Light‐Induced Enzyme Synthesis in Cell Suspension Cultures of Petroselinum hortense

Abstract

The conditions for protein synthesis in vitro with polyribosomes from cell suspension cultures of parsley (Petroselinum hortense) and a wheat-germ extract were investigated. Two different criteria were used as estimates of the translational activity: (a) the total rate of incorporation of [³⁵S]methionine into acid-insoluble material, (b) the ratio of large (molecular weight > 25000) to small (molecular weight < 25000) peptide products. Depending on which of the two criteria was employed, the pH optimum and the optimal concentrations for Tris-acetate, magnesium acetate, KCl, methionine and the wheat-germ extract differed considerably. The translational activity of the polyribosomes (both criteria) was efficiently protected by 0.1 M Mg²⁺ against degradation during the isolation procedure. The rate of synthesis of phenylalanine ammonia-lyase in vitro with the polyribosomes was determined by measuring the incorporation rate of l-[³⁵S]methionine into protein which was precipitable by a rabbit antiserum preparaed for the purified enzyme. The immunoprecipitate was analyzed by disc;gel electrophoresis in the presence of dodecylsulfate and was shown to contain small amounts of the complete enzyme subunits and relatively large amounts of shorter peptides which were also characteristic for the enzyme. The time courses of light-induced changes in the rate of phenylalanine ammonia-lyase synthesis in vitro were investigated during a period of 15 h under two different conditions of induction: the cell cultures were irradiated with ultraviolet light either (A) continuously or (B) for 2.5 h and then returned to darkness. Although the highest rate of enzyme synthesis was observed somewhat later in experiment A than in experiment B, the periods of time during which the rate of synthesis increased rapidly were limited in both cases to only a few hours. The results obtained in vitro were identical within the limits of the experimental error with theoretical calculations of the changes in the rate constant of phenylalanine ammonia-lyase synthesis in vivo. These changes were calculated from the corresponding curves for the changes in the enzyme activity under the two conditions of induction. The results are in agreement with previous observations suggesting that the induction of phenylalanine ammonia-lyase by light in the parsley cells was a short-term effect whose efficiency was greatly reduced within the 15 h of experimentation, even under continuous irradiation.