- Email: [email protected]
Nicotinamide metabolism in a nicotinamide-requiring yeast, Saccharomyces fragilis C351
ARCHIVES
OF
BIOCHEMISTRY
Nicotinamide
AND
BIOPHYSICS
Metabolism
118,
97-105
in a Nicotinamide-Requiring
Saccharomyces STERLING Department
CHAYKIN,
of Biochemistry
(1967)
fragilis
LANE KIKG,
and Biophysics,
Yeast,
G5:”
AND
RITA SCHINDLER
University of California,
Davis,
California
95616
Received June 10, 1966 Saccharomyces fragilis C&,1 was grown on Difco vitamin-free yeast base that had been supplemented with inositol, biotin, calcium pantothenate, and nicotinamide. It would not grow when nicotinamide was omitted from the medium. The metabolic fate of nicotinamide in this organism has been traced using nicotinamide-7-W. Nicotinamide was rapidly taken up by the cells and converted to DPN. No intermediates were detected. The appearance of TPN in the cells lagged behind that of DPN. During the period of maximum formation of TPN, the level of DPN in the cells decreased in what appeared to be a direct relationship to the increase in the level of TPN. Both DPN and TPN leaked out of the cells when one or more factors present in the vitamin-free yeast base were exhausted. The singular fate of nicotinamide in S. fragilis C 351offered a simple means for the preparation of DPN labeled with W in its nicotinamide moiety. A procedure has been worked out that employs S. fragilis C 351for the synthesis of small quantities of DPNW of high specific activity. TPN-W was an important by-product of the method. The nucleotides were isolated and separated by DEAE-cellulose chromatography.
The study of the metabolism of the pyridine nucleotides in mammals, in vivo, is a technically difficult procedure. The problems range from poor incorporation of labeled precursors into these compounds, to experimentally uncontrollable factors such as diet, hormone levels, and the potential of the different cell types present in an organism for individuality in their metabolic processes. Interpretation of data is oft’en impossible to make in the face of such a number of interacting variables. Thus at the time this investigation was begun, there was clearly a need for a model system in which the generalities of pyridine nucleo-
tide metabolism could be studied. Yeast have a number of attributes suited to such a model: They can be grown as a homogeneous cell type in a defined environment. The use of a nicotinamide-requiring strain would insure good incorporation of labeled precursors into the pyridine nucleotides. Furthermore, the principle organelles of mammalian cells have their counterparts in yeast. In particular, the study of the reactivities of the pyridine nucleotides in mitochondria might be possible if yeast were used as the experimental material. This presentation is a first report of t’he metabolism of the pyridine nucleotides in yeast.
1 This investigation was supported in part by Grants AM03464 and AM05741 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service. 2 A brief account of this work was presented at the Ninth Symposium on Advances in Tracer Methodology that was held in San Francisco on October 23, 1964.
fragilis CSS~was maintained for as long as a year at 4” under mineral oil on slants containing 1% Difco yeast extract, 2% peptone, 2yo dextrose, and 2% agar. Cells were transferred to and grown overnight at 37” on a liquid medium before experimental use. The liquid medium contained Difco vitamin free yeast base, 16.7 gm/liter
MATERIALS Saccharomyces
97
AND METHODS
98
CHAYKIN,
KING,
(prepared in a lo-times concentrated form and filtered before dilution), which had been supplemented with inositol, 4.8 mg/liter; biotin, 4.8 fig/liter; calcium pantothenate, 4.8 mg/liter; and nicotinamide 32 rg/liter. Nicotinamide was the growth-limiting factor in the liquid medium. Cells grown overnight on this medium were in stationary phase and nicotinamide-starved. Experimental flasks were inoculated with a onetenth volume of such a culture. All cultures were shaken vigorously in order to maintain aerobic growth conditions. Unless specified otherwise, cultures were incubated at 37”. Growth was estimated by following changes in optical density at 600 rnp. When optical density readings exceeded 0.500 the samples were diluted with 0.9% NaCl until the optical density readings fell below 0.500. Samples of culture taken as part of the kinet)ic studies were treated as follows. Five-ml samples were placed in cold 12-ml centrifuge tubes and quickly cooled by swirling the tubes in a mixture of ice and water. The cells were recovered by centrifugation for 2 minutes at 1500 g. Appropriate aliquots of the supernatant fluid were taken for counting of radioactive materials or paper chromatographic analysis, or both. The remaining supernatant fluid was removed by decantation. The cells were resuspended in 1.5 ml of cold 0.9% NaCl and again recovered by centrifugation. After removal of the supernatant fluid, the cells were suspended in 0.4 ml of 0.01 M phosphate buffer, pH 7.5, and the centrifuge tube containing the cells was placed in a boiling water bath for 3 minutes. The tubes were quickly cooled again and frozen overnight. Seven minutes