"On protein synthesis"

Date:
1958
Reference:
PP/CRI/I/1/16
Catalogue details

Licence: In copyright

Credit: "On protein synthesis". Source: Wellcome Collection.

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    in all cells engaged in protein synthesis. Recently Cole, Coote & Work(i957) have reported their presence in a variety of tissues from a number of animals. So far good evidence has been found for this reaction for about half the standard twenty amino acids, but it is believed that further research will reveal the full set. Meanwhile Davie, Koningsberger & Lipmann (1956) have purified the tryptophan-activating enzyme. It is specific for trypto phan (and certain tryptophan analogues) and will only handle the l -isomer. Isolation of the tyrosine enzyme has also been briefly reported (Konings berger, van de Ven & Overbeck, 1957; Schweet, 1957). The properties of these enzymes are obviously of the greatest interest, and much work along these lines may be expected in the near future. For example, it has been shown that the tryptophan-activating enzyme contains what is probably a derivative of guanine (perhaps GMP) very tightly bound. It is possible to remove it, however, and to show that its presence is not necessary for the primary activation step. Since the enzyme is probably involved in the next step in protein synthesis it is naturally suspected that the guanine derivative is also required for this reaction, whatever it may be. In vitro incorporation In order to study the relationship between the activating enzymes and the microsomal particles it has proved necessary to break open the cells and work with certain partly purified fractions. Unfortunately it is rare to obtain substantial net protein synthesis from such systems, and there is a very real danger that the incorporation of the radioactivity does not represent true synthesis but is some kind of partial synthesis or exchange reaction. This distinction has been clearly brought out by Gale (1953). The work to be described, therefore, has to be accepted with reservations. (See the remarks of Simkin & Work, this Symposium.) It has been shown, however, in the work described below, that the amino acid is incorporated into true peptide linkage. Again the significant results were first obtained by Zamecnik and his co workers (reviewed in Zamecnik et al. 1956). The requirements so far known appear to fall into two parts: (1) The activation of the amino acids for which, in addition to the labelled amino acid, one requires the ' pH 5 ' fraction, containing the activating enzymes, ATP and (usually) an ATP-generating system. There appears to be no requirement for any of the pyrimidine or guanine nucleotides. (2) The transfer to the microsomal particles. For this one requires the previous system plus GTP or GDP (Keller & Zamecnik, 1956) and of course the microsomal particles; the endoplasmic reticulum does not appear to be necessary (Littlefield & Keller, 1957). Hultin & Beskow (1956) have reported an experiment which shows clearly that the amino acids become bound in some way. They first incubate the mixture described in (1) above. They then add a great excess of ««labelled amino acid before adding the microsomal particles. Nevertheless some of the labelled amino acid is incorporated into protein, showing that it was in some place where it could not readily be diluted. Very recently an intermediate reaction has been suggested by the work of Hoagland, Zamecnik & Stephenson (1957), who have discovered that in the first step the 'soluble' RNA contained in the 'pH 5' fraction became labelled with the radioactive amino acid. The bond between the amino acid and the RNA appears to be a covalent one. This labelled RNA can be extracted, purified, and then added to the microsomal fraction. In the presence of GTP the labelled amino acid is transferred from the soluble RNA to microsomal protein. This very exciting lead is being actively pursued. Many other experiments have been carried out on cell-free systems, in particular by Gale & Folkes (1955) and by Spiegelman (see his review, 1957), but I shall not describe them here as their interpretation is difficult. It should be mentioned that Gale (reviewed in Gale, 1956) has isolated from hydrolysates of commercial-yeast RNA a series of fractions which greatly increase amino acid incorporation. One of them, the so-called 'glycine incorporation factor' has been purified considerably, and an attempt is being made to discover its structure. RNA turnover and protein synthesis From many points of view it seems highly likely that the presence of RNA is essential for cytoplasmic protein synthesis, or at least for specific protein synthesis. It is by no means clear, however, that the turnover of RNA is required. In discussing this a strong distinction must be made between cells which are growing, and therefore producing new microsomal particles, and cells which are synthesizing without growth, and in which few new microsomal particles are being produced. This is a difficult aspect of the subject as the evidence is to some extent conflicting. It appears reasonably certain that not all the RNA in the cytoplasm is turning over very rapidly—this has been shown, for example, by the Hokins (1954) working on amylase synthesis in slices of pigeon pancreas, though in the light of the recent work of Straub (this Symposium) the choice of amylase was unfortunate. On the other hand Pardee (1954)