although alkaline phosphatase has been long associated with the mineralization process, its exact function remains to be elucidated. To clarify its possible role in matrix vesicle-mediated mineralization, we tested the effect of vanadate, a phosphate analogue and powerful competitive inhibitor of alkaline phosphatase activity, on calcium and phosphate uptakes by a matrix vesicle-enriched microsomal fraction. Vanadate was also tested in a hydroxyapatite-seeded ion uptake system to determine possible direct effects on mineral formation. The effect of vanadate on vesicle mineral ion uptake was complex; low dosages of vanadate (2-20 microM) were stimulatory to Ca2+ uptake, but were inhibitory to Pi. Higher dosages (greater than 67 microM) were inhibitory to both ions. The effect of vanadate on ion uptake was strongly influenced by the stage of vesicle loading; major effects were seen during the lag and early uptake phases, and minimal effects were seen in the terminal stages. Concentrations of vanadate highly inhibitory to vesicle ion uptake had minimal effects on ion accretion by a hydroxyapatite-seeded system. Inhibition of alkaline phosphatase activity by vanadate broadly paralleled inhibition of Pi and Ca2+ uptake; however, at low vanadate concentrations, inhibition of Pi uptake closely paralleled that of alkaline phosphatase. The data indicate that vanadate binds with high affinity to Pi-loading sites, blocking initial Pi uptake. Complexation between vanadate and Ca2+ may be responsible for the stimulation of Ca2+ uptake at early stages of vesicle ion loading with low levels of vanadate by enhancing binding of Ca2+ to the vesicles. It may also account for the selective inhibition of Ca2+ uptake during the rapid stage of vesicle ion loading with high levels of vanadate by reducing Ca2+ ion activity. The close parallelism between inhibition of early Pi uptake and of alkaline phosphatase activity supports the concept that alkaline phosphatase is involved in Pi transport during the early stages of matrix vesicle ion loading. However, the fact that only about half of the Pi uptake was affected by vanadate, despite the progressive inhibition of alkaline phosphatase activity, indicates that alkaline phosphatase is not solely responsible for Pi uptake by the matrix vesicle-enriched fraction.     

Disposition of preformed mineral in matrix vesicles. Internal localization and association with alkaline phosphatase

Studies were made on the disposition of mineral ions in matrix vesicles (MV) and their relationship to alkaline phosphatase by treatment of MV-enriched microsomes (MVEM) with graded levels of Ca2+-chelating agents to complex accessible ions, fractionation of MVEM on hypertonic sucrose gradients at two different pH values (7.5 and 8.0) to evaluate for the presence of calcium phosphate mineral, and passage of MVEM through cation-exchange columns to determine the accessibility of the Ca2+. The effect of removal of Ca2+ and Pi on subsequent ability of MVEM to induce mineral formation from synthetic cartilage lymph was also determined. Passage through cation-exchange columns revealed that MV Ca2+ was not freely exchangeable, but coeluted in the void volume with alkaline phosphatase. However, upon incubation in synthetic cartilage lymph, progressively more Ca2+ was retained by the column. These findings indicate that, initially, the majority of Ca2+ in MVEM is internal and not readily exchangeable, but as Ca2+ accumulates, progressively more becomes external. The mineral in MV is labile and readily susceptible to loss; treatment with graded levels of EGTA removed major portions of the original Ca2+ and Pi. 45Ca uptake by these mineral-depleted MV was markedly reduced, even in the presence of alkaline phosphatase substrates. Sucrose gradient fractionation of MVEM caused extensive loss of Pi, but not Ca2+, from the low-density alkaline phosphatase-rich fractions. This reveals that Ca2+ and Pi are not initially coupled together: Pi is largely soluble, whereas Ca2+ must be tightly bound. In the high-density vesicles, large amounts of both Ca2+ and Pi are present. The slightly enhanced recovery at higher pH suggests the presence of a solid mineral phase. During mineralization by MV, Ca2+ became externalized, and concomitantly alkaline phosphatase activity declined. This suggests that a direct association exists between the enzyme and the developing mineral.     

Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

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.     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

Extracellular matrix vesicles in rat bone after parathyroidectomy

Summary The enzymatic activity of bone matrix vesicles from parathyroidectomized rats was determined and compared to the activity of vesicles from sham operated and normal animals. The vesicles were isolated from the alveolar bone by collagenase digestion and differential centrifugation and further purified on a discontinuous sucrose density gradient. The amount of extractable protein and the activity of alkaline phosphatase, acid phosphatase, and ATPase in the vesicle fractions thus obtained did not differ significantly from the values characteristic of preparations from control rats. It may therefore be suggested that parathyroid hormone depletion and the associated hypocalcemia have no significant effect on the occurrence and phosphatase activity of bone matrix vesicles.     

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.     

Membrane interactions of rat intestinal alkaline phosphatase: role of polar head groups

Lipid-protein interactions with purified membranous intestinal alkaline phosphatase have been studied by using rat intestine. The enzyme was incorporated equally well into neutral lecithin and anionic liposomes, including those made from phosphatidic acid alone. It could not be solubilized with chaotropic salts nor by phospholipases C and D from either native membranes or phospholipid vesicles. Detergents effected nearly complete release of enzyme from the vesicles. Phosphatase activity was lost upon treatment with phospholipase D alone. The activity was restored with free choline, or choline containing phospholipids, but not by the addition of other phospholipids or amines. The catalytic activity was also lower when the enzyme was bound to a phosphatidylcholine vesicle containing additional phosphatidic acid. Neither phosphatidylserine nor phosphatidylinositol addition altered enzyme activity. These results show that the enzyme binds to the membrane by a primary hydrophobic interaction with membrane phospholipids without requiring the polar head group and that the enzyme activity is affected via a secondary interaction with choline. We suggest that choline protects the active site of brush border alkaline phosphatase from inhibition by endogenous membrane phosphate groups.     

Preferential utilization of organic and inorganic sources of phosphorus by wheat plant

On soils of low P supply organic P (Po) makes up a similar or even larger part in soil solution than inorganic P (Pi). The ability of wheat (Triticum aestivum L., cv. Star) plants to hydrolyze and absorb this Po in comparison to similar concentrations of Pi was studied. Four concentration levels of Pi and Po were obtained by extracting two soils with deionized water in a ratio of 1:1 and concentrating the resulting filtrate by freeze drying to different degrees. The concentration of Pi varied between 5 and 36 μM and Po between 3 and 22 μM. Wheat seedlings were grown in these solutions for 12 and 24 h and acid and alkaline phosphatase activity determined. The reduction of Po concentration in solution expressed on a root length basis gave the rate of Po hydrolysis and the reduction in concentration of Pi and Po gave the P inflow into the roots. No alkaline phosphatase activity was detected. The activity of wheat root acid phosphatase increased with Po concentration in solution. Phosphorus uptake was 2 to 6 fold higher from Pi than from Po at similar concentrations of both. The rate of uptake from Pi, the inflow, as well as the rate of hydrolysis of Po increased linearly with concentration but at similar concentration the inflow was 2 to 4 times higher than the rate of Po hydrolysis. Results suggest that plants can utilize Po after hydrolysis by phosphatase, but Pi is more important and preferentially used by plants; Po may be essential for plant nutrition especially in high P-fixing soils.     

Expression of alkaline phosphatase induces rapid and artificial mineralization in specific transformed Escherichia coli

Matrix vesicles (MV) having high alkaline phosphatase (ALP) activity act as initiators of biological mineralization. Although bacteria have similar membranous structures to MV, ALP mediated mineralization has not been studied in bacterial cells. Escherichia coli was transformed with a bacterial ALP gene in this study. Recombinant E. coli overproducing ALP induced mineralization through hydrolysis of calcium-glycerophosphate (Ca-GP). Fourier transform infrared spectroscopy and electron microscopy combined with electron diffraction revealed newly formed hydroxyapatite mineral deposits. These findings suggest that hydrolysis of Ca-GP through ALP induced high Ca and Pi concentrations within bacterial cells followed by complete bacterial mineralization.     

