Enzymatic activity and in vivo distribution of 5′-nucleotidase, an extracellular matrix binding glycoprotein, during the development of chicken striated muscle

The ecto-enzyme 5′-nucleotidase isolated from chicken gizzard has previously been shown to be a potent ligand of two glycoproteins of the extracellular matrix, namely fibronectin and laminin. Using immunofluorescent labeling techniques we observed that 5′-nucleotidase codistributed with laminin during the development of chicken striated muscle. In contrast, ecto-5′-nucleotidase was only faintly detectable on cells surrounded by a matrix expressing high levels of fibronectin. This distribution pattern distinguished 5′-nucleotidase from the pluripotent extracellular matrix receptors, chicken β1-integrins, which are expressed equally well in muscle and connective tissue. In addition, the specific activity of striated muscle ecto-5′-nucleotidase was stable during development and increased markedly posthatching. At each age considered, this specific activity corresponded to an 80-kDa enzyme which was inhibited by α,β-methyleneadenosine diphosphate or by a monoclonal antibody directed against the smooth muscle isoform of the enzyme. Previous in vitro studies have revealed that 5′-nucleotidase is involved in the spreading of various mesenchyme-derived cells, such as chicken embryonic fibroblasts and myoblasts, on a laminin substrate. A prerequisite to examining a potential in vivo role for 5′-nucleotidase as an extracellular matrix ligand was to study its distribution. In adult muscle, 5′-nucleotidase displayed a more restricted distribution than in embryo. Results show that, in vivo, 5′-nucleotidase is revealed by immunofluorescent labeling using poly- and monoclonal antibodies to chicken gizzard 5′-nucleotidase in two structures, the costameres and myotendinous junctions, which are closely related to the focal adhesion sites observed in cell culture.