Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA.

We have developed monoclonal antibody 5109 against a unique highly acidic sequence in type II collagen. When paired with previously reported monoclonal antibody 9A4, 5109 can be used as the capture antibody in an ELISA assay for the neoepitope generated by collagenase-cleavage of type II collagen. The assay detects the sequence ZGlyGluX(759)GlyAspAspGlyProSerGlyAlaGluGlyProX(771)GlyProGlnGly(775) where Z is a variable length polypeptide, X is proline or hydroxyproline, and Gly(775) corresponds to C-terminal amino acid of the 3/4 piece after collagenase cleavage. Antibody 5109 detects the first and 9A4 the second underlined sequence. Antibody 5109 recognizes its epitope with a K=1.2x10(-8) M independently of hydroxylation of X(759). When X(771) is proline, the sequence is 90x more sensitively detected by this ELISA than when it is hydroxyproline. Type II collagen of human articular cartilage was fragmented by cyanogen bromide (CNBr) and trypsin. The immunoreactive fragment was captured with 5109 and sequenced. Proline(771) averaged 81% hydroxylated. Other 3rd position prolines were >97% hydroxylated. In urine of control individuals of 50-70 years of age, we failed to detect the presence of the collagen fragment in a majority (8/10) of specimens. The two controls with measurable levels averaged 123 pM. In a similar age cohort of osteoarthritic patients, the majority (9/10) showed measurable values of urinary collagen fragments averaging 312 pM. This assay can be used for monitoring type II collagen metabolism in patients with osteoarthritis.

Pyruvate dehydrogenase complex from ribbed mussel gill mitochondria.

The pyruvate dehydrogenase complex has been demonstrated in high speed pellet preparations from sonicated ribbed mussel gill mitochondria. The activity of the complex is inhibited by low chloride (less than 100 mM) concentrations, EDTA (1 mM), succinate, ATP, and NAD/NADH ratios below 4. Inhibition by EDTA is relieved by addition of 10 mM MgCl2-1 mM CaCl2. ATP inhibition was enhanced by NaF and reversed by high Mg++ concentrations in the absence of NaF. Pyruvate and thiamine pyrophosphate inhibited the inactivation by ATP. The nonhydrolyzable ATP analog AMP-PNP caused inhibition of the overall catalytic activity that was identical to ATP. Factors involved in the ATP inhibition and Mg++ reversal are lost with freezing or cold storage. Preliminary results using gamma-32P-ATP indicate that a protein kinase that phosphorylates the alpha subunit of E1 (pyruvate dehydrogenase) from the mammalian PDC is associated with the gill PDC. The activity of the complex may be regulated by a phosphorylation/dephosphorylation mechanism and by the relative levels of substrates, products, and other metabolites in the mitochondria.

Subcellular distribution of aminotransferases, and pyruvate branch point enzymes in gill tissue from four bivalves.

Aspartate aminotransferase (AAT), alanine aminotransferase (ALAT), malic enzyme (ME), malate dehydrogenase (MDH), pyruvate kinase (PK), and phosphoenolpyruvate carboxykinase (PEPCK) activities in cytosolic and mitochondrial fractions of gill tissue from Modiolus demissus (ribbed mussel), Mytilus edulis (sea mussel), Crassostrea virginica (oyster) and Mercenaria mercenaria (quahog) were determined using enzyme assay and starch gel electrophoresis combined with subcellular fractionation. AAT showed distinct mitochondrial and cytosolic isozymes in gills of all these animals. Although ALAT showed distinct mitochondrial and cytosolic isozymes in the gills of oysters, sea mussels and quahogs, only the mitochondrial ALAT was evident in ribbed mussel gill tissue. PK and PEPCK were cytosolic in all these preparations. ME was found only in the mitochondrial fraction of ribbed mussel and quahog gill tissue whereas sea mussel gills showed distinct cytosolic and mitochondrial ME isozymes. With oyster gills, the "cytosolic ME" was electrophoretically identical to the mitochondrial ME indicating that in vivo, the ME is probably mitochondrial. MDH showed distinct cytosolic and mitochondrial isozymes in all bivalve gills tested.

A rapid method for the purification of fatty acid synthetase from the yeast Saccharomyces cerevisiae.

A rapid method for the isolation and purification of small quantities of highly active fatty acid synthetase (FAS) from several strains of the yeast Saccharomyces cerevisiae, is presented. The purification procedure which is the shortest reported to this date (18 hr), involves the release of the enzyme by either cell wall digestion with Zymolyase 60000 or cell wall disruption by glass beads, followed by 35-50% ammonium sulfate fractionation, desalting by Sephadex G-25 chromatography, then calcium phosphate gel treatment, concentration by 50% ammonium sulfate precipitation, sedimentation of the enzyme in the ultracentrifuge and finally, column chromatography on DEAE Bio-Gel A. Fatty acid synthetase prepared by the cell breakage method, was found to be homogeneous according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-Tris-glycine disc gel electrophoresis and immunoelectrophoresis criteria. However, enzyme prepared from Zymolyase treated cells showed several proteolytic bands in addition to FAS bands, on SDS-PAGE. Enzyme obtained by both methods of cell breakage, showed a similar behavior throughout the purification procedure and gave a similar yield of enzyme of high specific activity (4800-7200 nmol/min/mg) that remained stable for several months at -85 degrees C.