Actinomycin preparation

In addition to the actinomycins hsted above the alphabetical designations AA, AC, J, M, K and Z have been employed by other investigators to designate actinomycin preparations isolated by them (Sarlet, 1950 Sarlet, Toussaint and Brasseur, 1951 Hirata and Nakanishi, 1949 Craveri, 1956 Bossi, Hutter, Keller-Schierlein, Neipp and Zahner, 1958). Many of these are probably identical with previously described actinomycin preparations despite the use of different alphabetical appellations. The great number of actinomycins and the synonomy of many of the names have made their descriptions difficult. The various actinomycins, their synonomy and their amino acid composition are given in Table 1. 

From Australian rutaceae, Melicope tereana, several alkaloids were isolated. From melicopine and melicopidine, after demethylation and nitric acid oxidation, two quinones, 248 and 249, were obtained (49MI1). Meli-copicine is similarly transformed into both quinones. Chemical degradation of actinomycin yielded a peptide-free quinone, actinomycinol (250). The mechanism of conversion of the phenoxazine skeleton in actinomycin into the acridonequinone has been outlined (58JCS469). The compound was synthesized and several derivatives were prepared (56CB1397 57CB44 58JCS496). 

C-HAAF (N-Hydroxy-N-acetyl-2-amino-fluorene was from the ICN Hadlochemlcals Division, and -actinomycin D was from Amersham. Calf thymus DNA (Worthington), was prepared according to the procedure of Muller and Crothers (15.). Poly (dAdT)" poly (dAdT) and poly(dGdC)-poly(dCdC) were purchased from PL Blochemlcals. DNA concentrations were determined absorptlometrlcally, and expressed in basepalrs. 

For the synthesis of the heterodet cyclic depsipeptides, the acid chloride which had been prepared after the removal of the iV-protecting groups was most used. Recently, a mixture of acetylchlqride and acetylimidazole has been used for the cyclization of actinomycin acid to actinomycin Ci . Heterocyclic peptides containing disulphide rings have been cyclized by oxidation of the corresponding disulphydryl compounds . 

Actinomycin.—Further exploration of actinomycin biosynthesis has involved the preparation of protoplasts of Streptomyces species. 

When cells are exposed to doses of a water-soluble form of actinomycin D, Act D-mannitol (Sigma), at concentrations of O.l-l.O /i.g/ml (made from a 1 mg/ ml stock solution dissolved in distilled water) for times of 1-4 hr it is possible to obtain segregated nucleoli with fibrillar caps separated from granular regions (Fig. 7a). These optimally segregated nucleoli can then be fixed and prepared for fluorescence microscopy or immunoelectron microscopy in order to localize... 

In table 1 the present state of characterization of several enzyme systems discussed in this publication is shown. While synthetases for gramicidin S, alamethicin or enniatin are available in purified or partially purified forms, the biosynthesis of actinomycins and valinomycin cannot yet be studied in vitro presumably,because the preparation of a cell free extract of these membrane bound systems needs more subtle and advanced techniques of membrane isolation and fragmentation. A promising approach for the investigation of such systems has been found in the study of protoplasts from the producer organism of these antibiotics. 

Nuclear and polysomal fractions were prepared from embryos labeled in vitro with [ HJuridine in the presence of actinomycin D at a concentration (0.04 /ig/ml) sufficient to inhibit rRNA synthesis as described by Schultz et al. (1973a). 

Martin, D.G. Peltonen, R.E. Nielsen, J.W. Preparative resolution of an actinomycin complex by countercurrent chromatography in the ito coil planet centrifuge. J. Antihiot. 1986, 39, 721. 

The mechanism by which interferon treatment increases the enzymatic phosphorylation of protein 67 and eIF-2 was investigated. The increase is seen at 5-6 hours after addition of interferon to mouse L cells and is blocked by actinomycin D. Since protein 67 is found in purified preparations of PK-i we wondered whether this protein is induced by interferon. Not having protein 67 substrate free of PK-i, we made use of the fact that in himian cells the interferon- and dsENA-induced equivalent of protein 67 migrates more slowly on SDS polyaciylamide gels (protein 69) (16). Addition... 

Keller and coworkers succeeded in the preparation of several aromatic carboxylic acid-activating starter enzymes involved in the biosynthesis of actinomycin (4-methyl-3-hydroxyanthranilic acid) (105,113,114), triostin (qninoxaline-2-carboxylic acid) (105,115), and mikamycin (3-hydroxypicolinic acid) (105,116). More recently, related enzymes have been identified in the Actinomycetes that form the thiopeptides nosihep-tide and thiostreptone (117,118), and in the pristinamyctn biosynthetic system (de Crecy-Lagard V, personal communication). Whether direct acylation of the pantetheine-attached starter amino acid occurs, or an additional transferase frinction is required, has not been settled. 

Fig. 4. The time dependence of cell-free enzyme synthesis uninhibited and inhibited at varying times by actinomycin or rifampicin. Synthesis was carried out as described in Table 1 except for the following modifications. In the control experiment, samples were removed from the synthesis system and assayed at the times indicated on the abscissa. In the actinomycin and rifampicin experiments, the inhibitor (2 jitg of actinomycin or 1 ju,g of rifampicin per milliliter) was added at the time indicated on the abscissa, and the incubation was continued for another 60 minutes before assay. All values have been normalized to an assay time of 200 minutes. The S-30 used was prepared from strain 514 which contains a deletion of the entire lac region including the repressor gene.

Yudkin [86] offered evidence that the messenger half-life for penicillinase differed in induced and constitutive mutant strains of B. licheniformis 749, based on penicillinase synthesis after treatment with actinomycin. In induced cultures the half-life was about 2 to 3 minutes, while in the constitutive mutant the half-hfe appeared to be 10 minutes. Yudkin [87] did not obtain similar results with other constitutive strains, and this high value is therefore not substantiated. Davies [84,88], using both actinomycin and rifampicin, inferred messenger half-lives of about 4.7 minutes for both induced and constitutive penicillinase synthesis. Subsequent experiments (Davies and Collins, in preparation) have given shorter estimates of 2 to 4 minutes for penicillinase messenger half-life during vanadate-induced synthesis. 

What holds the ribosomes together to form polysomes From Rich s original preparation, one would suspect it to be messenger RNA. Ribonuclease separates polysomes that are effective in protein synthesis into ineffective smaller fragments. Furthermore, actinomycin, which blocks the synthesis of messenger RNA, also breaks the polyribosomes into smaller units in the organs of animals injected with the antimetabolite. 

There are arguments for interpreting the action of aldosterone on active sodium transport by toad bladder, colon and skin as due to a hormone-induced multiplication of the sites of penetration of sodium into the cell, across its apical border (Crabbe, 1967) this requires the intervention of nucleic acid metabolism and the effect of aldosterone is blocked by actinomycin D. This drug actually fails to interfere significantly with the reaction of toad skin to insulin before paired preparations were stimulated with insulin, short-circuit current (uk/cm S.E.) averaged 42.4 + 2.6 for the control, 36.1 1.4 for the matched fragments exposed for 2 hours to actinomycin D, 10 2 hours after addition of insulin to both... 

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. 

Actinomycin A, thus prepared, is in the form of bright red crystalline platelets, melting at 250° with decomposition. The pigment is easily soluble in chloroform, benzene, or ethanol, less soluble in acetone and hot ethyl acetate, and slightly soluble in water and ether. Actinomycin A is levorotatory [ ]d = —320° (c = 0.25% in ethanol). It possesses a characteristic absorption spectrum in visible and in ultraviolet light (612), with maxima at 4500 A and between 2300 and 2500 A. 

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