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 > 25 000) 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 MgZf 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 ~-[~~S]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 shortterm effect whose efficiency was greatly reduced within the 15 h of experimentation, even under continuous irradiation.Phenylalanine ammonia-lyase catalyzes the conversion of phenylalanine to cinnamic acid and is the first of a sequence of more than 10 enzymes which are involved in the light-induced formation of flavone
A specific antiserum, raised against puified phenylaanine ammonialyase from irradiated ce spenio cultur of parsey ( The diution of cell cultus had no sigificant effect on the total rate of incorporation of radioactvity into protein in vivo or on the general template activity of the polyibosomal RNA in vitro throughout at least 25 hours.In dark-grown cell suspension cultures of parsley, the activity of PAL2 could be induced either by irradiation with UV light (12) or in the dark by dilution of the cell suspensions into fresh medium (13) or into water (11). Previous reports have shown that irradiation caused large increases in the activities of more than 10 enzymes which were all involved in the biosynthesis of flavonoid glycosides (8). The effect of dilution was restricted to increases in the activities of the first three of these enzymes, namely PAL, cinnamate 4-hydroxylase, and 4-coumarate:CoA ligase (13). It seemed therefore possible that the two modes of induction resulted in the formation of two different molecular species of PAL.
Testosterone, Androst-4-en-3,17-dione, Enzyme Induction, S trep to m yces hydrogenans After cultivation of S trep to m yces h ydrogenan s in the presence of 3H-labelled testosterone, radio active steroids were extracted separately from the cytosolic, ribosomal and cell wall-membrane fraction of the cells and from the culture medium, respectively.. The separation of the steroids was performed by one-and two-dimensional thin layer chromatography (TLC). The identification of the main metabolites was achieved by crystallization to constant specific radioactivity, specific staining procedures and acetylation. The oxidation of testosterone to androst-4-en-3,17-dione is by far the predominating reaction, which is almost finished after 3 h cultivation. Androst-4-en-3,17-dione is mainly transferred into the culture medium and partly accumulated within the cell wallmembrane fraction. High polar steroid metabolites and androstane derivatives are present in very small amounts only.
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