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Improved production of a chromophore component of antitumor antibiotic neocarzinostatin (NCS) by addition of threonine in a chemically defined medium
JOURNAL OF FERMENTATION AND BIOENGINEERING
Vol. 75, No. 3,223-225. 1993
Improved Production of a Chromophore Component of Antitumor Antibiotic Neocarzinostatin (NCS) by Addition of Threonine in a Chemically Defined Medium KOZO KUDO,I*TSUNEHISA SUTO, I YUKO YOSHIDA, 1 KIYOTO EDO, 2 ~ D NAKAO ISHIDA 3 Department of Microbiology, Akita University School of Medicine, Akita 010,1 Department o f Pharmacy, Fukushima Prefectural College o f Medicine, Hospital, Fukushima 960, 2 Institute o f Sendai Microbiology, Sendal 980, 3 Japan Received 1 June 1992/Accepted 6 November 1992 Production in a chemically defined medium of a free chromophore component (free NCS-chr) of an antitumor antibiotic neocarzinostatin (NCS) by Streptomyces carzinostaticus var. F-41 was improved by the use of L-threonine or L-asparagine as a sole nitrogen source. Under the conditions established, the yield of free NCSchr was comparable to that obtained in a control medium containing casamino acids.
An antitumor antibiotic, neocarzinostatin (NCS), produced by Streptomyces carzinostaticus var. F-41, was initially regarded as a single polypeptide with molecular weight of 11,000 (1, 2). Clinically, NCS has been shown to be effective against pancreas, stomach and bladder cancers in a number of patients, as reviewed by Maeda (3). Later, Ohtsuki et al. (4), and Goldberg et al. (5) showed that a non-protein chromophore component (NCS-chr) was present in the NCS molecule. The isolation and characterization of the NCS-chr were achieved independently by Koide et al. (6), Goldberg et al. (5) and Suzuki et al. (7). The NCS-chr isolated from clinical grade preparations of NCS exhibits an optical rotation of [a]D20-171o, a molecular weight of 659, and a molecular formula of C35Ha30~2 by mass spectrometry (8). Two components (2-hydroxy5-methoxy-7-methyl-l-naphthalencarboxylic acid and Nmethylfucosamine) have been isolated from the NCS-chr and characterized (8). Infrared spectrometry, polarographic analyses and other chemical data suggested the presence of peroxy acid, a highly strained ether ring (possibly an epoxide) and a bicyclo[7,3,0]dodecadiyne system in its structure (9). Further studies by Jung et al. and Koide et al. have shown that in vitro mixing of the NCS-chr with the NCS-protein moiety resulted in the regeneration of both the biological activities and chemical properties of NCS (6, 10). As noted for the holo-NCS molecule, the NCS-chr also inhibits DNA synthesis selectively without a concomitant effect on RNA or protein synthesis (11). In the present report, the biosynthesis of the NCS-chr is characterized. Previously we were successful in producing the free form of the NCS-chr (free NCS-chr) in a simple semi-defined medium containing either MgSO4 or MgCI2 at 1,000 times higher concentration than in the original NCS production medium (12). NCS-producing S. carzinostaticus var. F-41 was maintalned as previously described (2). For production of free NCS-chr, a semi-defined medium containing 4% glucose, 1.5% casamino acids, 1.25% MgSO4, 0.25% NaCI, 0.2% CaCO3 and 0.1% K2HPO4 (pH 7.2) was used. A casamino acids-free medium (basal medium) was employed to examine the effect of a variety of amino acids upon free NCS-chr synthesis. MgSO4 and K2HPO4 were sterilized