Phosphodiesterase activity of alkaline phosphatase in ATP-initiated Ca(2+) and phosphate deposition in isolated chicken matrix vesicles

Inorganic pyrophosphate is a potent inhibitor of bone mineralization by preventing the seeding of calcium-phosphate complexes. Plasma cell membrane glycoprotein-1 and tissue nonspecific alkaline phosphatase were reported to be antagonistic regulators of mineralization toward inorganic pyrophosphate formation (by plasma cell membrane glycoprotein-1) and degradation (by tissue nonspecific alkaline phosphatase) under physiological conditions. In addition, they possess broad overlapping enzymatic functions. Therefore, we examined the roles of tissue nonspecific alkaline phosphatase within matrix vesicles isolated from femurs of 17-day-old chick embryos, under conditions where these both antagonistic and overlapping functions could be evidenced. Addition of 25 microM ATP significantly increased duration of mineralization process mediated by matrix vesicles, while supplementation of mineralization medium with levamisole, an alkaline phosphatase inhibitor, reduces the ATP-induced retardation of mineral formation. Phosphodiesterase activity of tissue nonspecific alkaline phosphatase for bis-p-nitrophenyl phosphate was confirmed, the rate of this phosphodiesterase activity is in the same range as that of phosphomonoesterase activity for p-nitrophenyl phosphate under physiological pH. In addition, tissue nonspecific alkaline phosphatase at pH 7.4 can hydrolyze ADPR. On the basis of these observations, it can be concluded that tissue nonspecific alkaline phosphatase, acting as a phosphomonoesterase, could hydrolyze free phosphate esters such as pyrophosphate and ATP, while as phosphodiesterase could contribute, together with plasma cell membrane glycoprotein-1, in the production of pyrophosphate from ATP.     

The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method.

The ultrastructural localization of alkaline phosphatase (A1P) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl2 to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl2 and CeCl3. A1P activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method

Summary The ultrastructural localization of alkaline phosphatase (AlP) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl₂ to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl₂ and CeCl₃. AlP activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

2-Mercaptoacetate administration depresses the beta-oxidation pathway through an inhibition of long-chain acyl-CoA dehydrogenase activity.

In an investigation of the link between Pi transport and alkaline phosphatase in mammalian small intestine, the characteristics of Pi uptake by brush-border membrane vesicles prepared from rat intestine were compared with the properties of the tissue alkaline phosphatase. The NaCl-dependent Pi uptake had a Km of 0.1 mM at pH 7.5 and was inhibited totally by 1 mM-arsenate and by 1 mM-vanadate. These compounds are also potent competitive inhibitors of the alkaline phosphatase activity of the vesicles, with Ki values less than 5 microM at pH 7.5. When the effect on Pi uptake of several other potent inhibitors of alkaline phosphatase, including phosphonates and phosphate analogues, was tested, however, it was found that there was little, if any, inhibition of transport under conditions in which the inhibition of phosphatase activity was total. Incubation of the vesicles for 20 min with oxidized adenosine 5'-[beta gamma-imido]triphosphate followed by rapid gel filtration to remove the inhibitor resulted in an irreversible loss of phosphatase activity, but left Pi transport unimpaired. Conversely, a similar prolonged incubation with adenosine 5'-[beta-thio]diphosphate or adenosine 5'-[gamma-thio]triphosphate had no effect on alkaline phosphatase activity but resulted in a permanent partial loss of transport capability. The failure to demonstrate an inhibition of Pi transport resulting from inhibition of alkaline phosphatase and the different responses of enzymic activity and Pi transport to irreversible inhibition make it very unlikely that the enzyme is directly involved in the transport system.     

Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca²⁺ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro–osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.     

