We have undertaken a site directed mutational analysis of two of the preserved regions in the amino acid sequence of Dam methylase in order to characterize their role. Mutations in region IV (sequence DPPY) abolish catalytic activity and greatly affect AdoMet crosslinking. Mutants in region III display a lowered specific activity with an unchanged AdoMet crosslinking capacity. We have also made a series of deletions both at the N and C terminal parts of the protein, which have been found to provide inactive enzyme. We discuss the significance of these results for the understanding of the functional properties of the enzyme.
Primary cultivated rabbit articular chondrocytes were immobilized in calcium alginate beads. Both free and entrapped cells were allowed to grow under normal conditions. After long-term immobilization, the cells still exhibited metabolic activities, patterns of division, synthesis and secretion of extracellular matrix macromolecules such as type II collagen and proteoglycans. After 38 days, immobilized rabbit articular chondrocytes predominantly expressed type II but not type I collagen. Thus, they maintained their cartilage pheno-type. After bead lysis, harvested cells showed normal growth patterns when resuspended in culture medium. On the basis of these results, long-duration storage and large-scale production of extracellular matrix components are being investigated.
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