separately and added aseptically to a double-strength basal medium in 200 ml Monod tubes to give a final volume of 100 ml. Quantitative analysis of free NCS-chr and the native NCS molecule has been described previously (12). Standard NCS in this experiment was a gift from Kayaku Co. Ltd., Tokyo. To analyse the free NCS-chr in the fermentaion broth, 100 ml of culture filtrate was first extracted with ethyl acetate (EtOAc). The EtOAc layer was evaporated in vacuo and dissolved in 1 ml of methanol to measure the antimicrobial and isotopic activities. The effect of single amino acids on free NCS-chr formation was examined as follows. S. carzinostaticus vat. F-41 was grown in 100 ml of a hot-water (pH 7.2) extract fraction of a soybean meal medium (12) at 28°C for 16 h on a reciprocal shaker (30rpm) and spun down at 3,000rpm for 15 min. The resulting mycelial pellet was aseptically washed 3 times with saline and resuspended in 100 ml of saline. Three ml of this suspension was transferred to 100 ml of the basal medium supplemented with a single amino acid. After the indicated cultivation time, free NCS-chr was isolated from the culture filtrate to measure the antimicrobial activity. Mycelia were harvested by centrifugation, washed, dried and then weighed. The addition of a single amino acid to the basal medium resulted in good growth of mycelium (dry weight: 300-500 mg/100 ml). As shown in Table 1, only L-threonine (Thr) or L-asparagine (Asn) among the various amino acids tested gave good yields of free NCS-chr comparable to that obtained in the basal medium supplemented with casamino acids. The other amino acids supported little or no NCS-chr synthesis. The time course of typical fermentation in the medium supplemented with Thr is shown in Fig. 1. The pH, 7.2 initially, declined to 6.0 during the rapid growth phase. Approximately 70% of free NCS-chr was synthesized during the first 36 h and the maximum accumulation was obtained at 72 h. When a crude preparation of free NCS-chr was analyzed by a high performance liquid chromatography, the major active principle was eluted with methanol-H20-formic acid (93 : 5 : 2), as in the case with the NCS-chr (12), and two significant peaks appeared at 3 min and 14.5 min (data not shown). To determine the time of maximum incorporation into free NCS-chr, mycelia of S. carzinostaticus grown in the basal medium containing Thr for 16, 20, 24, 28, 32 and
* Corresponding author. 223
224
KUDO ET AL.
7.0 c~
"'0"0"0"0.•.
J. FERMENT.BIOENO.,
• . . . . . . . .-0 . . . . . . . . . . .•. . . . .
•
TABLE 2. Incorporation efficiency of '4C-Thr into free NCS-chr during cultivation
"q~ •
5.0
7
2
200
400
z
200
_
24
71
i
i
2 120 Cultivation time(h)
11
i
168
FIG. 1. Time course of free NCS-chr production. Symbols: ~---o, free NCS-activity; O - - - o , NCS activity; O-----O,mycelial growth measured as dried cell weight; ©---©, pH monitored by a pH meter (Horiba F-12). 48 h were separated, transferred to basal m e d i u m containing ~4C-Thr (5 p C i / 1 0 0 ml), a n d cultivated. A f t e r 2 h pulse-labelling followed b y continued cultivation in the original culture fluid for 96 h in total, free NCS-chr was extracted f r o m the culture filtrate to measure the radioactivity as described above. The results (Table 2) showed that the most efficient i n c o r p o r a t i o n o f 14C-Thr into the E t O A c fraction was at 24 h. The relative i n c o r p o r a t i o n o f '4C-Thr into free NCS-chr, determined after 96 h cultivation and TABLE 1. Effect of various amino acids in the basal medium on free NCS-chr yield by S. carzinostaticus var. F-41 Amino acida Leucine IsoLeucine Arginine HC1 Dt-Alanine Asparagine Lysine Gintamine Cystine Methionine DL-Tryptophan Valine Phenylalanine Glycine Threonine Casamino acids
Free NCS-chrb Cultivation time (h) 28 123 ----23 2 2 -24 26 23 23 -2 -
-
--22 23 23 24 24
-
-
--2 -23 26 26
a The amount of each amino acid added to the basal medium contained 0.132% nitrogen, which was the same nitrogen concentration as that of 1.5°~ casamino acids. b The free NCS-chr obtained from the culture filtrate was dissolved in methanol, diluted 2-fold and assayed on anti-microbial activity. Free NCS-chr potency was indicated by the minimum inhibitory concentration (dilution titer) of the diluted solution.
