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In order to provide some insight into the mechanism of hyaluronate synthesis, the subcellular localization of the synthetase system for hyaluronate was determined in eukaryotic cells. The mouse oligodendroglioma cell line G26-24, which produces copious amounts of hyaluronate in culture, was chosen as a system for these studies. Protease treatment and homogenization of cells followed by hyaluronate synthetase assay suggested that nucleotide-binding sites and trypsin-sensitive synthetase sites were not exposed at the outer membrane surface. Protease treatment following homogenization did result in decreased activity. Membrane fragments, prepared by gentle homogenization in iso- and hypotonic buffers, were subjected to differential centrifugation followed by several continuous and discontinuous sucrose equilibrium and velocity gradient systems. Hyaluronate synthetase activity co-fractionated with a plasma membrane marker in all systems, including those in which Golgi markers were separable. Treatment of intact cells in culture with several hyaluronidases resulted in a marked stimulation of cell-free synthetase activity. The stimulated activity was also found exclusively in plasma membrane-enriched fractions.
Philipson et al. (Sun,) studied this question.