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Home , Xanthosine, Xanthosine monophosphate

Vol. XVI No. 4 THE JOURNAL OF ANTIBIOTICS, SER. A 163

MECHANISM OF ACTION OF ANGUSTMYCINS, ANTIBIOTICS

Noauo TAN AKA Institute of Applied Microbiology, University of Tokyo

(Received for publication February 25, 1963)

Angustmycins A and C, produced by Streptomyces hygroscopicus, are nucleoside anti­ biotics: angustmycin A is 6-amino-9-(L-l,2-fucopyranoseenyl)- and angustmycin C 6-amino-9-(~-o-psicofuranosyl)-purine.1-'> They were observed to exhibit an inhibitory

activity against experimental infections and transplantable tumors in animals8'. The activity of angustmycins A and C was reversed by , , and related substances. It indicated that both angustmycins may act as antimetabolites in purine . The inhibition index suggested that guanosine synthesis is more interfered than adenosine synthesis in the presence of angustmycins. &> This paper describes the results of further experiments concerning the mode of action of these nucleoside antibiotics, indicating that both angustmycins may inhibit the process of biosynthesis of from monophosphate. Materials and Methods

The medium used was a glutamate-glucose-mineral medium, consisting of sodium glutamate 4.0 g/L, glucose 5.0 g/L, KH 2PO, 1.0 g/L, MgSO, 0.7 g/L, NaCl 1.0 g/L, (NH,) 2HPO, 2.0 g/L and FeCl 3 0.03 g/L. The method for- the study of antagonism of and xanthosine to angustmycins principally followed the procedure described in the previous paper.Gl The samples of angustmycins were obtained from Dr. Hso YONTSEN of our laboratory.

Results Reversal of the Activity of Angustmycins against B. subtilis PCI 219 by Inosine and Xanthosine

As reported in the previous paper5', the activities of angustmycins A and C were re­ versed by adenosine, guanosine, and guanine. The inhibition indexes indicated that guanosine synthesis is more interfered than adenosine synthesis. For the purpose of determining the detailed sites of action, the influence of xanthosine and inosine on the activity of angustmycins was investigated. As summarized in Table l, the antibacterial activities of both antibiotics were antagonized by inosine and xanthosine. Inhibition index of inosine to the activity of angustmycin A was 5, that to angustmycin C 50~100, that of xanthosine to A 1~2, and that to C 10. These values are compared with those of adenosine, , guanosine and guanine; and are presented in Table 2. Higher in­ hibition index of guanosine than those of xanthosine, inosine and adenosine suggests that both angustmycins may act on the amination process of to guanosine monophosphate (Fig. 1). 164 THE JOURNAL OF ANTIBIOTICS, SER. A July 1963

Fig. 1. A plausible site of action of Table 1. Reversal of activity of angustmycins angustmycins A and C in the against B. subtilis by nuc!eosides pathway of nucleot1de biosynthesis. in liquid synthetic medium. Formate, Glycine I Concen- 1 Glutamine, Asparate, CO2 I I tration I Inhi­ Anti- I necessary bition biotics- _ [ mcg/ml I Metabolite for I I,antagonism index j ______rn _c~[!n_l ___ _ 5 AICAR Angust- 200 Inosine 40 100 I fl 20 5 ! rnycin A ,, I Inosine~IMP 50 10 5 fl 2 100 Angust- 200 ✓, fl rnycin C 100 1 100 Xanthosine~XMP ASR 50 fl 1 50 200 Xanthosine 100 2 IAngustmycin Al------.. Angust- 2 t ll mycin A 100 " 50 IAngustmycin c:/"J 50 " 50 1 20 10 Guanosine~GMP AMP~Adenosine Angust- 200 100 "fl 10 10 ! ! rnycin C 50 5 10 GDP ADP " GTP ATP ~ ✓ Nucleic acids Table 2. Reversal of activity of angustrnycins by purine bases and AICAR : 5-Amino-4-irnidazolecai boxamide (Inhibition index). ribonuc!eotide Metabolite I Angustrnycin A J Angustmycin C IMP : Inosine-5'-rnonophosphate XMP : Xanthosine-5'-rnonophosphate Adenine ASR : ribonuc!eotide Adenosine 2 100 GMP : Guanosine-5' -monophosphate Guanine 100 2,000 GDP : Guanosine 100 500 GTP : 5 100 AMP: Adenosine-5'-monophosphate Inosine ADP : Xanthosine 2 10 ATP :