Labelling time (h)
cpm x 103
16-18 20-22 24-26 28-32 32-34 48-50
8.3 10.9 12.4 11.7 11.0 5.7
Free NCS-chr Relative incorporation (%) 66.9 87.9 100 94.9 88.7 46.0
Isolation of radioactive free NCS-chr was the same as described in the footnote to Table 1. The ['4C]radioactivity was measured in a liquid scintillation counters (Aloka LSC 700). The incorporation ratio given in % compares the radioactivity at the indicated time with that obtained at 24 h, because all of the cultures had a similar free NCSchr titer. estimated as a percentage o f the i n c o r p o r a t i o n at 24 h, was 87.9, 94.9 and 88.7% at 20, 28 a n d 32 h, respectively. W h e n n4C-Thr was a d d e d during the stationary phase, the relative i n c o r p o r a t i o n was less than 50%. Thus, the highest i n c o r p o r a t i o n rate o f Thr into free NCS-chr was f o u n d to be parallel with free NCS-chr production. Table 3 shows the effect o f protein synthesis inhibitors on the synthesis o f free NCS-chr. Mycelia in the r a p i d growth phase (at 20, 24, 28 and 32 h) were transferred to the basal m e d i u m containing 20 pCi o f 14C-Thr and either one o f 50/~g/ml chloramphenicol or 25 ~ g / m l puromycin. A f t e r 2 h pulse labelling, mycelia were transferred to the original culture fluid and cultivated for 120 h. The culture filtrate (one liter) was then examined for free NCS-chr as described above. A n Avicel column (2.5 x 30 cm) a b s o r b e d with crude free NCS-chr was eluted with ethyl ether, acetone and m e t h a n o l , successively. Each eluate was concentrated in vacuo and residues were dissolved in 1 0 m l o f methanol. The NCS-chr was detected only in the methanol eluate; the ethyl ether eluate contained unidentified antimicrobial activity (which will be reported elsewhere). W h e n 14C-Thr was a d d e d at 24 h, the radioactivity in the fractions eluted with ethyl ether, acetone and methanol were 30.5 × 103, 51.5 x 103 a n d 11.2 × 10 a cpm, respectively. The effect o f inhibitors on the biosynthesis o f free NCSchr was investigated using the m e t h a n o l eluate (Table 3). The a d d i t i o n o f either one o f the inhibitors during the r a p i d growth phase was not significantly effective towards the growth o f S. carzinostaticus, except for the a d d i t i o n at 20 h. I n c o r p o r a t i o n o f '4C-Thr into free NCS-chr at the late logarithmic phase o f the culture continued in the presence o f the inhibitors. These observations indicate that f o r m a t i o n o f free NCS-chr is unaffected by the inhibitors used in this experiment if the drugs are a d d e d to the organism in the stationary phase o f growth. It was evident that free NCS-chr was synthesized during r a p i d growth o f S. carzinostaticus var. F-41 when Thr or A s h as a sole nitrogen source was used at a level equivalent to the nitrogen concentration o f casamino acids. Synthesis m a y occur even in the stationary growth phase after 20 h cultivation. However, when the organism was cultivated in the same m e d i u m containing less t h a n 1 . 2 5 ~ MgSO4, synthesis o f detectable quantities o f free N C S - c h r did not occur (12). This might result f r o m the enzyme (s) related to the biogenesis o f free NCS-chr being suppressed in S. carzinostaticus when the m e d i u m contained a s u b o p t i m a l Mg 2+ concentration. However, it should be noted that the Mg 2+ concentration required is unusually high for k n o w n
VoL. 75, 1993 TABLE 3.
NOTES
225
Effect of inhibitor of protein synthesis on the incorporation of J4C-Thr into free NCS-chr in the rapid growth phase Incorporation of t4C-Thr into free NCS-chr (methanol eluate) cpm × 10~ Relative incorporation
Time of addition
01)
Mycelial growth (dry weight: mg/100 ml)
None Chloramphenicol (50) Puromycin (25)
20 20 20
498 341 316
27.6 40.2 35.9
None Chloramphenicol (50) Puromycin (25)
24 24 24
502 485 466
32.3 51.5 47.3
None Chloramphenicol (50) Puromycin (25)
28 28 28
510 495 489
30.1 49.2 46.1
95.5 97.4
None Chloramphenicol (50) Puromycin (25)
32 32 32
495 500 510
28.1 39.8 38.4
77.2 81.1
Inhibitor (/zg/ml)
78.6 75.8 100 100
Experimental procedures were as for Table 2 except that pulse-labelling was carried out in 20/zCi of ~4C-Thr and drugs. As a control (none), mycelia was incubated with 20/~Ci of ~4C-Thr and without drug. Determination of the incorporation ratio (%) was the same as described in Table 2.
biochemical processes. Besides Thr, Asn was the other amino acid incorporated into free NCS-chr. It seems likely that Asn was incorporated through Thr in the A s n ~ h o m o serine~Thr metabolic pathway. REFERENCES
protein component and a protein component from neocarzinostatin (NCS) and their biological activity. J. Antibiot., 33, 342346 (1980).
