Effects of stratification on in vitro protein synthesis using components from embryos of Pinus lambertiana Dougl.

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Virginia Tech


An vitro protein synthesizing system using Sephadex G-25 gel filtration for supernatant purification was used to determine the ability of extracts from embryos of dormant and stratified sugar pine seeds to promote protein synthesis. It was found that the ribosomes from the embryos of dormant seeds were more active than those from stratified seeds but they also required a greater quantity of supernatant protein to produce this activity. The supernatant fraction from stratified embryos was more active than that from dormant embryos but there were indications that this increase was due to seed imbibition. Concentrations of other essential components of the system were the same for both dormant and stratified embryo systems. In each 0.5 m1 sample, maximum incorporation occurred using 0.03-0.06 mg ribosomal protein, 0.795 μmo1es ATP, 1.8 μmo1es PEP, 13.2 μgms pyruvate kinase, 0.090 μmoles GTP, and 0.0375 mg polyuridylic acid. Optimum incubation conditions were at 37°C for 60 minutes. Ribonuclease and protease inhibited phenylalanine incorporation.

Ribonuclease activity in the supernatant fraction increased with purification and was significantly higher in the dormant embryos than in the stratified. Protease activity decreased with purification of the supernatant and there was no significant difference between activity in the dormant and stratified embryo supernatant fractions. Protease activity was high in the ribosomal fraction. Ribosomes from dormant embryos appeared to bind more polyuridylic acid than did those from stratified embryos. Resedimented ribosomes consisted primarily of monoribosomes but some polyribosomes were present in both dormant and stratified embryos. Analysis of incubation mixtures produced similar results. The majority of the labelled polyphenylalanine was associated with the monoribosomes.