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Histochemical localization of alkaline phosphatase in rabbit ulnar growth plate
Abstracts from the IV International Conference
on Matrix Vesicles
that the particles were made up of strontium. (3) Most of the particles first appeared in the chondrocytes of the upper hypertrophic zone (HZ). (4) The particles were associated with intracellular lipid deposits, mitochondria, and cytoplasmic vesicles. (5) Many particle-containing vesicles were within or adjacent to mitochondria. (6) Numerous vesicles -some containing particles-were observed within the chondrocyte lacunae. (7) In the longer time periods, particles were found with decreasing frequency within HZ chondrocytes and with increasing frequency with HZ matrix vesicles. (8) In the matrix, most of the particles were associated with matrix vesicles rather than other structures, such as proteoglycan or collagen. This study suggests that the intracellular vesicles containing strontium-and presumably calcium-are precursors of matrix vesicles that become sites of hydroxyapatite nucleation.
MECHANISMS OF MATRIX MINERALIZATION
VESICLE-MEDIATED
Roy E. Wuthier Department of Chemistry, University of South Carolina, Columbia, South CarcJina, USA. Matrix vesicles (MV) are involved in the initiation of calcification in numerous tissues. They are rich in alkaline phosphatase (AP) and phospholipids and contain substantial amounts of Caz+ and P,, mainly present as insoluble amorphous CaP. but also as acidic phospholipid and proteolipid complexes. MV can induce mineral deposition in vitro from solutions containing physiological levels of Cap+ and P,. Depending on the method of preparation, isolated MV may or may not require organic phosphate substrates (OPS) to induce mineralization. Some MV preprations require mM levels of OPS (e.g., l-.2 mM ATP or AMP); others can induce mineral formation in the absence of OPS. Treatment of the latter with mild acidic buffers markedly reduces the rate of MV mineralization, inclicating that in these MV, preformed mineral is essential. Removal of AP has little effect on mineralization of these MV but greatly decreases that of OPSdependent MV. Thus, AP-rich MV in the absence of preformed mineral and in the presence of mM levels of OPS can induce mineral formation. Nonetheless, uptake of P, by these MV is not coupled to AP hydrolysis of OPS, indicating that AP is not a P, porter. Since OPS levels in cartilage extracellular fluid (ECF) are low (1 mM), the flux of OPS into the ECF also may be important to MV-mediated calcification. In addition, since acidic phospholipids, lipid-Ca-P, complexes and proteolipid-Ca-P, complexes can also induce de novo mineralization, their role in MV needs further study. Further, MV Ca*+ -bincling proteins (e.g., AP) may also contribute. Finally, althougln Ca 2+-dependent ATPase has been also considered, its function in MV calcification is doubtful.
ULTRASTRUCTURAL LOCALIZATION OF ALKALINE PHOSPHATASE IN DEVELOPING BONE, DENTIN, ENAMEL, AND CEMENTUM
Since 1923 when Robinson introduced the notion that alkaline phosphatase (APase) enhanced calcification in calcifying cartilage by increasing local ion-concentrating mechanisms, the role of APase has been debated, advocated, abandoned, and rediscovered. In 1978 Bernard showed that APase can be localized in woven bone to the exact ultrastructural microenvironment where calcification is occurring. The technique uses formic acid decalcification at pH 4.5 followed by the Hugon and Borgers method of lead precipitation of phosphates released from alkaline phosphatase by reaction with beta-glycerol phosphate as a substrate. This study reports on the electron microscope analog of bone APase localization in developing enamel, dentin, and cementum. It is found intracellularly within vesicles and extracellularly in matrix vesicles and calcification nodules of mantle dentin and woven bone during initial or primary calcification. Later with the advent of subsequential or secondary calcification (where no matrix vesicles are found) in circumpulpal dentin, enamel, cementum, and lamellar bone, APase is localized to the exact calcification front. There is little doubt that an intimate relationship exists in all mammalian calcifying tissues between APase and the mechanism of mineralization.
