Matrix Vesicle Fractions Research Articles

Partition of inorganic ions and phospholipids in isolated cell, membrane and matrix vesicle fractions: evidence for Ca-Pi-acidic phospholipid complexes.

Electrolytes and phospholipids of cartilage fractions were partitioned by extraction with organic and aqueous solvents into six solubility groups: Electrolytes I, II and III, and Lipids I, II and III. Of the total Ca, only 4% was water soluble (Electrolytes I); 4-12% was complexed with lipids (Electrolytes II); while the majority (84-92%) was insoluble (Electrolytes III). In contrast, nearly half of the Mg and Pi were water soluble. Of the neutral phospholipid, 95% was not complexed with mineral ions (Lipids I), but 30-45% of the acidic phospholipid was (Lipids II). Ca/Pi ratios were extremely low in the water-soluble phase, but were in the range of amorphous calcium phosphate (ACP) in the insoluble. Molar ratios of the lipid-mineral complex were: Ca:Mg:Pi:acidic phospholipid, 4:3:2:2. Mg/Ca ratios in the soluble fraction were high (5.5-8.9), sufficient to stabilize ACP. Kinetic studies revealed rapid turnover of soluble Ca, insoluble turning over much more slowly. Labeling of lipid-complexed Ca was rapid in cells, but occurred later in matrix vesicles, suggesting transfer. While lipid-Ca-Pi complexes can nucleate apatite in vitro, those present in vivo inside matrix vesicles apparently do not because of the excess Mg. We conclude therefore, that in vesicle-mediated calcification, lysis of the membrane may be essential to allow release of internal Mg.

Biosynthesis of matrix vesicles in epiphyseal cartilage

The in vivo metabolism of 32P orthophosphate into phospholipids of chondroxyte, matrix vesicle, and membrane fractions of chicken epiphyseal cartilage has been studied. Incorporation of radioactive phosphate into the total phospholipid fraction of matrix vesicles was rapid, the labeling of phosphatidylcholine and lysophosphatidylserine being even more rapid in matrix vesicles than in chondrocytes. These findings indicate that matrix vesicles are formed by a rapid, metabolically active process, and are not remnants of dead cells, as had previously been postulated by some workers. The rate of incorporation of 32P orthophosphate into phosphatidylserine and sphingomyelin of matrix vesicles was significantly slower than that of phosphatidylcholine and certain other vesicle phospholipids. These findings are paradoxical because, compared with chondrocytes, matrix vesicles were enriched in phosphatidylserine and sphingomyelin and depleted in phosphatidylcholine. These results indicate that in vesicle formation the rates of degradation of the various phospholipid classes must be markedly different: phosphatidylcholine must be degraded much more rapidly than either phosphatidylserine or sphingomyelin. Support for this comes from previous data which revealed that substantial phospholipase activity is present in epiphyseal cartilage, especially in the zone of hypertrophy where matrix vesicle formation appears to be particularly active.

Electrolytes of isolated epiphyseal chondrocytes, matrix vesicles, and extracellular fluid.

Chondrocyte, matrix vesicle, and membrane fractions, as well as interstitial fluid samples from the proliferating and hypertrophic zones of chicken epiphyseal cartilage were analyzed for electrolyte content. Intracellular Ca levels were 1.4–2.1 mM, over 90% of which was nondiffusible. Isolated hypertrophic chondrocytes had higher intracellular Na and lower K than proliferating cells. Matrix vesicles contained 25 to 50 times higher concentrations of Ca than the adjacent cells. Vesicles from the zone of hypertrophy contained twice as much Ca as did those from the proliferating area. Ca/P1 molar ratios of matrix vesicles were much higher than those of cells or of later mineral deposits. These findings indicate that Ca is concentrated in matrix vesicles during formation, but acuumulation of Ca and P1 must continue in the matrix. X-ray diffraction of freeze-dried vesicle and membrane fractions failed to detect crystalline apatite, suggesting that crystals seen in electron micrographs of matrix vesicles may be artifacts. Interstitial fluid expressed from epiphyseal cartilage was higher in K, Pi, Mg and nucleotides, and lower in Na and Cl, than blood plasma. Fluid from the hypertrophic zone was higher in K and nucleotides, but not Pi or Mg, than that from the proliferating layer. These data suggest that selective leakage or extrusion of these constituents, which are normally intracellular, must occur, especially in the hypertrophic zone. More of the Ca and Mg, and less of the Pi, was protein-bound in cartilage fluid than in blood plasma. There was more binding of the divalent cations in fluid from proliferating than from hypertrophic cartilage. The presence of greater amounts of ultrafilterable peptides in fluid from hypertrophic than from proliferating cartilage or blood plasma, suggests that proteolytic activity may release bound divalent cations during mineralization.

Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles

1. 1. Intact cells, cell fragments (membranes) and matrix vesicles were isolated from the proliferating and calcifying layers of epiphyseal cartilage by sequential hyaluronidase and collagenase digestion and differential centrifugation. Lipids were extracted and analyzed for various lipid classes and their fatty acid composition by column, thin-layer, paper and gas-liquid chromatography. 2. 2. On a protein basis the isolated matrix vesicles had more total lipid than either the membrane or cell fractions, the vesicles and membranes being richer in non-polar lipids and containing smaller quantities of phospholipids than whole cells. Expressed as a percentage of the total lipid, the cells were richer in triacylglycerols and lower in free fatty acids than the membrane or vesicle fractions. The proportion of free cholesterol and the cholesterol/phospholipid ratio were nearly twice as high in the matrix vesicles as in the other tissue fractions. Choline and ethanolamine phosphoglycerides progressively declined in the membrane and matrix vesicle fractions, whereas serine phosphoglycerides and sphingomyelin increased. Non-phosphorus-containing polar lipids were present in all fractions, the vesicles being richer in polyhexosyl ceramides, cerebrosides, glycosyldiacylglycerols and certain uncharacterized acidic polar lipids. 3. 3. Fatty acid patterns of the matrix vesicles were distinctive from those of isolated cells, being generally richer in 18 : 0 and 18 : 2, and lower in 16 : 1 and 18 : 1 fatty acids. Monoacyl forms were similarly increased in 16 : 0 and/or 18 : 0, and reduced in 16 : 1,18 : 1 or 20 : 2 fatty acids, depending on the lipid class. The fatty acid composition of diphosphatidylglycerol from cells and matrix vesicles was markedly different, providing evidence that the cardiolipin in the vesicles was not from mitochondrial components. 4. 4. Based on the fact that the matrix vesicles were significantly enriched in free cholesterol, sphingomyelin, glycolipids and serine-phosphoglycerides, it is concluded that they are derived from the plasma membrane of the cell, supporting earlier conclusions based upon morphological and enzymological evidence.

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 2+ + K +)-ATPase activity was not found in the cell fraction. All activities 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.