Stimulation of myoblast membrane protein synthesis by 25-hydroxy-vitamin D3

The effects of 25-hydroxy-vitamin D3 (25 OHD3) on myoblast protein synthesis were studied in connection with its role on muscle cell phosphate metabolism. The sterol markedly increased leucine incorporation into total cell proteins in cultured chick embryo myoblasts. This enhancement was greater than that produced by 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) and occurred prior to a significant stimulation of cell phosphate accumulation. Maximum effects of 25 OHD3 (8 h) on myoblast phosphate uptake were suppressed by cycloheximide indicating that they are mediated by de novo protein synthesis. At a similar treatment period, labelling of myoblasts with [3H]leucine (control) and [14C]leucine (+25 OHD3) followed by co-electrophoresis of total protein extracts on SDS-PAGE and isoelectrofocusing gels revealed that the sterol selectively affects the synthesis of proteins of 20 kDa and 50 kDa. These macromolecules were recovered in the microsomal fraction after differential centrifugation of homogenates. Further fractionation of myoblast microsomes on sucrose density gradients showed co-localization of the 50 kDa and 20 kDa proteins with microsomal subfractions which preferentially bind [3H-alpha]bungarotoxin, suggesting that the proteins induced by 25 OHD3 are associated to plasma membranes and may play a role in the effects of the sterol on cell phosphate uptake.