MATURATIONAL CHANGES IN THE ULTRASTRUCTURAL DISTRIBUTION OF Ca-ATPase IN EPIPHYSEAL GROWTH PLATE T. Akisaka and C.V. Gay The Pennsylvania State University, University Park, PA (USA), and Hiroshima University, Hiroshima, Japan The electron microscopic cytochemical localization of calcium-activated adenosine triphosphatase (Ca + + -ATPase) was determined in chick epiphyseal growth-plate cartilage using the one-step method of Ando et al. (Acta Histochem. Cytochem. f4:705, 1981). Specificity of the reaction was shown by its sensitivity to 10 mM vanadate, an inhibitor of Ca-ATPase, and its insensitivity to 2.5 mM levamisole, an inhibitor of alkaline phosphatase. In matrix vesicle and plasma membranes, reaction product was confined to the extracellular side of the membrane. The change in enzymatic activity of matrix vesicle membranes corresponded well with changes in the plasma membrane: levels were low in reserve cells, progressively increased in proliferative and early hypertrophic cells, and progressively decreased as fully hypertrophic cells approached the calcifying stages. Mitochondrial activity was observed at all cellular zones, an indication that mitochondria retain their ability to buffer intracellular calcium during the life span of the chondrocyte. A remarkable decrease in enzyme activity of matrix vesicles occurred at the onset of their calcification. It is possible that the disappearance of Ca-ATPase in matrix vesicles is a key event that brings about their mineralization. This loss of detectable enzyme may be related to the degradation of acidic lipids, known to occur in matrix vesicles.
HISTOCHEMICAL LOCALIZATION OF ALKALINE PHOSPHATASE IN RABBIT ULNAR GROWTH PLATE
George W. Bernard and Joseph Hsiu Department of Anatomy and Oral Biology, Center for the Health Sciences, University of California, Los Angeles, California, USA
J.R. Ralphs and S.Y. Ali Experimenfal Pathology Unit, Institute of Orthopaedics, Stanmore, Middlesex, UK
470
Abstracts from the IV International Conference
Alkaline phosphatase distribution was investigated using the lead precipitation technique of Lewinson et al. (1982). Phosphatase reaction product was present in two regions of the growth plate. First, there was the major region on cell membranes and matrix vesicles of longitudinal septae, extending from the final hypertrophic cell 25-30 cells (610pm) up the cell columns. A notable feature of this zone was that the onset of phosphatase activity was frequently polar, with reaction product being present only at the cell periphery. The first phosphatase-positive matrix vesicles were observed in the longitudinal septum adjacent to these cells and increased in numbers toward the hypertrophic zone. Apatite crystals were first observed in phosphatase-positive vesicles, clusters of needles inside vesicles being associated with reaction product outside. The second zone of activity was present at the opposite end of the plate, extending for 2-3 cells (50-60p1.m) only, from the resting zone to the secondary center of ossification, and included active cells and matrix vesicles. The role of matrix differences in mediating the differential onset of phosphatase activity in the two zones, possibly involving the mediation of diffusion of factors from the plasma, is discussed. Lewinson D., Toister Z. and Silberman M. J. Histochem. Cytochem 30:261-269, 1982.
Neonatal rat calvaria were cut into very small fragments and incubated in collagenase for 90 minutes at 38°C with constant shaking. The supernatant was centrifuged at 600 g, 12,000 g, and 127,000 g successively to obtain cell, membrane, and crude matrix vesicle fractions resp. The crude vesicle fraction was subfractionated on a sucrose density gradient to purify it further. The fractions were analyzed for alkaline phosphatase (ALP) and for several peptidases. The activities using amino acid naphthylamides and alanylglycylglycine correlated well with those of ALP, similar to our previous results on bovine fetal alveolar bone. Other peptidase activities that correlated with ALP in matrix vesicles from rat calvaria were shown with tetraalanine, alanylalanyl B-napthylamide, and trimethionine. On the other hand, NCBZ alanylglycylglycine and N-Actetraalanine showed much less activity, and there was poor correlation with ALP activity. Low activity was also found using leucylglycyl B-naphthylamide and lysylglycylglycine. There was moderate activity with methionylalanine and with alanylmethionine. The results with the substrates tested so far suggest that the neutral peptidases that we demonstrated in matrix vesicles are aminopeptidases. This may be related to the fact that aminopeptidases are usually found in cell membranes, from which the vesicles are probably formed.
