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H H Hsu

Publications and source records attributed to H H Hsu.

At least 73 records · Page 4Linked to original sources
PubMed1986

The deposition of calcium pyrophosphate by NTP pyrophosphohydrolase of matrix vesicles from fetal bovine epiphyseal cartilage.

About 5 mumol CaPPi/mg protein was deposited within 3 h in the presence of the reaction mixtures containing 1 mM ATP, 2 mM Ca2+, 1 mM Pi, and 17 micrograms of purified NTP pyrophosphohydrolase. At 1 mM ATP, 50% of the deposition was inhibited by 0.5-1 mM of various substrate and product analogues including AMP, ADP, and ethylene hydroxyl diphosphonate. The magnitude of inhibition on NTP pyrophosphohydrolase activity was in the order of AMP = CMP = ADP greater than adenosine greater than adenine greater than NAD = NADP. AMP, CMP, ADP, and adenosine are competitive inhibitors. The modes of inhibition by adenine, NAD, and NADP differ from the competitive inhibition. Ribose, 3'-AMP, 2'-AMP, and cAMP did not inhibit the enzyme activity.

Adenosine Triphosphate↗
PubMed1986

Development and cross-reactive properties of monoclonal antibodies to bovine matrix vesicle alkaline phosphatase.

Alkaline phosphatase (ALPase), concentrated in the membranes of matrix vesicles, is believed to play a role in initial calcification. To further purify, characterize, and identify this enzyme in tissue, a monoclonal antibody was developed against the ALPase of isolated fetal calf matrix vesicles. Splenic lymphocytes derived from mice immunized with Sepharose 6B-purified fetal calf matrix vesicle ALPase were fused with mouse plasmacytoma cells (line X63-Ag-8.653) using standard hybridoma technology. Hyperimmune sera and hybridoma culture supernatants were screened for the presence of specific antibody using a newly developed double-immunosorbent assay in which putative antibody is added to microtiter plate wells precoated with affinity-purified rabbit antimouse immunoglobulin. After incubation and washing, partially purified fetal calf matrix vesicle ALPase is added to each well. The enzyme adheres only to wells that contain specific anti-ALPase antibody. These wells are identified by adding the enzyme substrate p-nitrophenyl phosphate and reading the wells in a plate-reading spectrophotometer at 405 nm. A hybridoma-producing specific antibody was subsequently cloned and grown as ascities-producing tumors in pristane-primed mice. Ouchterlony analysis indicated that the cell line secretes an immunoglobulin of IgG1 class. This antibody reacts specifically with ALPase derived from calf matrix vesicles and cross-reacts with ALPase of bovine kidney, liver, and placental origin and human bone but does not cross-react with bovine intestinal ALPase or ALPase derived from matrix vesicles isolated from rachitic rat growth plate cartilage.

Alkaline Phosphatase↗
PubMed1985

Purification and partial characterization of alkaline phosphatase of matrix vesicles from fetal bovine epiphyseal cartilage. Purification by monoclonal antibody affinity chromatography.

Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.

Alkaline Phosphatase↗
PubMed1984

Isolation of a plasma membrane-enriched fraction from collagenase-suspended rachitic rat growth plate chondrocytes.

An attempt was made to concentrate plasma membranes of homogenized chondrocytes isolated by collagenase digestion of rachitic rat epiphyseal growth plate cartilage. This study reports the characterization of enzymes in the plasma membrane of isolated chondrocytes and their comparison with extracellular matrix vesicle components. The plasma membrane-enriched fractions that were obtained showed a sevenfold increase in 5'-nucleotidase and a 15-fold increase in alkaline phosphatase, both of which are regarded as plasma membrane markers. SDS-polyacrylamide gel electrophoretic profiles of proteins extracted from membrane fractions contained several major protein bands also seen in isolated matrix vesicles. These studies indicate the usefulness of concentrating plasma membrane components from isolated chondrocytes, after the chondrocytes have been enzymatically freed from investing matrix and other stromal components by collagenase.

Alkaline Phosphatase↗
PubMed1984

The deposition of calcium pyrophosphate and phosphate by matrix vesicles isolated from fetal bovine epiphyseal cartilage.

Since calcium (Ca) deposition by isolated fetal bovine matrix vesicles is selectively supported by nucleoside triphosphate, and since the Ca deposits appear to be amorphous by transmission electron microscopy, attempts were made to study further the nature of these Ca deposits. Calcification of isolated matrix vesicles was allowed to occur in a calcifying medium in which either inorganic phosphate (Pi) or [gamma-P]ATP was labeled with 32P. 32P in Ca P (pyrophosphate) deposits were analyzed by a Dowex 1 X 10 anion exchange chromatography. The results of the analysis indicate that the (32P) radioactivity was mainly associated with Pi when Pi in the calcifying media was labeled with 32P. In contrast, 32P was found to be associated with inorganic pyrophosphate (PPi) when [gamma-32P]ATP was used. Using a specific enzyme coupling assay for PPi, the presence of PPi in the Ca deposits was demonstrated. The amounts of Pi and PPi in the Ca deposits initiated by fetal calf matrix vesicles were found to be approximately equal. To exclude the possibility that the major part of PPi of Ca P deposit existed as adsorbed form, the deposition was performed under the conditions in which Pi was omitted from calcifying medium. The results of these experiments showed that substantial amount of PPi and Ca deposits remained the same and was not correlated to the amount of Pi in these deposits. In contrast, Pi of CaP was decreased if Pi was omitted from the calcifying medium. Thus, it appears that the major portion of PPi exists as mineral rather than adsorbed form.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1983

Purification and partial characterization of ATP pyrophosphohydrolase from fetal bovine epiphyseal cartilage.

