Actinomycin radioactive

L-proline isomer Actinomycin Radioactivity in peptide-bound imino acids ... 

Dry the disks and count in a liquid scintillation counter (see Note 1). The plot of radioactivity incorporated vs reaction time should be linear for at least 1.5-2 h and plateau after 3 1 h. Actinomycin D at a concentration of 100 pg/mL should inhibit DDDP activity by >80% and RDDP by <20% phosphonoformic acid at 100 pg/mL should strongly inhibit both DDDP and RDDP activities, but neither should be affected by 100 pg/mL of aphidicolin. 

Actinomydn.—The phenoxazinone skeleton of the actinomycins (181) has been shown to derive from two molecules of tryptophan via 3-hydroxy-4-methylanthranilic acid (185),methionine serving as the source of the aromatic methyl groups.Depression of the incorporation of radioactive tryptophan into actinomycin by kynurenine (182) and 3-hydroxy-4-methylkynurenine (184) indicated that (182) and (184) could well be biosynthetic intermediates. In contrast 3-hydroxykynurenine (183) was found to have little or no effect on tryptophan incorporation, thus casting doubt on its participation in actinomycin biosynthesis. However, both [ Hjkynurenine [as (182)] and [ H]-3-hydroxykynurenine [as (183)] have recently been shown to be efficient precursors for actinomycin in Streptomyces antibioticus. 3-Hydroxykynurenine (183) is therefore implicated in actinomycin biosynthesis and further substantive evidence is provided for the pathway tryptophan —> (182) — (183) — (184) —> (185) —> actinomycin (181). 

Another approach to the analysis of mRNA transfer to the ribosomes is the study of cytoplasmic particles containing newly formed cytoplasmic mRNA. Leytin et al. (1971) have found that the material containing newly formed mRNA is somewhat less dense in the CsCl gradient than polysomes formed earlier. For example, while the bulk of polysomes have a buoyant density of 1.55 to 1.56 g/cm, the newly formed material possesses a density of 1.47 to 1.53 g/cm. Newly formed particles comprise only a small fraction of the total material since they cannot be detected by routine UV absorption methods. With more detailed analysis two newly formed components may be observed component A with p = 1.49 g/cm and component B with p = 1.52 g/cm. After long-term incubation, or in the presence of actinomycin D (chase experiment), almost all radioactive material is transferred from A to B peaks to the main peak of the polysomes. Inhibition of protein synthesis by cycloheximide interferes with these polysome transformations, and the label representing newly synthesized material accumulates in component A (p = 1.49 g/cm ). Thus one can suggest that component A converts to B and the latter is transformed into mature polysomes. It was found further than... 

Under suitable Conditions, the protoplasts from Streptomyces anti-bioticus readily incorporated radioactively-labelled threonine, valine, proline, glycine and methionine into the relevant positions of actinomycins. Administration of radioactive tryptophan yielded actinomycins labelled in the chromophore. When the oxygen pressure was low, trace amounts of 4-methyl-3-hydroxy-anthranilic acid, which is an intermediate in the biosynthesis of the chromophoric part of actinomycin, could be detected. 

Experiments using hepatoma cells have shown that several discrete steps follow their exposure to corticosteroids. First, the steroid molecules pass across the cell membrane and bind to specific cytoplasmic receptor proteins. These steroid-receptor complexes then rapidly migrate into the nucleus, where they bind to sites containing DNA (for references, see Baxter et al., 1972). After about 30 minutes RNA required for TAT synthesis (presumed to be mRNA) begins to accumulate, and an increase in the rate of TAT synthesis (as measured by specific immunoprecipita-tion of radioactively labeled enzyme) can be detected after about 60 minutes (Granner et al., 1970). The initial increase in TAT synthesis is inhibited by low doses of actinomycin D (AMD) (e.g., 0.1 jug/ml), presumably because of an inhibition of mRNA synthesis. Under normal circumstances, however, TAT synthesis continues to increase until about... 

Infection with picornaviruses results in a strong (though selective) inhibition of the cellular ENA synthesis (5, 4> 85> 84), and the appearance of a new, virus-induced ENA synthesizing activity. The latter was soon shown to be insensitive to the action of Actinomycin D (5), an antibiotic which prevent DNA-dependent ENA synthesis by intercalating in the dG-dC sequences of the template MA (6, 7) These observations suggested the possibility of treating infected cultures with the antibiotic in order to suppress host-cell activity and measxiring the incorporation of radioactive precursors into viral ENA. Since the precursors added to the medium do not equilibrate instantaneously with the intracellular pool of nucleotides, a correction for this fact must be introduced, at least for the very early times. 