elapsed from the time the culture sample was taken until the cells were heat-treated. The radioactive metabolites of nicotinamide7J4C were separated and identified by descending paper chromatography. Samples (0.3 ml) of the cell-free culture medium or 0.2.ml samples of the cell extracts were applied to 1.5 X 22-inch strips of Whatman No. 1 filter paper in preparation for paper chromatographic analysis. Appropriate chromatographic markers were applied to the chromatograms to aid in the identification of radioactive materials. They included DPN, TPN, nicot,inamide, and nicotinic acid. Each chromatogram was run in n-butanol saturated with water (1) and then re-chromatographed in an ethanol: ammonium acetate solvent system (5 parts of 95(% ethanol + 2 parts of 1 M ammonium acetate; adjusted to pH 7.5 with concentrated NHdOH). The chromatogram was developed in the first solvent for 18 hours. The first solvent was used to separate nicotinamide and nicotinic acid from the pyridine nucleotides. Their RF values were 0.69,
AND
SCHINDLER
0.11, and 0.00, respectively. After the determination of radioactivity associated with the various radioactive components separated by the first solvent had been completed, the chromatograms were developed in the second solvent, for 5(r60 hours. Serrated edges were cut on the lower ends of the chromatograms in preparation for the second chromatographic development. This procedure was used in order to prevent skewing of the materials being chromatographed by uneven dripping of the solvent. Nicotinamide and nicotinic acid ran off the chromatograms during the second chromatographic procedure; the pyridine nucleotides, DPN and TPN, had RF values of 0.50 and 0.25, respectively. The nicotinamide-7-l% and nicotinic acid-7-l% used in these studies were purchased from New England Nuclear Corp.; their specific activit’ies were 42.1 mC/mmole and 10 mC/mmole, respectively. Radioactive substances in aqueous solution were counted on a liquid scintillation counter in the solvent developed by Bray (2). Radioactive materials on paper chromatograms were quantitated with a Vanguard paper strip counter with integrator attachment. When cells were grown as a source of small quantities of DPN-1% and TPN-I%, 500 ml of the liquid medium described above was used. The culture medium was cooled to 0” before the cells were collected by centrifugation for 15 minutes at 17,000 g. The cells (1.3-1.5 gm, wet weight) were suspended in 10 ml of 0.01 M phosphate buffer, pH 7.5, heated in a boiling water bath for 3 minutes, and cooled quickly. Cell debris was removed by centrifugation; the pH of t,he extract was 7.0. The pyridine nucleotides were isolated by chromatography of the extract on DEAE-cellulose using a linear gradient of ammonium bicarbonate (3) RESULTS
AND
DISCUSSION
Figure 1 shows the time course of the growth of S. fragilis and the rate of the uptake of nicotinamide upon which that growth depends. The rate of disappearance of nicotinamide from the medium was only slightly affected by the temperature at which the cells were incubated. However, there was a considerable temperature effect on the length of the lag period before the cells began to grow rapidly. Since the cells entered the rapid growth phase fast’er when grown at 37”, that temperature was adopted for routine use. Figure 2 presents data that support the contention that nicotinamide is the growth-
SACCHAROMYCES
FRAGILIS
99
Cm
8 t w’ HOURS
FIG. 1. The effect of temperature on growth and nicot,inamide-7-l% uptake. The concentration of nicotinamide in the medium was 15Opg/liter. All other conditions were as described in the text. n ando; 24”;M andA, 37”;(---) nicotinamide-T-14C uptake; (-) I&.
limiting nutrient in the culture medium. The optical density at 600 rnp was more t,han tripled when the nicotinamide concentration was quadrupled. The final optical density was essentially the same when either nicotinamide or nicotinic acid was used to satisfy t,he growth requirement. The lag phase was slightly short’er when nicotinic acid was used as growth factor. When the distribution of radioactivity between the cells and the medium was examined over a 24-hour period, an interesting phenomenon came to light (Fig. 3). Cells crown at, low concentrations of nicotinamide-7-14C or nicotinic acid-7-14C took up the radioactive material and retained most of it throughout the 24-hour duration of the experiment’. The distribution of radioactivity at higher concentrations of nicotinamide and nicot,inic acid had a complex time course. Radioactivity first moved into the cells and then back into the medium again. The radioactivit,y in t,he cells was restricted t,o a relatively few compounds. The time course of the appearance of radioactivity
in the various
compounds
in cells
grown on nicotinamide-7-14C is shown in Figs. 4 and 5. When cells grown on nicotinic acid were examined the spectrum of compounds
was
qualitat’ively
identical
to the
2
0
6
12
16
24
HOURS FIG. 2. Growth at high and low concentrations of nicot,inamide and nicotinic acid. The low concentration was 33 pg/liter and the high concentration was 130 pg/liter. (- - -) Nicotinic acid; (-) nicotinamide.