ATP pyrophosphohydrolase was partially purified from fetal bovine epiphyseal cartilage. The purification was about 10- and 100-fold over the enzyme activities of matrix vesicle fraction and cell homogenate, respectively. The pyrophosphohydrolase and alkaline phosphatase were separated by a sequential application of Sepharose CL-6B and DEAE-cellulose column chromatographies. The purified enzyme migrated as a single band corresponding to the molecular weight of 230,000 in sodium dodecyl sulfate-polyacrylamide disc gel by electrophoresis. The enzyme absolutely required Zn2+ for its activity and appeared to bind Zn2+ strongly with an apparent affinity of p[Zn2+]0.5 = 13.4. The apparent Km for ATP was 0.18 mM. The enzyme was also reactive toward various nucleoside triphosphates including GTP, CTP, and UTP. In contrast, various phosphodiesters including RNA, UDP-glucose, NAD, and bis-p-nitrophenylphosphate were 5% or less as reactive as the nucleoside triphosphates. The pyrophosphohydrolase was inactive toward adenosine 3':5'-monophosphate or various phosphonates. UDP-glucose (1 mM), NAD (1 mM), or RNA (1 mg/ml) failed to inhibit the ATP pyrophosphohydrolase activity. These observations suggest that the ATP pyrophosphohydrolase of the cartilage is probably not a phosphodiesterase I. The matrix vesicle fraction, which probably also included some plasma membrane vesiculated during collagenase digestion, contained the highest specific activity of the enzyme as compared to other subcellular fractions of either epiphyseal or articular cartilage.

Adenosine Triphosphatases↗
PubMed1983

Electron microscopic localization of adenosine triphosphate (ATP)-hydrolyzing activity in isolated matrix vesicles and reconstituted vesicles from calf cartilage.

The presence and distribution of adenosine triphosphatase (ATPase) activity in isolated matrix vesicles and reconstituted vesicles from fetal calf epiphyseal growth plate cartilage was studied by electron microscopic cytochemical methods to determine whether phosphatase activity would be found concentrated on the inside or the outside of matrix vesicle membranes or on both sides, and whether reconstitution of vesicles from deoxycholate-solubilized substituents would lead to the reassembly of membranes with ATPase incorporated. ATPase activity was observed on both the outer and inner surfaces of the investing membranes of isolated matrix vesicles and reconstituted vesicles. A transmembrane location of ATPase could indicate phosphate transfer across the vesicle membrane. Orthophosphate released by phosphatase activity within the protected microenvironment of the matrix vesicle could combine with membrane- or lipid-bound calcium, known to be present in vesicles, to form the first hydroxyapatite mineral during calcification.

Adenosine Triphosphatases↗
PubMed1978

Calcification of isolated matrix vesicles and reconstituted vesicles from fetal bovine cartilage.

Ca deposition by isolated matrix vesicles from fetal calf growth plate cartilage and by a deoxycholate extract from matrix vesicles that included their phosphatase was studied under defined in vitro conditions. Electron microscopy showed that after removal of deoxycholate and lyophilization of the vesicle extract, new vesicles were reconstituted, often with multiple membrane layers. Both intact calf vesicles and reconstituted vesicles initiated Ca deposition maximally when supplied with ATP, GTP, CTP, or UTP. Only nucleoside triphosphates supported Ca deposition well; mono- and diphosphoesters, although hydrolyzed, were ineffective as substrates. Nucleoside triphosphates supported Ca deposition even if the final [Ca] X [P] reached in the reaction mixture was below a metastable level (3.5 mM2), suggesting that matrix vesicles or reconstituted vesicles promote calcification by localizing Ca or PO4 or both. ATP or GTP supported Ca deposition readily at concentrations ranging from 0.25 to 1.0 mM but, at 2.5 and 5.0 mM, Ca deposition was inhibited. The ATPase of intact matrix vesicles and reconstituted vesicles was stimulated by addition of Ca2+ and Mg2+. Ca deposition did not require additional Mg2+. These results lend support to the hypothesis that matrix vesicles and their phosphatases play an important role in mineralization.

Adenosine Triphosphatases↗
PubMed1977

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.

Adenine Nucleotides↗
PubMed1975

Specific adenosine binding proteins from rat liver.

Specific adenosine-binding proteins from homogenates of rat liver have been fractionated on a DEAE-cellulose column. Three major peaks have been identified with respect to histone phosphokinase and cAMP and adenosine-binding activities. Peak I contains only histone phosphokinase activity not stimulated by cAMP. Peak II contains histone phosphokinase slightly stimulated by cAMP. Both cAMP- and adenosine-binding activities are found in this fraction. The major adenosine-binding protein is associated with Peak III. Histone phosphokinase in Peak III which also binds cAMP is stimulated 2-fold by 2.5 muM cAMP whereas adenosine at 2.5 X 10(-4)M inhibits these enzymes equally well in each of three peaks. The specificity of adenosine binding is discussed.

Adenosine↗