REGULATION OF ALKALINE PHOSPHATASE Al THE TRANSCRIPTIONAL AND TRANSLATIONAL LEVEL
FURTHER CHARACTERIZATION VESICLE PROTEASE
Howard H. T. Hsu, Ricky Hsu, Teresa Stewart, and H. Clarke Anderson
N. Katsura,’
Department of Pathology, University of Kansas Medical Center, Kansas City, Kansas, USA Alkaline phosphatase (ALP) is a multigene-controlled enzyme consisting of placental, intestinal, and liver-kidneybone isoenzymes. Affinity chromatography with monoclonal antibovine cartilage ALP was used to purify liver and kidney ALPS, and thus, to confirm that cartilage ALP is, indeed, a liver-kidney-bone ALP. Both ALPS were further purified to apparent homogeneity by ion exchange and gel filtration chromatography. SDS-mercaptoethanol gel electrophoresis of matrix vesicle, liver, and kidney ALPS indicated a molecular weight of about 80,000. The culturing of bovine epiphyseal chondrocytes resulted in a loss of alkaline phosphatase with multiple passages. To see whether the decrease in ALP was controlled by transcription or translation, mRNAs were isolated from cultured chondrocytes at different time intervals and tested for their translation activity. .Fluorography analysis of in vitro 35S-methionine-labeled translates indicates that anti-ALP immunoreactive polypeptides were more abundant in chondrocytes prior to culturing than after 4-day culture. Several discrete bands of immunoreactive polypeptides with molecular weights of less than 43,000 were observed, suggesting incomplete biosynthesis of ALP during in vitro translation condition. Thus, it appears that the decrease in biosynthesis with prolonged culture resulted from reduced level of transcription. Supported by DE05262. NEUTRAL PEPTIDASE VESICLES OF BONE
ACTIVITIES
IN MATRIX
Albert Hirschman, Department of Anatomy and Cell Biology, Downstate Medical Center, SUNY, Brooklyn, NY, USA.
on Matrix Vesicles
OF MATRIX
K. Yamada
‘Department of Oral Biochemistry, School of Dentistry, Department of Orthopaedics, School of Medicine, Nagasaki University, Nagasaki, Japan Matrix vesicle protease was isolated and purified from matrix vesicles (MV) of bovine epiphyseal cartilage (Endocrine Control of Bone and Calcium Metabolism, D.V. Cohn, J.T. Potts Jr., and T. Fujita, eds. Excerpta Medica, Amsterdam, pp. 4 18-420, 1984). Similar protease was isolated from MV of chicken epiphyseal cartilage. Heavy fraction of MV in the sucrose gradient was abundant in the protease. EDTA and o-phenanthlorine inhibited the activity completely. Phosphoramidon and some of the phosphate compounds inhibited it to a lesser extent. The inhibition was reversed by Co, Zn, and ferrous ions. This protease degrades cartilage proteoglycan in a trypsinlike mode. The protease released from MV at the mineralization site could degrade proteoglycans (as well as other noncollagenous proteins), allowing calcium phosphate nucleation and calcification to proceed. ROLE OF THE CAa+-BINDING ALKALINE PHOSPHATASE IN THE MECHANISM OF CARTILAGE CALCIFICATION B. de Bernard Department Biochimica, Biofisica e Chimica de//e Macromolecole, University of Trieste (Italy) Among the noncollagenous proteins of cartilage extracellular matrix, alkaline phosphatase is one of the most interesting compounds in the process of calcification. Evidence is presented that alkaline phosphatase activity of matrix vesicles, considered the early loci of calcification, is associated to a Caz+ -binding glycoprotein (Ca2+-binding APase). From isolated matrix vesicles, the enzyme was extracted with deoxycholate-butanol and purified by Fast Protein Liquid chro-
on Matrix Vesicles
that the particles were made up of strontium. (3) Most of the particles first appeared in the chondrocytes of the upper hypertrophic zone (HZ). (4) The particles were associated with intracellular lipid deposits, mitochondria, and cytoplasmic vesicles. (5) Many particle-containing vesicles were within or adjacent to mitochondria. (6) Numerous vesicles -some containing particles-were observed within the chondrocyte lacunae. (7) In the longer time periods, particles were found with decreasing frequency within HZ chondrocytes and with increasing frequency with HZ matrix vesicles. (8) In the matrix, most of the particles were associated with matrix vesicles rather than other structures, such as proteoglycan or collagen. This study suggests that the intracellular vesicles containing strontium-and presumably calcium-are precursors of matrix vesicles that become sites of hydroxyapatite nucleation.
