Glial-released proteins: II. Two-dimensional electrophoretic identification of proteins regulated by hydrocortisone

The proteins released into the culture medium (CM) by confluent C6 glioma cell monolayers were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). These cells synthesize and release a complex set of proteins which can be resolved on the basis of molecular charge and size. Over 400 spots on fluorograms corresponding to radio-labeled glial-released protein (GRP) were detected and classified according to their positions in 2-D gels. The marked species charge heterogeneity of the 2-D pattern was used as a criterion to assign the majority of GRP components to "series'. Series are composed of families of related proteins or glycoproteins distributed in a line of evenly spaced members in the isoelectric focusing dimension. Long-term exposure of monolayers to 2 microM hydrocortisone influenced the accumulation in the culture medium of half of the classified GRP species. Five classes of GRP were identified based on their steroid-responsiveness as seen in the deviation of GRP radiolabel ratios from control and hormone-treated culture CM. Hormonal response, verified by reverse-label experiments, showed a number of GRP in CM are either consistently increased 1- to 7- fold (Classes I and II) or decreased 1- to 3-fold (Classes IV and V). The remaining GRP (Class III) included those proteins which were found to be uninfluenced or to change in a week or inconsistent manner. Some GRP series were coordinately induced while other series gave graded responses. These results represent the first high-resolution classification of GRP by physical and biological properties.