Polypeptides, leucine incorporation into

Further the effects of thialysine on lysine or leucine incorporation into polypeptides were studied,using polypeptide synthesizing systems from E.coli,rat liver or rabbit reticulocytes. It was shown that in all three systems thialysine inhibits incorporation of labelled lysine into polypeptides,whereas it is without effect on leucine incorporation. The results obtained are reported in Table II. 

Per cent of inhibition of lysine or leucine incorporation into polypeptides by thialysine.Experimental details in... 

Per cent of inhibition of C-proline or C-leucine incorporation into polypeptides by j -thiaproline(jJ-T) and y-thiaproline (y-T). Polypeptide synthesizing systems from E.coli,rat liver and rabbit reticulocytes. Experimental details in (56). 

Molnar et al. (1964) found that puromydn administered to intact rats almost completely inhibited incorporation of both glucosamine-l- C and DL-leucine-l- C into liver and plasma proteins. Inhibition of protein synthesis by puromycin indicates operation of the usual ribosomal mechanism of protein synthesis. Since the great bulk of carbohydrate incorporation into glycoprotein appears to occur by postribosomal mechanisms (see Section IV), inhibition of carbohydrate incorporation by puromycin suggests that there is no appreciable reservoir of nonglycosylated polypeptide in either rat liver or sheep submaxillary gland. 

The possibility that J-thiaproline could be incorporated into polypeptides in place of proline was then investigated by studying its effect on the incorporation of labelled proline or leucine.Protein synthesizing systems from E.coli,rat liver and rabbit reticulocytes were... 

Further,studying the effect of puromycin on growing polypeptide chains labelled with I C-leucine in the presence or absence of J-thiaproline,data were obtained indicating that j3-thiaproline once incorporated into polypep-... 

Their studies involved the partial polymerization of NCAs of mixtures of specific amino adds having known e.e.s, followed by determination of the e.e.s of the amino adds in both the resulting polypeptides and in the residual unreacted NCA monomers. [94] In a typical experiment it was found that when an optically impure leucine NCA monomer having an l > d e.e. of 31.2% was polymerized to the extent of 52 % to the helical polyleucine peptide, the e.e. of the polymer was enhanced to 45.4 %, an increase of 14.2 %. In the same experiment the e.e. of the unreacted leucine NCA monomer was depleted to a similar extent. Analogous experiments with valine NCAs of known e.e.s, however, led to a reverse effect, namely, the preferential incorporation of the racemate rather than one enantiomer into the growing polyvaline peptide. This finding was interpreted to be the result of the fact that polyvaline consists of (3-sheets rather than a-helices, emphasizing that the Wald mechanism applies only to a-helix polymers. At about the same time Brach and Spach [95] showed that, under proper conditions, (3-sheet polymers could also be implicated in the amplification of amino add e.e.s. 

These results indicated that thiaisoleucine may be incorporated in place of isoleucine into polypeptides,in competition with isoleucine,and that its incorporation does not impair the polypeptide synthesis. This was confirmed by the analysis of the polysomal pattern and of the polysomal-bound radioactivity. Rabbit reticulocyte lysates were incubated,in the presence of all the factors necessary for protein synthesis,with labelled isoleucine or leucine, and with or without thiaisoleucine. As shown in Fig.3 the polysomal profile remained unchanged in the presence or absence of thiaisoleucine,while the polysomal-bound radioactivity due to isoleucine was highly reduced in the presence of thiaisoleucine.On the other hand the polysomal-boiind radioactivity due to leucine was unaffected by the presence of thiaisoleucine. These results showed that the incorporation of thiaisoleucine into the polypeptide chain does not modify the ribosome run-off nor the elongation rate of the growing polypeptide chain. 

In the mammalian systems the differences between the two analogs are less evident as regards the affinity for aminoacyl-tENA synthetases and the capacity to bind tENA. On the other hand -thiaproline inhibits to a higher extent the incorporation of both proline and leucine into polypeptides,impairing further chain elongation once incorporated. 

An essential part of the rationale presented above under (a) consists of the identification of altered products of mitochondrial protein synthesis as a result of the mutation. Although this is not a sufficient criterion for mitochondrial specification (since an altered protein might arise as a result of a mutational alteration in a component of the mitochondrial protein-synthesizing machinery, i.e., one of the mt r- or tRNAs, (as in poky Neu-rospora), it is a necessary one. We have therefore devoted considerable effort to demonstrating the capability of the mitochondria in the mutant to perform some form of protein synthesis. We did this by showing that they were capable of incorporating (1) labeled formate into formylmethionyl-puromycin as a measure of mitochondrial polypeptide-chain initiations (see also next section) (2) labeled leucine into mitochondrial membrane proteins in a reaction that is insensitive to cycloheximide (CHX), but sensitive to chloramphenicol (CAP) and (3) that continued exposure of cells to the latter led to their conversion to petite phenocopies, s5 of characteristic aspects of their phenotype, such as the presence of cytochrome b, and that this change was reversed on removal of the inhibitor (see also Table I). 

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