MECHANISMS OF MATRIX MINERALIZATION
VESICLE-MEDIATED
Roy E. Wuthier Department of Chemistry, University of South Carolina, Columbia, South CarcJina, USA. Matrix vesicles (MV) are involved in the initiation of calcification in numerous tissues. They are rich in alkaline phosphatase (AP) and phospholipids and contain substantial amounts of Caz+ and P,, mainly present as insoluble amorphous CaP. but also as acidic phospholipid and proteolipid complexes. MV can induce mineral deposition in vitro from solutions containing physiological levels of Cap+ and P,. Depending on the method of preparation, isolated MV may or may not require organic phosphate substrates (OPS) to induce mineralization. Some MV preprations require mM levels of OPS (e.g., l-.2 mM ATP or AMP); others can induce mineral formation in the absence of OPS. Treatment of the latter with mild acidic buffers markedly reduces the rate of MV mineralization, inclicating that in these MV, preformed mineral is essential. Removal of AP has little effect on mineralization of these MV but greatly decreases that of OPSdependent MV. Thus, AP-rich MV in the absence of preformed mineral and in the presence of mM levels of OPS can induce mineral formation. Nonetheless, uptake of P, by these MV is not coupled to AP hydrolysis of OPS, indicating that AP is not a P, porter. Since OPS levels in cartilage extracellular fluid (ECF) are low (1 mM), the flux of OPS into the ECF also may be important to MV-mediated calcification. In addition, since acidic phospholipids, lipid-Ca-P, complexes and proteolipid-Ca-P, complexes can also induce de novo mineralization, their role in MV needs further study. Further, MV Ca*+ -bincling proteins (e.g., AP) may also contribute. Finally, althougln Ca 2+-dependent ATPase has been also considered, its function in MV calcification is doubtful.
ULTRASTRUCTURAL LOCALIZATION OF ALKALINE PHOSPHATASE IN DEVELOPING BONE, DENTIN, ENAMEL, AND CEMENTUM
Since 1923 when Robinson introduced the notion that alkaline phosphatase (APase) enhanced calcification in calcifying cartilage by increasing local ion-concentrating mechanisms, the role of APase has been debated, advocated, abandoned, and rediscovered. In 1978 Bernard showed that APase can be localized in woven bone to the exact ultrastructural microenvironment where calcification is occurring. The technique uses formic acid decalcification at pH 4.5 followed by the Hugon and Borgers method of lead precipitation of phosphates released from alkaline phosphatase by reaction with beta-glycerol phosphate as a substrate. This study reports on the electron microscope analog of bone APase localization in developing enamel, dentin, and cementum. It is found intracellularly within vesicles and extracellularly in matrix vesicles and calcification nodules of mantle dentin and woven bone during initial or primary calcification. Later with the advent of subsequential or secondary calcification (where no matrix vesicles are found) in circumpulpal dentin, enamel, cementum, and lamellar bone, APase is localized to the exact calcification front. There is little doubt that an intimate relationship exists in all mammalian calcifying tissues between APase and the mechanism of mineralization.
