Human articular chondrocytes with higher aldehyde dehydrogenase activity have stronger expression of COL2A1 and SOX9.

OBJECTIVE: To determine in human articular chondrocytes the activity of Aldehyde dehydrogenase (ALDH), which are reported as stem/progenitor cell marker in various adult tissues and evaluate gene expression of ALDH1A isoforms. DESIGN: ALDH activity was evaluated by flow cytometry with Aldefluor™ assay in cells, isolated from human osteoarthritic (OA) cartilage. Its coexpression with surface markers was identified. Cells were sorted according to ALDH activity, and gene expression in sorted populations (ALDH(+) and ALDH(-)) was analyzed by RTq-PCR with Taqman(®) assay. RESULTS: About 40% of freshly isolated chondrocytes demonstrated ALDH activity that remarkably declined during monolayer culture. Markers CD54 and CD55 were significantly stronger expressed, while CD47, CD140b, CD146 and CD166 were depleted in ALDH-expressing (ALDH(pos)) cells. Gene expression analysis revealed significantly higher expression of chondrocyte-specific genes COL2A1, SOX9 and SERPINA1 and lower expression of osteogenic markers RUNX2 and osteocalcin (BGLAP) in sorted ALDH(+) fraction. COL1A1, ACAN, ALPL and stem cell markers NANOG, OCT4, SOX2 and ABCG2 did not differ remarkably between the populations. Genes of isoenzymes ALDH1A2, ALDH1A3 and ALDH2 were strongly expressed, while ALDH1A1 was weakly expressed in chondrocytes. Only ALDH1A2 and ALDH1A3 were significantly enriched in ALDH(+) fraction. CONCLUSIONS: We identified ALDH activity with significantly stronger expression of CD54 and CD55 in human articular chondrocytes. Gene expression of isotypes ALDH1A2, ALDH1A3 and ALDH2 was identified. Coexpression of ALDH activity with chondrogenic markers suggests its association with collagen II producing chondrocyte phenotype. Isotypes ALDH1A2 and ALDH1A3 can be associated with the ALDH activity in these cells.

Inhibition of inorganic pyrophosphatase of animal mitochondria by calcium.

Calcium ion is an uncompetitive inhibitor of the inorganic pyrophosphatases of bovine heart and rat liver mitochondria with respect to substrate MgPPi at pH 8.5 and a non-competitive inhibitor of the former enzyme at pH 7.2. The concentration of Ca2+ required to decrease the maximal velocities for both enzymes at pH 8.5, 0.4 mM Mg2+ was about 10 microM. The inhibition results from the binding of two Ca2+ ions to both free enzymes and their complexes with the substrate. The results suggest that Ca2+ regulates pyrophosphatase activity and hence PPi level in mammalian mitochondria.

Kinetic models for the action of cytosolic and mitochondrial inorganic pyrophosphatases of rat liver.

Initial rates of PPi hydrolysis by cytosolic and mitochondrial inorganic pyrophosphatases of rat liver have been measured in the presence of 0.2-100 microM MgPPi and 0.01-50 mM Mg2+ at pH 7.2 to 9.3. The apparently simplest model consistent with the data for both enzymes implies that they bind substrate, in the form of MgPPi, and three Mg2+ ions, of which two are absolutely required for activity. The third metal ion facilitates substrate binding but decreases maximal velocity for the cytosolic enzyme, while substrate binding is only modulated for the mitochondrial enzyme. The model is also applicable to bovine heart mitochondrial pyrophosphatases. The active form of the substrate for the cytosolic pyrophosphatase is MgP2O7(-2); the catalytic and metal-binding steps require a protonated group with pKa = 9.2 and an unprotonated group with pKa = 8.8, respectively. The results indicate that the mitochondrial pyrophosphatase is more sensitive to variations of Mg2+ concentration in rat liver cells than is the cytosolic one.