Phosphate binding in the active site of alkaline phosphatase and the interactions of 2-nitrosoacetophenone with alkaline phosphatase-induced small structural changes

To monitor structural changes during the binding of Pi to the active site of mammalian alkaline phosphatase in water medium, reaction-induced infrared spectroscopy was used. The interaction of Pi with alkaline phosphatase was triggered by a photorelease of ATP from the inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP. After photorelease, ATP was sequentially hydrolyzed by alkaline phosphatase giving rise to adenosine and three Pi. Although a phosphodiesterase activity was detected prior the photorelease of ATP, it was possible to monitor the structural effects induced by Pi binding to alkaline phosphatase. Interactions of Pi with alkaline phosphatase were evidenced by weak infrared changes around 1631 and at 1639 cm(-1), suggesting a small distortion of peptide carbonyl backbone. This result indicates that the motion required for the formation of the enzyme-phosphate complex is minimal on the part of alkaline phosphatase, consistent with alkaline phosphatase being an almost perfect enzyme. Photoproduct 2-nitrosoacetophenone may bind to alkaline phosphatase in a site other than the active site of bovine intestinal alkaline phosphatase and than the uncompetitive binding site of L-Phe in bovine intestinal alkaline phosphatase, affecting one-two amino acid residues.     

Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization

Activity of the bone/liver/kidney isozyme of alkaline phosphatase (AP) is known to be critical for mineralization in developing bone, although its role is unclear. The work now reported explores changes in the activity of this Zn2+-containing enzyme that occur during Ca2+ accumulation by matrix vesicles (MV). A marked loss (up to 65-70%) in AP activity was found to accompany Ca2+ accumulation by MV. These two events were highly correlated, both temporally and quantitatively. Investigation into possible causes revealed that the decline in AP activity during Ca2+ uptake was not due to action of proteases but rather resulted from interaction with the developing mineral phase, loss of metal ions (Zn2+ and Mg2+) from the active site of the enzyme, and concomitant irreversible denaturation of the enzyme. Protease inhibitors did not protect AP from loss of activity during mineralization; in contrast, protease treatments, which progressively destroyed the ability of MV to accumulate Ca2+ actually reduced loss of AP activity. These findings clearly demonstrate that AP is present at the site of MV mineralization and that its catalytic activity is profoundly reduced by the mineralization process.     

Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.

Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix vesicle fraction were studied in detail. Mg-2-+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent. Ca-2-+ stimulated all activities weakly in the absence of Mg-2-+. However, when Mg-2-+ was present, Ca-2-+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca-2-+, and hence would not seem to be involved with active Ca-2-+ transport in the typical manner. The distribution of alkaline phosphatase, pyrophosphatase, and Mg-2-+ ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na-+ + K-+)-ATPase activity was found in the cell fraction. All activites showed nearly identical sensitivities to levamisole (4 - 10-3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10-15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg-2-+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.     

Vesicles with lactate dehydrogenase and without alkaline phosphatase present in the resting zone of epiphyseal cartilage.

Matrix vesicles are membrane-invested vesicles that initiate mineralization in the extracellular matrix of calcifying tissues. The epiphyseal cartilages of young-rat rib bones were divided into the growth zone and the resting zone, followed by the isolation of matrix vesicles after collagenase treatment. Matrix vesicles with both alkaline phosphatase and lactate dehydrogenase were detected in the growth cartilage found in the epiphyseal growth plates of young rabbits [Hosokawa, Uchida, Fujiwara & Noguchi (1988) J. Biol. Chem. 263, 10045-10047], but were not detected in the resting zone. By contrast, and surprisingly, lactate dehydrogenase-containing vesicles without alkaline phosphatase were found in the resting zone, but not in the growth zone. In both the growth and resting zones, isoenzyme patterns of lactate dehydrogenase in the two different vesicles were identical with those of cytosolic lactate dehydrogenase of chondrocytes, suggesting the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase. The same results as for young-rat rib bones were obtained with the resting and growth cartilages of young-dog and monkey rib bones.