patterns obtained with cells grown on nicotinamide. Since the only differences noted were small quantitative ones, the experiments in which nicotinic acid was used are not presented in detail. The order of appearance of radioactive compounds in cells grown at both high and low concentrations of either nicotinamide-7J4C or nicotinic acid-7J4C was nicotinamide, DPN, nicotinic acid, and TPN. No other radioactive materials were observed. Nicotinamide and nicotinic acid had a transient existence in cells grown at both high and low concent#rations of the growth-limiting nutrient. The levels of the pyridine nucleotides, on the other hand, were quite stable in nicobinamide-limited
cells,
but,
fluctuated
in
cells grown at high concentrations of nicotinamide. Although it is not obvious from a ronsideration of Figs. 4 and 5 that DPN - -~~.~-.-~~
100
CHAYKIN,
KING,
AND SCHINDLER
IO
6.
A. t 5
‘5- z,a*-, -O‘----$----CELLS,.
, 0
I
I
6
12
I6
24
/---“” I 6
1 I2
-
I 18
HOURS
FIQ. 3 Distribution of radioactivity between the cells and the medium. These data were collected in the same experiment as the data shown in Fig. 2. A and B were at the high and low concentrations of nicotinamide and C and D at the high and low concentrations of nicotinic acid, respectively.
1
TPN
--------
--__________
---a-_
0
DPN
8\c.-8------0
6
I 12
--
--
-I 18
NICOTINIC ----m
ACID
I 24
HOURS
FIG. 4. Radioactive compounds found in cells grown at a low concentration of nicotinamide. The data were collected in the same experiment as shown in Fig. 3B. The radioactive compounds were derived from the cells found in a &ml sample of the culture at the times specified in the figure.
SACCHAROMYCES
FRAGILIS
Gas,
101
FIG. 5. Radioactive compounds found in cells grown at a high concentration of nicotinamide. The data were collected in the same experiment as shown in Fig. 3A. The radioactive compounds were derived from the cells found in a 5-ml sample of the culture at the times specified in the figure. and TPN had a precursor-product relationship, presentation of the data in an alternate form makes that relationship clearly visible (Fig. 6). Increases in the amounts of TPN in the cells were directly compensated for by equivalent decreases in the amounts of DPN present. The relative amounts of DPN and TPN in the cells at the conclusion of the experiment were a function of the original nicotinamide concentration in the medium. Cells grown on nicotinamide-deficient media produced more TPN than DPN; those grown on nicotinamide-rich media contained more DPN than TPN. The shift in radioactivity from the cells to the medium (Fig. 3A), which occurred as the cells grown at high concentrations of nicotinamide entered the stationary phase (Fig. 2), appeared to be most closely related to the decrease in the cellular pyridine nucleotide levels. It was therefore not surprising to find that the radioactivity that
began to appear in the medium after the sixth hour of incubation was associated with DPN and TPN. The time course of the appearance of the nucleotides is shown in Fig. 7. The identity of the pyridine nucleotides in both the cells and the medium was established by enzymic conversion to their reduced forms and identification of the reduced forms by chromatography on DEAE-cellulose (3). The DPN and TPN that appeared in the medium did not seem to be subject to degradation. When nicotinic acid was used as the growth factor it was the only radioactive material present in the medium during the first 2 hours of incubation of the cells. When nicotinamide was used as the growth factor both nicotinamide and nicotinic acid appeared in the medium (Fig. 7). Approximately 60% of the growth factor was in the form of nicotinic acid in the first sample taken (30 minutes) in the experiment shown
CHAYKIN,
KING,
TPN
.----------”
I
6
I
12
AND SCHINDLER
4
I
I6
24
I
I
I
6
12
16
2I
HOURS
HOURS
FIG. 6. Percentage of total cellular pyridine nucleotide present as DPN and TPN. (A) Derived from data presented in Fig. 4; (B) derived from data presented in Fig. 5.
