H. Clarke Anderson

Pyrophosphate stimulation of calcium uptake into cultured embryonic bones. Fine structure of matrix vesicles and their role in calcification

Abstract The onset of mineralization in embryonic chick femurs was studied as a model for the initiation of biological calcification. Electron microscopy confirmed the presence of calcifying matrix vesicles within newly formed bone, and showed that these vesicles were the initial site of crystal deposition. Matrix vesicles were first seen on day 6 of embryonic development, and already were present in considerable numbers on day 7, at which time mineral deposition was just beginning. As a reflection of initial mineralization the uptake of 45Ca and 40Ca into 7-day-old bones was studied during 2 days in organ culture. A control level of uptake was established using a defined culture medium, P-6. Addition of inorganic pyrophosphate (PPi) to this medium caused a marked increase in calcium uptake into areas of matrix which normally calcify in vivo. The maximal 45Ca uptake, greater than 4-fold, was achieved with 4 μg of P per milliliter of PPi and was partially heat-inhibitable. Since the matrix vesicles are known to be rich in inorganic pyrophosphatase, it is proposed that mineralization is promoted in vesicles by the enzymatic hydrolysis of pyrophosphate. The membrane-bounded matrix vesicles appear to provide the necessary enzymes and environment to concentrate calcium and phosphate for initiating crystal formation.

The lipids of matrix vesicles from bovine fetal epiphyseal cartilage

Matrix vesicles are extracellular, membrane-bounded particles which are abundant in the matrix of calcifying cartilage and which are morphologically related to the deposition of mineral in this tissue. The matrix vesicles and cells of the epiphyses of the long bones of fetal calves were liberated by digestion with collagenase and separated by differential centrifugation. Vesicle preparations were found to be lipid-rich and to contain significantly more sphingomyelin and phosphatidyl serine than cellular fractions. The ratio, moles cholesterol to moles total phospholipid, was higher in vesicles than in cells, a finding which is consistent with vesicles having their origins in the plasma membranes of the chondrocytes.RésuméLes vésicules matricielles sont des particules extracellulaires, entourées par une membrane, qui sont nombreuses dans la matrice du cartilage en voie de calcification et qui sont en rapport morphologique avec le dépôt minéral. Les vésicules matricielles et les cellules de lépiphyse dos longs dembryons de veaux sont libérées par digestion grâce à la collagénase et séparées par centrifugation différentielle. Les vésicules ainsi obtenues sont riches en lipides et contiennent significativement plus de sphingomyéline et phosphatidyl sérine que les fractions cellulaires. Le rapport moles de cholestérol sur moles de phospholipides totaux est plus élevé dans les vésicules que dans les cellules; ce résultat est en accord avec lorigine des vésicules au niveau des membranes cellulaires des chondrocytes.ZusammenfassungMatrixvesikel sind extrazelluläre, membrangebundene Teilchen, welche in der Matrix von verkalkendem Knorpel gehäuft vorkommen und welche in morphologischem Zusammenhang mit der Mineralablagerung in diesem Gewebe stehen. Matrixvesikel und Epiphysenzellen aus Röhrenknochen von Kalbsembryonen wurden mittels Collagenaseverdauung freigesetzt und durch Differential-Zentrifugierung getrennt. Die Vesikelpräparate waren reich an Lipidien und enthielten bedeutend mehr Sphingomyelin und Phosphatidylserin als die Zellfraktionen. Das molare Verhältnis Cholesterol/Gesamtlipide war in den Vesikeln höher als in den Zellen; dieser Befund stimmt mit der Tatsache überein, daß die Vesikel ihren Ursprung in den Plasmamembranen der Chondrozyten haben.

A simple and defined method to study calcification by isolated matrix vesicles. Effect of ATP and vesicle phosphatase.

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 microgram of protein, were incubated for 2 h in a 45Ca-labelled solution with 2.2 mM Ca2+, 1.6 mM PO 3/4-and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO4 hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca2+ or Mg2+ was found to stimulate matrix vesicle ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, beta-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, vesicles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.

Enzymatic Characterization of the Matrix Vesicle Alkaline Phosphatase Isolated from Bovine Fetal Epiphyseal Cartilage

SummaryPurified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PPi. Optimal activities forp-nitrophenyl phosphate, ATP, and PPi are found at pH 10.5, 10.0, and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations.p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasing substrate concentration. Heat inactivation studies indicate that both phosphorohydrolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ and Hg2+ ions inhibit thep-nitrophenyl phosphatase activity at pH 10.5 while Mn2+ ions show no effect. Pi, levamisole, CN−, Zn2+, Ca2+ ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. Pi is a linear noncompetitive inhibitor with a Ki of 8.0 mM. Levamisole and L-phenylalanine are uncompetitive inhibitors with inhibition constants of 0.02 and 39.4 mM, respectively. Ca2+ ions inhibit noncompetitively with a Ki of 9.3 mM. Zn2+ ion is a potent noncompetitive inhibitor with an inhibition constant of 0.026 mM. The enzyme is inhibited irreversibly by Be2+ ion, EDTA, EGTA, ethane-l-hydroxydiphosphonate, dichloromethanediphosphonate, L-cysteine, and N-ethylmaleimide. NaCl, KCl, and Na2SO4 at 0.5–1.0 M inhibit the enzyme.At pH 8.5, the cleavage of PPi by the matrix vesicle enzyme is inhibited by Mg2+ and Ca2+ ions at concentrations greater than 0.5 mM. Mg2+ ions in the range of 0.1–4 mM stimulate the matrix vesicle ATPase whereas higher concentrations produce inhibition. Ca2+ ion does not affect the ATPase activity between 0.1 and 10 mM at either pH 7.5 or 10.0. With 5′-AMP as substrate, the 3.5-fold decrease in the maximum velocity of the matrix vesicle relative to the chondrocyte enzyme may be accounted for in terms of a small excess enthalpy of activation (1160 cal/mole) partially compensated by an increased entropy of activation.

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.

Cartilage induction in vitro: Ultrastructural studies☆

Abstract When 19-day fetal rat triceps muscle was cultured for 7 to 14 days upon decalcified, sequentially extracted adult rat bone, cartilage formed within clefts and vascular spaces of the decalcified bone. The bone substrata were prepared by extracting tibias and femurs of Sprague-Dawley rats with 1:1 chloroform:methanol, 0.6 N HCl, 2 M CaCl2, 0.6 M EDTA, 8 M LiCl, and H2O at 56°C. The culture medium used was CMRL 1066 with 15% newborn calf serum. During cultivation, fibroblastic mesenchymal cells migrated out of muscle and into bone crevices where they secreted a cartilaginous matrix composed of thin, randomly dispersed collagen fibrils and proteoglycan granules. The latter are characteristic for cartilage matrix. Extracted bone matrix contained mature collagen fibrils, some of which retained their typical 640-A banding. Other collagen fibrils were partially disaggregated and expanded to reveal component 50-A-thick, beaded micro fibrils. Such an expansion of collagen fibrils is known to result from exposure to proteoglycan solvents such as 2 M CaCl2. The decalcified bone matrix contained many residual devitalized cells and cell fragments which often were seen in close proximity to chondrifying mesenchymal cells. This finding indicates the possibility that residual cellular material could play a role in stimulating cartilage development.