Radioactive RF has been used to infect cells and search for the modifications of the parental molecules in each sub-cellular compartment. Such studies revealed that soon after infection with RF, the input d-s molecules are found in the cytoplasm as part of a Replicative-Intermediate-like structure (18). Treatment of the host-cell with interferon abolishes the infectivity of RF, but does not prevent the intracellular processing of RF into RI. In contrast, exposure of the cells to Actinomycin D inhibits the infectivity of RF and the intracellular transition to RI as well (36). As it was found that a cellular RNA polymerase specifically binds to RF (37)> it was postulated that RF served as abnormal template for a cellular RNA polymerase to transcribe the first viral messenger (18, 57). 

Studies on the incorporation of labeled amino acids in thyroid slices have provided a more detailed description of the mechanism of synthesis of the hormone. The radioactivity first appears in soluble polypeptides with sedimentation coefficients of 3, 8, or 12. Puromy-cin or actinomycin blocks the incorporation of the precursor into the soluble polypeptides. The half-life of the messenger RNA for thyroglobulin polypeptide was estimated to be 15-20 hours. Indeed, after inclusion of actinomycin in the incubation mixture, thyroglobulin synthesis continues for several hours. The subunits are transferred from the site of synthesis to an assembly center, in which the subunits are iodinated, carbohydrate units are included in the molecule, and subunits are condensed into a finished protein. Puromycin fails to interfere with the formation of 19 S units. 

On the basis of the early observation that the increases in microsomal enzyme activity produced by phenobarbital and 3-methylcholanthrene were blocked by actinomycin-D, it was suggested that enzyme induction resulted from the synthesis of new enzyme protein which was, in turn, dependent upon the DNA-directed synthesis of a messenger-like RNA. Treatment of rats with 3-methylcholanthrene causes an increase of about 40% in the level of RNA in rat liver nuclei and the nuclear RNA from 3-methylcholanthrene-treated rats is more active in directing protein synthesis than RNA from control animals. Moreover, the in vitro incorporation of radioactive precursors such as orotic acid or cytidine triphosphate into nuclear RNA is 50 to 100% greater in preparations from 3-methylcholanthrene-treated animals than controls. It is of interest that treatment of rats with phenobarbital has been recently reported to result in a marked suppression of endogenous hepatic ribonuclease activity. 

A stimulus which alters the steady-state level of an endogenous cellular component may do so by influencing its rate of synthesis, its rate of break-down, or both. When administered to intact animals, phenobarbital or 3-methylcholanthrene increase (20-50%) the steady-state level of microsomal protein. Similarly, micro-somes from animals pretreated with phenobarbital or 3-methylcholanthrene incorporate radioactive amino acids into protein more rapidly than microsomes from control animals and this effect is blocked by co-administration of actinomycin-D. It was therefore assumed that the increased levels of microsomal protein and enzyme activity after inducers were the result of enhanced synthesis. However, turnover studies have revealed that phenobarbital in particular has a profound effect upon microsomal protein catabolism. Proteins of the endoplasmic reticulum were labelled by injection of radioactive amino acids and the rate at which radioactivity disappeared from the microsomes was compared in control and phenobarbital-treated animals. Assuming a comparable degree of isotope re-utilization in the two groups, this approach provides a relative measure of microsomal-protein turnover. In control animals, radioactivity of total microsomal protein decreases with time with a half-time of about 3 days. In phenobarbital-treated animals, however, there is a marked stabilization of microsomal protein so that almost no radioactivity is lost over a S-day period. The reduced protein catabolism is observed both in total microsomes and in a purified microsomal protein, NADPH cytochrome c reductase. Thus, repeated administration of phenobarbital to animals evokes an increase in... 

Taylor (1965) showed that the amount of radioactivity incorporated into RNA is not significantly different in SF virus-infected CEF cells and in mock-infected cells early in infection. During the exponential phase of viral replication, however, about 70-90% of the newly synthesized RNA is virus specific, since it can be synthesized in the presence of actinomycin D. Mussgay et al. (1970) demonstrated almost the same inhibition pattern in CEF cells infected with SIN virus. Moreover, infection of BHK cells with WEE virus did not enhance cellular RNA degradation (M. Wagatsuma and B. Simizu, unpublished data). 

More direct evidence that polymyxin synthesis proceeds by a mechanism that differs from that of protein synthesis comes from experiments with growing cultures of B. polymyxa in which the effects of inhibitors of protein and nucleic acid synthesis on the incorporation of radioactive precursors into protein and polymyxin B were studied (Paulus and Gray, 1964). As shown in Table 7, chloramphenicol, actinomycin D, and to a lesser extent puromycin inhibit the incorporation of L-threonine- C into protein but stimulate its incorporation into polymyxin B. This differential effect of inhibitors on protein and polymyxin synthesis strongly supports the hypothesis that polymyxin synthesis does not proceed by the kind of template mechanism that operates in protein synthesis. 