MATURATIONAL CHANGES IN THE ULTRASTRUCTURAL DISTRIBUTION OF Ca-ATPase IN EPIPHYSEAL GROWTH PLATE T. Akisaka and C.V. Gay The Pennsylvania State University, University Park, PA (USA), and Hiroshima University, Hiroshima, Japan The electron microscopic cytochemical localization of calcium-activated adenosine triphosphatase (Ca + + -ATPase) was determined in chick epiphyseal growth-plate cartilage using the one-step method of Ando et al. (Acta Histochem. Cytochem. f4:705, 1981). Specificity of the reaction was shown by its sensitivity to 10 mM vanadate, an inhibitor of Ca-ATPase, and its insensitivity to 2.5 mM levamisole, an inhibitor of alkaline phosphatase. In matrix vesicle and plasma membranes, reaction product was confined to the extracellular side of the membrane. The change in enzymatic activity of matrix vesicle membranes corresponded well with changes in the plasma membrane: levels were low in reserve cells, progressively increased in proliferative and early hypertrophic cells, and progressively decreased as fully hypertrophic cells approached the calcifying stages. Mitochondrial activity was observed at all cellular zones, an indication that mitochondria retain their ability to buffer intracellular calcium during the life span of the chondrocyte. A remarkable decrease in enzyme activity of matrix vesicles occurred at the onset of their calcification. It is possible that the disappearance of Ca-ATPase in matrix vesicles is a key event that brings about their mineralization. This loss of detectable enzyme may be related to the degradation of acidic lipids, known to occur in matrix vesicles.
HISTOCHEMICAL LOCALIZATION OF ALKALINE PHOSPHATASE IN RABBIT ULNAR GROWTH PLATE
George W. Bernard and Joseph Hsiu Department of Anatomy and Oral Biology, Center for the Health Sciences, University of California, Los Angeles, California, USA
J.R. Ralphs and S.Y. Ali Experimenfal Pathology Unit, Institute of Orthopaedics, Stanmore, Middlesex, UK
470
Abstracts from the IV International Conference
Alkaline phosphatase distribution was investigated using the lead precipitation technique of Lewinson et al. (1982). Phosphatase reaction product was present in two regions of the growth plate. First, there was the major region on cell membranes and matrix vesicles of longitudinal septae, extending from the final hypertrophic cell 25-30 cells (610pm) up the cell columns. A notable feature of this zone was that the onset of phosphatase activity was frequently polar, with reaction product being present only at the cell periphery. The first phosphatase-positive matrix vesicles were observed in the longitudinal septum adjacent to these cells and increased in numbers toward the hypertrophic zone. Apatite crystals were first observed in phosphatase-positive vesicles, clusters of needles inside vesicles being associated with reaction product outside. The second zone of activity was present at the opposite end of the plate, extending for 2-3 cells (50-60p1.m) only, from the resting zone to the secondary center of ossification, and included active cells and matrix vesicles. The role of matrix differences in mediating the differential onset of phosphatase activity in the two zones, possibly involving the mediation of diffusion of factors from the plasma, is discussed. Lewinson D., Toister Z. and Silberman M. J. Histochem. Cytochem 30:261-269, 1982.
Neonatal rat calvaria were cut into very small fragments and incubated in collagenase for 90 minutes at 38°C with constant shaking. The supernatant was centrifuged at 600 g, 12,000 g, and 127,000 g successively to obtain cell, membrane, and crude matrix vesicle fractions resp. The crude vesicle fraction was subfractionated on a sucrose density gradient to purify it further. The fractions were analyzed for alkaline phosphatase (ALP) and for several peptidases. The activities using amino acid naphthylamides and alanylglycylglycine correlated well with those of ALP, similar to our previous results on bovine fetal alveolar bone. Other peptidase activities that correlated with ALP in matrix vesicles from rat calvaria were shown with tetraalanine, alanylalanyl B-napthylamide, and trimethionine. On the other hand, NCBZ alanylglycylglycine and N-Actetraalanine showed much less activity, and there was poor correlation with ALP activity. Low activity was also found using leucylglycyl B-naphthylamide and lysylglycylglycine. There was moderate activity with methionylalanine and with alanylmethionine. The results with the substrates tested so far suggest that the neutral peptidases that we demonstrated in matrix vesicles are aminopeptidases. This may be related to the fact that aminopeptidases are usually found in cell membranes, from which the vesicles are probably formed.
