The Incorporation of 14 C-Proline into the Proteins of Growing Cells: II. A NOTE ON EVIDENCE FROM CULTURED CARROT EXPLANTS BY ACRYLAMIDE GEL ELECTROPHORESIS

Abstract

The method of acrylamide gel electrophoresis has been applied to the separation of the proteins of growing carrot explants which are soluble in a buffer solution at pH 8.3. At least nine distinct bands were detectable in this way. When carrot tissue had absorbed ¹⁴C-proline it entered into the composition of all these bands and, in all except one, it was converted to hydroxyproline to different degrees which characterized the band in question. Thus, in the soluble protein the ¹⁴C-hydroxyproline: ¹⁴C-proline ratio varied from 0 to 0.53. The bulk of the protein in the tissue was insoluble in buffer at pH 8.3; it contained the bulk of the radioactivity absorbed from proline (84.8 per cent.); and its average ¹⁴C-hydroxyproline : ¹⁴C-proline ratio was the highest of all (1.28). A particulate protein preparation, separated at 25,000 g. from the soluble protein, had an intermediate ratio (0.643) of ¹⁴C-hydroxyproline to ¹⁴C-proline. Therefore, there are in cultured carrot explants many distinct protein moieties which incorporate ¹⁴C-proline, and they convert it to ¹⁴C-hydroxyproline to very different degrees. The evidence is consistent with the incorporation of the proline, first into the various soluble proteins which are electrophoretically separable and subsequently, with progressively greater hydroxylation, into the more insoluble protein that constitutes the bulk of the protein of the cell and its organelles. It is, therefore, quite incorrect to say (as some have done) that all of the hydroxyproline-containing protein is in very close association with the cell wall, for part of it is present in the cell in soluble and electrophoretically separable forms.