7. Suzuki, K., Miura, K., Kumada, Y., Takeuchi, T., and Tanaka, N.: Biological activities of non-protein chromophore of antitumor protein antibiotics: auromomycin and neocarzinostatin. Biochem. Biophys. Res. Commun., 94, 255-261 (1980).
8. Edo, K., Katamine, S., lshlda, N., Koide, Y., and Nozoe, S.:
1. Ishida, N., Miyazaki, K., Kumagai, K., and Rikimaru, M.: Neocarzinostatin, an antitumor antibiotic of high molecular weight: isolation, physicochemical properties and biological activities. J. Antibiot., Set. A, 18, 68-76 (1965). 2. Kudo, K., Kikuchi, M., and Ishida, N.: Biogenesis of an antitumor antibiotic protein, neocarzinostatin. Antimicrob. Agents & Chemother., 1, 289-295 (1972). 3. Maeda, H.: Neocarzinostatin in cancer chemother. (review). Anticancer Rev., 1, 165-186 (1981). 4. Ohtsuki, K. and Ishida, N.: Mechanism of action of neecarzinostatin (NCS) and NCS-associated non-protein chromophore. Protein, Nucleic Acid & Enzyme, 26, 937-949 (1981).
9.
10.
11.
5. Napier, M.A., Holmquist, B., Stroydom, D., and Goldberg, I. H.: Neocarzinostatin: spectral characterization and separation of a non-protein chromophore. Biochem. Biophys. Res. Commun., 89, 635--642 (1979).
6. Koide, Y., Ishii, F., Hasnda, K., Koyama, Y., Edo, K., Katamine, S., Kltame, F., and lshida, N.: Isolation of a non-
12.
Naphthalenecarboxylic acid from neocarzinostatin (NCS) and their biological activity. J. Antibiot., 33, 347-350 (1980). Edo, K., Mlzugaki, M., Koide, Y., and Ishida, N.: The structure of neocarzinostatin chromophore possessing a novel bicyclo[7,3,0]dodecadiyne system. Tetrahedron Lett., 26, 331-334 (1985). Jung, G., Kohlein, W., and Lander, G.: Biological activity of the antitumor protein neocarzinostatin coupled to a monoclonal antibody by N-succimidyl-3-[2-pyridylthio]propionate. Biochem. Biophys. Res. Commun., 101, 599-606 (1980). Ohtsuki, K., Koike, T., Sato, T., Koyamn, Y., and lshidn, N.: The inhibitory mechanism of in vitro protein phosphorylation by a non-protein chromophore removed from neocarzinostatin. Biochim. Biophys. Acta, 673, 147-156 (1980). Kudo, K., Snto, T., Koide, Y., Edo, K., and lshlda, N.: Production of a free chromophre component of neocarzinostatin (NCS) in the culture filtrate of Streptomyces carzinostaticus var. F-41. J. Antibiot., 35, 1111-1115 (1982).
Vol. 75, No. 3,223-225. 1993
Improved Production of a Chromophore Component of Antitumor Antibiotic Neocarzinostatin (NCS) by Addition of Threonine in a Chemically Defined Medium KOZO KUDO,I*TSUNEHISA SUTO, I YUKO YOSHIDA, 1 KIYOTO EDO, 2 ~ D NAKAO ISHIDA 3 Department of Microbiology, Akita University School of Medicine, Akita 010,1 Department o f Pharmacy, Fukushima Prefectural College o f Medicine, Hospital, Fukushima 960, 2 Institute o f Sendai Microbiology, Sendal 980, 3 Japan Received 1 June 1992/Accepted 6 November 1992 Production in a chemically defined medium of a free chromophore component (free NCS-chr) of an antitumor antibiotic neocarzinostatin (NCS) by Streptomyces carzinostaticus var. F-41 was improved by the use of L-threonine or L-asparagine as a sole nitrogen source. Under the conditions established, the yield of free NCSchr was comparable to that obtained in a control medium containing casamino acids.