REGULATION OF ALKALINE PHOSPHATASE Al THE TRANSCRIPTIONAL AND TRANSLATIONAL LEVEL
FURTHER CHARACTERIZATION VESICLE PROTEASE
Howard H. T. Hsu, Ricky Hsu, Teresa Stewart, and H. Clarke Anderson
N. Katsura,’
Department of Pathology, University of Kansas Medical Center, Kansas City, Kansas, USA Alkaline phosphatase (ALP) is a multigene-controlled enzyme consisting of placental, intestinal, and liver-kidneybone isoenzymes. Affinity chromatography with monoclonal antibovine cartilage ALP was used to purify liver and kidney ALPS, and thus, to confirm that cartilage ALP is, indeed, a liver-kidney-bone ALP. Both ALPS were further purified to apparent homogeneity by ion exchange and gel filtration chromatography. SDS-mercaptoethanol gel electrophoresis of matrix vesicle, liver, and kidney ALPS indicated a molecular weight of about 80,000. The culturing of bovine epiphyseal chondrocytes resulted in a loss of alkaline phosphatase with multiple passages. To see whether the decrease in ALP was controlled by transcription or translation, mRNAs were isolated from cultured chondrocytes at different time intervals and tested for their translation activity. .Fluorography analysis of in vitro 35S-methionine-labeled translates indicates that anti-ALP immunoreactive polypeptides were more abundant in chondrocytes prior to culturing than after 4-day culture. Several discrete bands of immunoreactive polypeptides with molecular weights of less than 43,000 were observed, suggesting incomplete biosynthesis of ALP during in vitro translation condition. Thus, it appears that the decrease in biosynthesis with prolonged culture resulted from reduced level of transcription. Supported by DE05262. NEUTRAL PEPTIDASE VESICLES OF BONE
ACTIVITIES
IN MATRIX
Albert Hirschman, Department of Anatomy and Cell Biology, Downstate Medical Center, SUNY, Brooklyn, NY, USA.
on Matrix Vesicles
OF MATRIX
K. Yamada
‘Department of Oral Biochemistry, School of Dentistry, Department of Orthopaedics, School of Medicine, Nagasaki University, Nagasaki, Japan Matrix vesicle protease was isolated and purified from matrix vesicles (MV) of bovine epiphyseal cartilage (Endocrine Control of Bone and Calcium Metabolism, D.V. Cohn, J.T. Potts Jr., and T. Fujita, eds. Excerpta Medica, Amsterdam, pp. 4 18-420, 1984). Similar protease was isolated from MV of chicken epiphyseal cartilage. Heavy fraction of MV in the sucrose gradient was abundant in the protease. EDTA and o-phenanthlorine inhibited the activity completely. Phosphoramidon and some of the phosphate compounds inhibited it to a lesser extent. The inhibition was reversed by Co, Zn, and ferrous ions. This protease degrades cartilage proteoglycan in a trypsinlike mode. The protease released from MV at the mineralization site could degrade proteoglycans (as well as other noncollagenous proteins), allowing calcium phosphate nucleation and calcification to proceed. ROLE OF THE CAa+-BINDING ALKALINE PHOSPHATASE IN THE MECHANISM OF CARTILAGE CALCIFICATION B. de Bernard Department Biochimica, Biofisica e Chimica de//e Macromolecole, University of Trieste (Italy) Among the noncollagenous proteins of cartilage extracellular matrix, alkaline phosphatase is one of the most interesting compounds in the process of calcification. Evidence is presented that alkaline phosphatase activity of matrix vesicles, considered the early loci of calcification, is associated to a Caz+ -binding glycoprotein (Ca2+-binding APase). From isolated matrix vesicles, the enzyme was extracted with deoxycholate-butanol and purified by Fast Protein Liquid chro-