Direct incorporation of hydroxyproline into protein of sycamore cells incubated at growth-inhibitory levels of hydroxyproline.

Free hydroxyproline inhibits the growth of animals1' I and plants.3-7 The mechanism of this inhibition is not known. Among the theoretically possible effects of hydroxyproline are: (1) reduction of protein synthesis by lowering proline incorporation, (2) interference with proline hydroxylation, and (3) direct incorporation of hydroxyproline resulting in nonfunctional proteins. Hydroxyproline residues in collagen arise from an hydroxylation of peptide-bound proline8 (although data suggesting a very slight direct incorporation of hydroxyproline into chick embryo tissues have been reported).I A similar origin has been proposed for the hydroxyproline present in plant cell-wall protein.10 11 Thus any direct incorporation of hydroxyproline might result in nonfunctional proteins. Hitherto the major difficulty in demonstrating a direct incorporation of hydroxyproline arose from the alternative possibility of an indirect incorporation, through conversion of hydroxyproline to proline, followed by incorporation into protein and subsequent hydroxylation. This possibility can now be eliminated by the use of aax'-dipyridyl, a recently discovered inhibitor of proline hydroxylation.'2 Materials and Methods. A 10-ml portion taken from suspension cultures of exponentially growing sycamore cells (Acer pseudoplatanus) was used for each treatment.13 The cultures were grown in a 20 per cent coconut-milk medium14 with constant agitation by a gyratory shaker. After treatment with or without various inhibitors as described (Tables 1, 2, and 3), the cells were drained over a sinteredglass filter, rinsed with distilled water, disrupted by sonication, and separated by low-speed centrifugation into pellet and supernatant fractions. The cell-wall fraction was obtained from the pellet by extensive washing with 1 M1 NaCl, containing 200 ,ig/ml of L-proline and of L-hydroxyproline, followed by water washings. After each washing the cell-wall fragments were resedimented by a low-speed, oneminute centrifugation. Finally the pellet was suspended in water and portions taken for dry-weight determination and for hydrolysis (6 N HCl, 105°, 36 hr). The supernatant fraction from the sonicated cells was made to 10 per cent trichloroacetic acid (TCA) and the precipitate centrifuged down after one hour at 2°. The pellet was dissolved in 0.5 N NaOH containing 200 ,g/ml of L-proline and of L-hydroxyproline. Sufficient TCA was added to reprecipitate the protein which was again collected by centrifugation, twice washed with 5 per cent TCA, and then dissolved in 2 N NH40H. Portions were taken for quantitative assay1 and for hydrolysis. All hydrolysates were applied to Whatman no. 4 paper and separated electrophoretically at pH 1.9.16 Labeled amino acids, detected by radioautography, were eluted off the paper, and their radioactivity was determined by liquid scintillation counting. Portions of the filtered incubation media, collected earlier, were cleared by centrifugation and also counted. Results and Discussion.-Sycamore cells incubated either with proline-C14 or