An antitumor antibiotic, neocarzinostatin (NCS), produced by Streptomyces carzinostaticus var. F-41, was initially regarded as a single polypeptide with molecular weight of 11,000 (1, 2). Clinically, NCS has been shown to be effective against pancreas, stomach and bladder cancers in a number of patients, as reviewed by Maeda (3). Later, Ohtsuki et al. (4), and Goldberg et al. (5) showed that a non-protein chromophore component (NCS-chr) was present in the NCS molecule. The isolation and characterization of the NCS-chr were achieved independently by Koide et al. (6), Goldberg et al. (5) and Suzuki et al. (7). The NCS-chr isolated from clinical grade preparations of NCS exhibits an optical rotation of [a]D20-171o, a molecular weight of 659, and a molecular formula of C35Ha30~2 by mass spectrometry (8). Two components (2-hydroxy5-methoxy-7-methyl-l-naphthalencarboxylic acid and Nmethylfucosamine) have been isolated from the NCS-chr and characterized (8). Infrared spectrometry, polarographic analyses and other chemical data suggested the presence of peroxy acid, a highly strained ether ring (possibly an epoxide) and a bicyclo[7,3,0]dodecadiyne system in its structure (9). Further studies by Jung et al. and Koide et al. have shown that in vitro mixing of the NCS-chr with the NCS-protein moiety resulted in the regeneration of both the biological activities and chemical properties of NCS (6, 10). As noted for the holo-NCS molecule, the NCS-chr also inhibits DNA synthesis selectively without a concomitant effect on RNA or protein synthesis (11). In the present report, the biosynthesis of the NCS-chr is characterized. Previously we were successful in producing the free form of the NCS-chr (free NCS-chr) in a simple semi-defined medium containing either MgSO4 or MgCI2 at 1,000 times higher concentration than in the original NCS production medium (12). NCS-producing S. carzinostaticus var. F-41 was maintalned as previously described (2). For production of free NCS-chr, a semi-defined medium containing 4% glucose, 1.5% casamino acids, 1.25% MgSO4, 0.25% NaCI, 0.2% CaCO3 and 0.1% K2HPO4 (pH 7.2) was used. A casamino acids-free medium (basal medium) was employed to examine the effect of a variety of amino acids upon free NCS-chr synthesis. MgSO4 and K2HPO4 were sterilized
separately and added aseptically to a double-strength basal medium in 200 ml Monod tubes to give a final volume of 100 ml. Quantitative analysis of free NCS-chr and the native NCS molecule has been described previously (12). Standard NCS in this experiment was a gift from Kayaku Co. Ltd., Tokyo. To analyse the free NCS-chr in the fermentaion broth, 100 ml of culture filtrate was first extracted with ethyl acetate (EtOAc). The EtOAc layer was evaporated in vacuo and dissolved in 1 ml of methanol to measure the antimicrobial and isotopic activities. The effect of single amino acids on free NCS-chr formation was examined as follows. S. carzinostaticus vat. F-41 was grown in 100 ml of a hot-water (pH 7.2) extract fraction of a soybean meal medium (12) at 28°C for 16 h on a reciprocal shaker (30rpm) and spun down at 3,000rpm for 15 min. The resulting mycelial pellet was aseptically washed 3 times with saline and resuspended in 100 ml of saline. Three ml of this suspension was transferred to 100 ml of the basal medium supplemented with a single amino acid. After the indicated cultivation time, free NCS-chr was isolated from the culture filtrate to measure the antimicrobial activity. Mycelia were harvested by centrifugation, washed, dried and then weighed. The addition of a single amino acid to the basal medium resulted in good growth of mycelium (dry weight: 300-500 mg/100 ml). As shown in Table 1, only L-threonine (Thr) or L-asparagine (Asn) among the various amino acids tested gave good yields of free NCS-chr comparable to that obtained in the basal medium supplemented with casamino acids. The other amino acids supported little or no NCS-chr synthesis. The time course of typical fermentation in the medium supplemented with Thr is shown in Fig. 1. The pH, 7.2 initially, declined to 6.0 during the rapid growth phase. Approximately 70% of free NCS-chr was synthesized during the first 36 h and the maximum accumulation was obtained at 72 h. When a crude preparation of free NCS-chr was analyzed by a high performance liquid chromatography, the major active principle was eluted with methanol-H20-formic acid (93 : 5 : 2), as in the case with the NCS-chr (12), and two significant peaks appeared at 3 min and 14.5 min (data not shown). To determine the time of maximum incorporation into free NCS-chr, mycelia of S. carzinostaticus grown in the basal medium containing Thr for 16, 20, 24, 28, 32 and
* Corresponding author. 223
224
KUDO ET AL.