The precursor relationships of the peptide imino acids in the actinomycins was studied with the use of C-labeled L-proline and hydroxy-L-proline (Katz, Prockop and Udenfriend, 1962 Katz and Weissbach, I963). The data revealed that C-proline was rapidly incorporated into the actinomycins produced by 5. antibioticus and that the radioactivity in a given actinomycin was located almost exclusively in the imino acids. The ratio of hydroxyproline/proline in actinomycin I and of 4-oxoproline/prohne in actinomycin V in the C-experi-ments approached 1. 

By contrast, the amino acids of actinomycin were found to be devoid of radioactivity. In recent studies it has been found that incorporation of the C-label of benzene ring labeled tryptophan into actinomycin can be exceptionally high. During optimum synthesis as much as 25 to 35% of the radioisotope supplied is introduced into the actinomycin chromophore. Tryptophan, labeled in the alanine side chain, is not incorporated into the actinomycin chromophore to any significant extent (unpublished observations) suggesting that the benzenoid moiety of tryptophan is the precursor of the actinomycin chro-... 

Birch (1958) had suggested that acetate rather than tryptophan may play an important role for s mthesis of the actinomycin chromophore. He postulated that the 3-hydroxy-4-methylanthranilic acid precursor might arise from four acetate molcules through a process of oxidation, reduction and amination. If acetate constituted the source of the anthranilate precursor, eight moles of acetate would be required for synthesis of one mole of the actinomycin chromophore and it would be expected that the actinocin formed would possess appreciable C-label. The results of an experiment with sodium acetate-2- C indicate that acetate is not directly involved as a precursor of the actinomycin chromophore (Sivak, Meloni, Nobili and Katz, I962). Of the total radioactivity incorporated into actinomycin by 5. antihioticus following addition of acetate- C, only 4% was present in the chromophore. On the other hand, the amino acid, proline, in the peptide portion of the actinomycin molecule possessed appreciable radioactivity. 

The preceding studies with radioisotopes have provided information concerning the amino acids which serve as precursors for the biogenesis of the actinomycins. Kinetic studies (Fig. 4) of the rate of incorporation of C-labeled amino acids into the antibiotic during short incubations (30 to 120 minute intervals) have also provided considerable information concerning actinomycin s mthesis (Katz and Weissbach 1962, 1963). This has been possible because actinomycin is readily soluble in organic solvents, e.g., ethyl acetate, whereas the amino acid precursors are not. Once the antibiotic is extracted into ethyl acetate, the radioactivity incorporated into the antibiotic as a function of time can be determined by conventional techniques of liquid scintillation counting. 

With L-threonine- C as precursor it can be noted (Fig. 13 a) that uptake of the C-labeled compound into the cell occurs extremely rapidly requiring less than 1 to 2 minutes. No significant difference in the rate of uptake was observed with or without chloramphenicol however, the maximum size of the C-labeled pool is greater and its utilization slower when chloramphenicol is present. Uptake of the amino acid into the pool actually precedes its appearance into either actinomycin or protein. Both the rate and extent of incorporation of L-threonine into actinomycin were enhanced by chloramphenicol. The effect was particularly striking after the first 20 to 30 minutes of incubation when incorporation of threonine into actinomycin (in the absence of chloramphenicol) had leveled off. Incorporation of the C-amino acid into protein is inhibited 90% or more with chloramphenicol present whereas, in its absence, the C-labeled compound was incorporated quite extensively. In experiments with L-proline- C (Fig. the uptake of the imino acid into the cell proceeded more slowly than was observed with L-threonine. Moreover, no difference was observed in the rate of incorporation of the labeled amino acid into actinomycin until after the incorporation of the radioisotope in the absence of chloramphenicol had reached a plateau. Data similar to that found with threonine and proline were obtained also with glycine and L-vahne. Although chloramphenicol enhances the rate and extent of incorporation of radioactivity from tryptophan and methionine into actinomycin it was not possible to observe any significant increase in the size of the C-labeled pool in the presence of chloramphenicol. Since these compounds, particularly tryptophan, are metabolized extensively it is possible that the radioactivity in the pool was distributed in a number of different metabolites and, therefore, did not reflect the actual level of the amino acid present in the pool. 

L-Phenylalanine-l- C, an amino acid which is not a precursor of the actinomycin molecule, was also suppUed to S. antibioticus in the presence and absence of chloramphenicol. As would be expected, the incorporation of radioactivity into protein was inhibited by over 90%. The C-labeled pool proved to be twice the size of the corresponding one formed in the absence of the inhibitor. Negligible... 

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