1. Peptide-elongation factors were purified from rat liver and hurnan tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzytne from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes. Cholesteryl 14-methylhexadecanoate seems to play an important role in protein synthesis. This compound stimulates the incorporation of labelled amino acids into tRNA of rat liver in vitro (Hradec & Dolejg, 1968). Purified aminoacyl-tRNA synthetases from mammalian tissues lost all or most of their activity after extraction with organic solvents (Hradec & Duiek, 1969). Only about 50% of the normal charging of tRNA was obtained with extracted pH 5 enzymes from rat liver (Hradec & Dugek, 1968b). In both these instances, a complete reactivation of extracted enzymes could be induced by the addition of cholesteryl 14-methylhexadecanoate into incubation mixtures. These results indicated that the presence of this ester in the molecule of aminoacyl-tRNA synthetases is apparently essential for their normal function. However, in contrast with only partially deactivated pH 5 enzyrnes no incorporation of labelled amino acids into ribosomal proteins was found in reaction mixtures containing extracted cell sap (Hradec & Duiek, 1968b) although normal incorporation was again obtained after the addition of cholesteryl 14-methylhexadecanoate. This apparent discrepancy indicated that the ester affects not only the function of aminoacyl-tRNA ligases but also that of some other soluble enzymes involved in protein synthesis.Enzymes participating in protein synthesis that are present in the soluble fraction of the cell may be divided into three main groups: those required for peptide-chain initiation, elongation and termination (Matthaei et al. 1968). Relatively little is known about the nature and formation of the initiation complex in mamma...

Horseradish peroxidase in the presence of hydrogen peroxide mediates the activation of carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I) to DNA-bound products in vitro. The peroxidase activating system is greater than 10 times more effective with respect to DNA modification by Sudan I than the microsomal enzymes containing cytochrome P450. The DNA-binding reaction of the Sudan I metabolite(s) formed by the peroxidase system is dependent on Sudan I and H2O2 concentration and pH. Reactive intermediate(s) or product(s) of the Sudan I oxidation by peroxidase with a short half-life are responsible for the DNA modification. DNA modified by peroxidase-activated Sudan I becomes colored and has an absorption maximum at approximately 480 nm. The modification of DNA by Sudan I metabolites(s) formed by the peroxidase system is inhibited by some compounds of physiological importance (ascorbate, glutathione, Mg2+ ions) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-Postlabeling assay of the DNA modified by Sudan I activated by the peroxidase system indicates that the covalent DNA adduct formation is the principal type of the DNA modification. Four major and several minor adducts of deoxyribonucleotide 3',5'-bisphosphate from DNA with Sudan I metabolite(s) were detected by the classical Randerath 32P-postlabelling assay as well as by the nuclease P1 version of the same method.

Horseradish peroxidase in the presence of hydrogen peroxide has the ability to mediate the activation of carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I) to DNA- and transfer RNA (tRNA)-bound products in vitro. tRNA is more accessible for modification by the activated carcinogen studied. tRNA modified by activated Sudan I becomes colored and has an absorption maximum of approximately 480 nm. Binding of metabolite(s) to tRNA is inhibited by ascorbate, glutathione, Mg2+ ions and nitrosobenzene. The mechanism of these protections was shown to be different for the different agents. tRNA modified by activated Sudan I exhibits a significantly increased acceptance for L-methionine. Enzymatic hydrolysis of modified tRNA with subsequent separation of nucleosides by HPLC suggests that the covalent modification of tRNA originating from the formation of more than one adduct with the nucleosides in tRNA is the predominant interaction of the activated Sudan I with tRNA.

Highly purified peptide elongation factor 1 from rabbit reticulocytes liberates the terminal phosphate from [ Y -~~P I G T P and incorporates it into its own protein. Approximately one phosphate residue becomes bound by one molecule of the factor. Only the eEF-lcr subunit of the factor ( M , 53000) becomes phosphorylated as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by autoradiography and by the incubation of [ Y -~~P I G T P with individual subunits of the elongation factor separated by chromatofocusing in the presence of 5 M urea. The phosphorylation also takes place, though to a lesser extent, if the factor is incubated with Na2H32P04, probably due to the presence of endogenous GTP bound in the molecule of the factor. The content of endogenous GTP in various factor preparations was 0.21 -0.43 mol/mol factor. Phosphorylation of the peptide elongation factor is ribosome-independent, acid-labile and apparently autocatalytic since no other proteins are required for this reaction. Preincubation of the factor with GTP or with inorganic phosphate results in the phosphorylation of the factor and is followed by an enhanced binding of phenylalanyl-tRNA to 80s ribosomes in the presence of poly(U). This is accompanied by a dephosphorylation of the factor protein and thus the reversible autophosphorylation of the factor apparently activates its binding site for aminoacyl-tRNA. This is supported by the observation that sodium fluoride, which inhibits the dephosphorylation of the factor, blocks the factor-catalyzed binding of aminoacyl-tRNA to ribosomes. The incorporation of phosphate into factor protein also inhibits the formation of an eEF-1 . GDP complex, which is inactive in protein synthesis. Thus GDP liberated by the GTPase activity of the factor cannot affect its binding site for aminoacyl-tRNA. This may be the other reason for the enhanced activity of the phosphorylated factor. The autocatalytic GTP-dependent phosphorylation of the peptide elongation factor 1 apparently modifies its function and may thus play a regulatory role in protein synthesis.Several cellular functions in mammalian tissues are modulated by reversible protein phosphorylation, which is considered to be a major general mechanism of metabolic regulation [ 11. Many enzymes and also protein-synthesis factors [2 -41 are phosphorylated by different protein kinases, which bind phosphate groups by ester linkages to serine, threonine and tyrosine residues in the protein, and the resulting bond is acid-stable (see [5] for a review).Autophosphorylation occurring in the absence of kinases represents another mechanism of protein phosphorylation. It results in an acid-labile binding of phosphate to histidine or lysine residues by phosphoamide linkage [6, 71. Such a phosphorylating activity, utilizing phosphate liberated from GTP, has been found to be associated with eIF-2 [8].The prokaryotic EF-Tu, a functional analogue of eukaryotic eEF-1, reveals a significant GTPase activity, which is involved in the el...

1. The neutral portion of the molecule of carcinolipin was found to be cholesterol by comparison of mixed melting points with cholesterol, its dibromide and its acetate. 2. The fatty acid present in carcinolipin was subjected to oxidative degradation by chromic acid and permanganate. Butan-2-one was the main neutral degradation product resulting from both these procedures. A mixture of dibasic acids was obtained after the oxidation with chromic acid. Permanganate oxidation yielded a complete homologous series of branched-chain C(5)-C(17) fatty acids. 3. The mass spectrum of the acid was characteristic for a saturated C(17) acid. The alcohol prepared by lithium aluminium hydride reduction of the original acid showed a mass spectrum typical for an anteiso compound. 4. Comparison of mixed melting points, gas-liquid-chromatographic behaviour and mass spectra of the fatty acid isolated from carcinolipin with an authentic sample of 14-methylhexadecanoic acid demonstrated the identity of these compounds. Cholesterol esters synthesized from authentic cholesterol and the fatty acid isolated from carcinolipin or synthetic 14-methylhexadecanoic acid showed an identical stimulating effect on the incorporation of labelled algal-protein hydrolysate into rat liver transfer RNA in vitro. 5. Mass spectra, results of oxidative degradations and comparisons with an authentic sample, as well as biological activity of the synthetic cholesterol 14-methylhexadecanoate, provided good evidence that carcinolipin is cholesterol (+)-14-methylhexadecanoate.