7.0 c~
"'0"0"0"0.•.
J. FERMENT.BIOENO.,
• . . . . . . . .-0 . . . . . . . . . . .•. . . . .
•
TABLE 2. Incorporation efficiency of '4C-Thr into free NCS-chr during cultivation
"q~ •
5.0
7
2
200
400
z
200
_
24
71
i
i
2 120 Cultivation time(h)
11
i
168
FIG. 1. Time course of free NCS-chr production. Symbols: ~---o, free NCS-activity; O - - - o , NCS activity; O-----O,mycelial growth measured as dried cell weight; ©---©, pH monitored by a pH meter (Horiba F-12). 48 h were separated, transferred to basal m e d i u m containing ~4C-Thr (5 p C i / 1 0 0 ml), a n d cultivated. A f t e r 2 h pulse-labelling followed b y continued cultivation in the original culture fluid for 96 h in total, free NCS-chr was extracted f r o m the culture filtrate to measure the radioactivity as described above. The results (Table 2) showed that the most efficient i n c o r p o r a t i o n o f 14C-Thr into the E t O A c fraction was at 24 h. The relative i n c o r p o r a t i o n o f '4C-Thr into free NCS-chr, determined after 96 h cultivation and TABLE 1. Effect of various amino acids in the basal medium on free NCS-chr yield by S. carzinostaticus var. F-41 Amino acida Leucine IsoLeucine Arginine HC1 Dt-Alanine Asparagine Lysine Gintamine Cystine Methionine DL-Tryptophan Valine Phenylalanine Glycine Threonine Casamino acids
Free NCS-chrb Cultivation time (h) 28 123 ----23 2 2 -24 26 23 23 -2 -
-
--22 23 23 24 24
-
-
--2 -23 26 26
a The amount of each amino acid added to the basal medium contained 0.132% nitrogen, which was the same nitrogen concentration as that of 1.5°~ casamino acids. b The free NCS-chr obtained from the culture filtrate was dissolved in methanol, diluted 2-fold and assayed on anti-microbial activity. Free NCS-chr potency was indicated by the minimum inhibitory concentration (dilution titer) of the diluted solution.
Labelling time (h)
cpm x 103
16-18 20-22 24-26 28-32 32-34 48-50
8.3 10.9 12.4 11.7 11.0 5.7
Free NCS-chr Relative incorporation (%) 66.9 87.9 100 94.9 88.7 46.0
Isolation of radioactive free NCS-chr was the same as described in the footnote to Table 1. The ['4C]radioactivity was measured in a liquid scintillation counters (Aloka LSC 700). The incorporation ratio given in % compares the radioactivity at the indicated time with that obtained at 24 h, because all of the cultures had a similar free NCSchr titer. estimated as a percentage o f the i n c o r p o r a t i o n at 24 h, was 87.9, 94.9 and 88.7% at 20, 28 a n d 32 h, respectively. W h e n n4C-Thr was a d d e d during the stationary phase, the relative i n c o r p o r a t i o n was less than 50%. Thus, the highest i n c o r p o r a t i o n rate o f Thr into free NCS-chr was f o u n d to be parallel with free NCS-chr production. Table 3 shows the effect o f protein synthesis inhibitors on the synthesis o f free NCS-chr. Mycelia in the r a p i d growth phase (at 20, 24, 28 and 32 h) were transferred to the basal m e d i u m containing 20 pCi o f 14C-Thr and either one o f 50/~g/ml chloramphenicol or 25 ~ g / m l puromycin. A f t e r 2 h pulse labelling, mycelia were transferred to the original culture fluid and cultivated for 120 h. The culture filtrate (one liter) was then examined for free NCS-chr as described above. A n Avicel column (2.5 x 30 cm) a b s o r b e d with crude free NCS-chr was eluted with ethyl ether, acetone and m e t h a n o l , successively. Each eluate was concentrated in vacuo and residues were dissolved in 1 0 m l o f methanol. The NCS-chr was detected only in the methanol eluate; the ethyl ether eluate contained unidentified antimicrobial activity (which will be reported elsewhere). W h e n 14C-Thr was a d d e d at 24 h, the radioactivity in the fractions eluted with ethyl ether, acetone and methanol were 30.5 × 103, 51.5 x 103 a n d 11.2 × 10 a cpm, respectively. The