Accumulation of Xanthosine in the Culture Medium of B. subtilis containing Angustmycins The 18-hour culture of B. subtilis was washed, resuspended in a glutamate-glucose­ mineral medium with or without angustmycin (A 200 mcg/ml or C 1,000 mcg/ml), and incubated at 30° for 16 hours. After removal of bacterial cells by centrifugation, the culture fluid was absorbed on carbon and eluted with 50 % ethanol containing 1 % ammonia. After evaporation in vacuo to small volumes, the eluates were applied to Dowex 1-X8 (formate) columns (25 cm x 1.3 cm) and eluted successively with ammonium formate

according to the gradient method of HuRLBERT et al. 7> The optical density at 260 mµ of each fraction is illustrated in Fig. 2. The peaks of ultraviolet absorption of fractions No. 6, 14, 25 and 33 were not significantly shifted in acidic and alkaline reactions, indicating that the materials involved are angustmycins or related nucleosides and . However, the maximum of ultraviolet absorption of fraction No. 33 of angustmycin A or C-containing culture was changed from 260 mµ to 278 mµ, when sodium hydroxide was added. The fractions No. 31 to No. 35 were collected, absorbed on carbon and eluted with 50 % ethanol containing 1 % ammonia. The eluates were concentrated in vacuo, and Vol. XVI No. 4 THE JOURNAL OF ANTIBIOTICS, SER. A 165

examined for ultraviolet absorption, orcinol reaction, paper chromatography and paper electrophoresis. Both solutions, thus obtained from angustmycin A-containing culture and from angustmycin C-containing culture gave a single spot in paper chromatography and paper electrophoresis. The Rf value was 0.27, identical with that of xanthosine. The solvent

system used was tertiary butanol-HCl-H20 (70: 13.2: 16.8). Each showed ultraviolet

Table 3. Comparison of the substance in the fractions Fig. 2 Column-chromatogram of the No. 31 to No. 35 (Fig. 2), obtained from the culture of B. subtilis containing culture containing angustmycin A or C, angustmycin A or C and control with nucleosides by ultraviolet culture. absorption peaks. o.t M 16 M 16M NH,formale * Nff, /Orlll11le •· Nlt,-jOrmote* Obt formic•

Materials in 0.1 N HCl in 0.1 N NaOH carbon eluate from Fr. 31 to 35 from Angusfmljcirz A· contoirzinq angustmycin A- 262 mµ, 238 mµ 278 mµ, 248 mµ culture containing culture Fr. 31 to 35 from lO 262 mµ, 238 mµ 278 mµ, 248 mµ angustmycin C-

Discussion

The activity of angustmycins A and C was reversed by guanosine, xanthosine, inosine and adenosine. Inhibition indexes suggested that both antibiotics may act on the biosynthesis of guano­ sine monophosphate from xanthosine monophosphate. The assumption was further supported by the finding of accumulation of xanthosine in the culture of B. subtilis containing angustmycin A or C. As far as our experiments concerned, no difference was observed between the activity of angustmycin A and that of angustmycin C. Psicofuranine, an antibiotic identical with angustmycin C, was reported to inhibit the biosynthe­ sis of guanosine-5'-phosphate from xanthosine-5'-phosphate8,9l. It is in accordance with the observa­ tion of the present work. 166 THE JOURNAL OF ANTIBIOTICS, SER. A July 1963

Summary

The activity of angustmycins A and C, nucleoside antibiotics, was reversed by inosine and xanthosine as well as by guanosine and adenosine. Inhibition indexes of guanosine to both antibiotics were higher than those of adenosine, inosine and xanthosine, indicating that angustmycins may inhibit the biosynthesis of guanosine monophosphate from xan· thosine monophosphate. An accumulation of xanthosine was observed in the culture of B. subtilis containing angustmycin A or C, which appeared to support the assumption. No marked difference was~demonstrated between the activity of angustmycin A and that of angustmycin C.

This work was supported by the grant of U.S. Public Health Service (CY-05082). The author expresses a deep gratitude to Prof. HAMAO UMEZAWA for his thoughtful direction in this study.

Reference

1) Hso YilNTSEN, H. YoNEHARA & H. Ur: Studies on a new antibiotic, angustmycin. I. J. Anti­ biotics, Ser. A 7(4) : 113~115, Aug. 1954 2) Hso Y0NTSEN; K. OHKURA, Y. IsHir & H. YoNEHARA: Studies on angustmycin. III. J. Anti­ biotics, Ser. A 9(6) : 195~201, Dec. 1956 3) Hso YtlNTSEN: Studies on angustmycin. VII. J. Antibiotics, Ser. A 11(6) : 233~243, Nov. 1958 4) Hso YtlNTSEN: Studies on angustmycin. VIII. J. Antibiotics, Ser. A 11(6) : 244~249, Nov. 1958 5) TANAKA, N.; N. MrYAIRI & H. UMEZAWA: Studies on antagonists to angustmycins. J. Anti­ biotics, Ser. A 13(4) : 265~269, July 1960 6) TANAKA, N.; T. NrsHIMURA, H. YAMAGUCHI & H. UMEZAWA: Activity of angustmycins against experimental infections and transplantable tumors. J. Antibiotics, Ser. A 14(2) : 98~102, Mar. 1961 7) HURLBERT, R. B.; H. ScHMITZ, A. F. BRUMM & V. R. PoTTER : metabolism. II. Chro- matographic separation of acid-soluble nucleotides. J. Biol. Chem. 209(1) : 23~40, July 1954 8) HANKA, L. J. : Mechanism of action of psicofuranine. J. Bact. 80(1) : 30~36, July 1960 9) SLECHTA, L. : Inhibition of xanthosine-5'-phosphate aminase by psicofuranine. Biochem. Bio­ phys. Res. Comm.3: 596~598, Nov. 1960