Abraham Marcus

[68] The wheat embryo cell-free system☆

Publisher Summary Cell-free extracts of wheat embryos catalyze the incorporation of radioactive amino acids into protein in response to several mRNAs. The translational fidelity of the system has been established by the analysis of the products of reactions catalyzed by STNV-RNA, by BMV-RNA and by rabbit globin mRNA. The system is most active with eucaryotic mRNAs but Q β -RNA can also be translated in the wheat system yielding authentic phage coat protein. This chapter describes the methods used for isolating viable wheat embryos and for carrying out a typical TMV-RNA catalyzed amino acid incorporation reaction.

[11] Protein chain initiation in wheat embryo

Publisher Summary Wheat embryo extracts contain four soluble factors that are required for the incorporation of radioactive amino acids into protein in response to plant viral mRNA. Two of these factors function in initiation and two in elongation. This chapter describes the resolution of the factors from wheat embryo supernatant and some of the characteristics of the initiation reactions.

The effect of phenazine methosulfate and pyocyanine on the L-amino acid oxidase reaction

Abstract 1. Phenazine methosulfate stimulates the oxidation of certain amino acids by L -amino acid oxidase. A study of the conditions affording maximal stimulation indicates that the mode of action of phenazine methosulfate involves the reoxidation of the reduced enzyme-flavin. 2. Pyocyanine inhibits the oxidation of certain amino acids by L -amino acid oxidase. A study of conditions required for maximal inhibition, suggests that the mode of action of pyocyanine involves reduction of the enzyme-flavin to a form reacting poorly with oxygen. This concept is discussed in the light of a suggested mechanism for the L -amino acid oxidase reaction. 3. Spectrophotometric assays for L -amino acid oxidase are described, using phenazine methosulfate as a bridge between enzyme-flavin and indophenol or ferricyanide.

Initiation factors elF4A and C1 from wheat germ and the formation of mRNA · ribosome complexes

Abstract The binding of ribosomes to mRNA is analyzed in a fractionated system from wheat germ with [3H]uridine-labeled poly(A)+ RNA prepared from germinating wheat embryos. The reaction requires factors eIF3, eIF4C, and eIF5; Met-tRNA and the Met-tRNA binding system; either GTP or GMP-PNP; ATP; and factors C1 and eIF4A. These requirements are identical to those previously found to be necessary for formation of ribosome · Met-tRNAiMet complexes, with the exception of ATP, and factors C1 and eIF4A. The function of factors C1 and eIF4A is therefore specifically related to the mRNA attachment reaction. The presence of GTP in the mRNA binding reaction results in the formation of 80 S ribosome complexes, while with GMP-PNP only 40 S ribosome complexes are formed. Ribosome binding to native reovirus RNA in the fractionated wheat germ system is similar to the reaction with poly(A)+ RNA, strongly requiring ATP and factors C1 and eIF4A. Binding to inosine-substituted reovirus RNA, however, is only partially dependent upon ATP, and both the ATP-dependent and the ATP-independent binding reactions strongly require factor C1 and are substantially stimulated by factor eIF4A. The ATP-independent reaction is inhibited by pm7GDP, has a strong requirement for Met-tRNAiMet, and the 40 S ribosome complex is stable to RNase. These results indicate that the ATP-independent binding of ribosomes to inosine-substituted reovirus RNA proceeds through the normal initiation process. They further suggest that neither factor C1 nor eIF4A function exclusively to unwind mRNA secondary structure. Since eIF4A is required for the ATP-independent binding to inosine mRNA, and at the same time interacts with ATP in the reaction with ATP-requiring mRNAs, this factor may have two roles in protein chain initiation, one related to the mRNA · ribosome interaction, and one related to the function of ATP.

Functional Characterization of the Initiation Factors of Wheat Germ

Protein biosynthesis is one of the first biological processes “activated” by exposure of wheat embryos to water (1). A major facet of the “activation” process is the attachment of ribosomes to preformed mRNA (2), and the reaction can, in part, be reproduced in vitro (3). In further studies it was found that the ribosome attachment reaction could be carried out with a number of eucaryotic mRNAs, in particular with plant viral RNAs (4, 5, 6). Utilizing particularly TMV-RNA, we have studied aspects of the initiation reaction in vitro and the subsequent sections summarize the current status of these studies.

Preparation and properties of γ-methyleneglutamic acid

Abstract Procedures are described for the preparation of dl -γ-methylene glutamic acid, d -γ-methyleneglutamic acid, N -acetyl- dl -γ-methyleneglutamic acid, and γ-amino-α-methylenebutyric acid. A new method for determining γ-methyleneglutamic acid is described. The method involves heating with concentrated hydroxylamine and does not yield appreciable color with other monoaminodicarboxylic acids. Glutamine synthetase preparations from peanut cotyledons and liver glutamic dehydrogenase do not catalyze their respective reactions with γ-methyleneglutamate. Preliminary evidence suggests that peanut extracts catalyze a glutamic dehydrogenase type reaction with γ-methylene glutamate.