effect o f inhibitors on the biosynthesis o f free NCSchr was investigated using the m e t h a n o l eluate (Table 3). The a d d i t i o n o f either one o f the inhibitors during the r a p i d growth phase was not significantly effective towards the growth o f S. carzinostaticus, except for the a d d i t i o n at 20 h. I n c o r p o r a t i o n o f '4C-Thr into free NCS-chr at the late logarithmic phase o f the culture continued in the presence o f the inhibitors. These observations indicate that f o r m a t i o n o f free NCS-chr is unaffected by the inhibitors used in this experiment if the drugs are a d d e d to the organism in the stationary phase o f growth. It was evident that free NCS-chr was synthesized during r a p i d growth o f S. carzinostaticus var. F-41 when Thr or A s h as a sole nitrogen source was used at a level equivalent to the nitrogen concentration o f casamino acids. Synthesis m a y occur even in the stationary growth phase after 20 h cultivation. However, when the organism was cultivated in the same m e d i u m containing less t h a n 1 . 2 5 ~ MgSO4, synthesis o f detectable quantities o f free N C S - c h r did not occur (12). This might result f r o m the enzyme (s) related to the biogenesis o f free NCS-chr being suppressed in S. carzinostaticus when the m e d i u m contained a s u b o p t i m a l Mg 2+ concentration. However, it should be noted that the Mg 2+ concentration required is unusually high for k n o w n
VoL. 75, 1993 TABLE 3.
NOTES
225
Effect of inhibitor of protein synthesis on the incorporation of J4C-Thr into free NCS-chr in the rapid growth phase Incorporation of t4C-Thr into free NCS-chr (methanol eluate) cpm × 10~ Relative incorporation
Time of addition
01)
Mycelial growth (dry weight: mg/100 ml)
None Chloramphenicol (50) Puromycin (25)
20 20 20
498 341 316
27.6 40.2 35.9
None Chloramphenicol (50) Puromycin (25)
24 24 24
502 485 466
32.3 51.5 47.3
None Chloramphenicol (50) Puromycin (25)
28 28 28
510 495 489
30.1 49.2 46.1
95.5 97.4
None Chloramphenicol (50) Puromycin (25)
32 32 32
495 500 510
28.1 39.8 38.4
77.2 81.1
Inhibitor (/zg/ml)
78.6 75.8 100 100
Experimental procedures were as for Table 2 except that pulse-labelling was carried out in 20/zCi of ~4C-Thr and drugs. As a control (none), mycelia was incubated with 20/~Ci of ~4C-Thr and without drug. Determination of the incorporation ratio (%) was the same as described in Table 2.
biochemical processes. Besides Thr, Asn was the other amino acid incorporated into free NCS-chr. It seems likely that Asn was incorporated through Thr in the A s n ~ h o m o serine~Thr metabolic pathway. REFERENCES
protein component and a protein component from neocarzinostatin (NCS) and their biological activity. J. Antibiot., 33, 342346 (1980).
7. Suzuki, K., Miura, K., Kumada, Y., Takeuchi, T., and Tanaka, N.: Biological activities of non-protein chromophore of antitumor protein antibiotics: auromomycin and neocarzinostatin. Biochem. Biophys. Res. Commun., 94, 255-261 (1980).
8. Edo, K., Katamine, S., lshlda, N., Koide, Y., and Nozoe, S.:
1. Ishida, N., Miyazaki, K., Kumagai, K., and Rikimaru, M.: Neocarzinostatin, an antitumor antibiotic of high molecular weight: isolation, physicochemical properties and biological activities. J. Antibiot., Set. A, 18, 68-76 (1965). 2. Kudo, K., Kikuchi, M., and Ishida, N.: Biogenesis of an antitumor antibiotic protein, neocarzinostatin. Antimicrob. Agents & Chemother., 1, 289-295 (1972). 3. Maeda, H.: Neocarzinostatin in cancer chemother. (review). Anticancer Rev., 1, 165-186 (1981). 4. Ohtsuki, K. and Ishida, N.: Mechanism of action of neecarzinostatin (NCS) and NCS-associated non-protein chromophore. Protein, Nucleic Acid & Enzyme, 26, 937-949 (1981).
9.
10.
11.
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