HOURS FIG. 7. Radioactive compounds found in the culture medium when cells were grown at a high concentration of nicotinamide. The radioactivity is that found in a 5-ml sample of the culture medium at the times specified in the figure. (- - -) Nicotinamide; (-. . .-) nicotinic acid.
in Fig. 7. This observation is compatible with the high levels of nicotinamide deamidase activity known to be present in yeast (4). The experimental evidence presented above leads to the following picture of
nicotinamide metabolism in S. fro&s C&51. This strain of yeast can deamidate nicotinamide and cause nicotinic acid to appear in the medium. The enzyme does not appear to be an extracellular one since the cell-free medium lacks deamidase activity. The ap-
SACCHAROMYCES
25
50
FRAGILIS
C&
103
75 FRACTION
NUMBER
FIG. 8. The chromatograpbic isolation of DPN-i4C and TPN-i4C. The cell extract was applied to a 1.5 X 25-cm DEAF-cellulose (bicarbonate) column. The column was developed with a linear gradient of ammonium bicarbonate (O-O.4 M; 1500 ml). Five-ml fractions were collected. (-) Absorbance at 260 rnp; (- - -) radioactivity; peak I, nicotinamide; peak II, DPN; peak IV, TPN; peaks III and V, unidentified. pearance of the acid in the medium can be explained by either leakage from the cells or by a deamidase associated with the cell Both nicotinamide and nicotinic membrane.
acid appear inside the cells when C& is grown on either form of the growth factor. DPN is the first nucleotide form of the growth factor which is found in the cells; TPN appears to be derived from DPN. None of the possible intermediates in the synthesis of DPN was observed. The reduced nucleotides were not found either. The absence of these compounds may have been due to metabolism, both synthetic and oxidative, during the 7-minute period required to harvest, wash, and kill the cells. A more rapid harvesting procedure will have to be used if the biosynthetic intermediates and reduced nucleotides are to be detected. Cells grown under conditions in which a factor(s) present in the vitamin-free yeast base was limiting, leaked nucleotides into the medium. The vitamin-free yeast base was shown to be involved in the leak-
age phenomenon in the following way. A medium similar to the one used in Fig. 3A was supplemented with four. times the concentrations of the vitamin supplements (exclusive of nicotinamide). The leakage was the same as that shown in Fig. 3A. Tripling of the concentration of the vitamin-free yeast base reduced the leakage of the pyridine nucleotides. The distribution of radioactivity between the cells and the medium was very much like that shown in Fig. 3D. The observation of the leakage of nucleotides from yeast is not without precedent. Rose (5) found that yeast grown on a biotin-deficient medium leaked desamido DPN into the medium. Since the pyridine nucleotides were the unique end products of the met’abolism of nicotinamide in S. fragilis C& it seemed possible that this organism might be used for the biosynthesis of DPNJ4C and TPN14C from nicotinamide-14C. The system appeared to offer a means for the preparation of small quantities of the nucleotides,
104
CHAYKIN,
KING,
AND SCHINDLER
TABLE NUCLEOTIDE Tie
32 32 120
Stationary
I YIELDSO To Fbdioactivity
of harvest
TPN
phase
Intracellular Extracellular Intracellular Extracellular Intracellular Extracellular Total
Just before stationary phase Stationary phase
(1The amounts of radioactivity originally were recovered in DPN and TPN.
16 None 9 None 10 6 16
DPN
Total
26
42
None
None
26
35
None
None
28 14 42
38 20 58
placed in the culture medium as nicotinamide-7-14C which
at high specific activity and in good yield. Cells of S. frugilis C 361were therefore grown on a large scale. The nucleotides were extracted in much the same fashion as that used in the kinetic studies discussed above. (The detailed methodology is presented in Experimental Procedure.) The separation of the components of the cell extract is exemplified by the data presented in Fig. 8. The nucleotides recovered from such a column were homogeneous by radiochemical criteria. They were, however, contaminated with nonradioactive materials from yeast. Chemically pure DPN and TPN were obtained by freeing the fractions obtained from the first column from NHJIC03 by lyophilization, reducing the nucleotides by specific enzymic means, rechromatography of the reduced nucleotides and if necessary oxidation, and a third chromatographic procedure in order to isolate the nucleotides, once again, in their oxidized forms. The method, including the use of 1 X lo-cm DEAEcellulose columns with batchwise elution of the nucleotides, has been presented in an earlier publication (3). The method depends on the fact that the chromatographic mobilities of the nucleotides and not those of the impurities are altered by the specific enzymic reduction procedure. Thus during the second chromatographic separation the impurities were eluted before the reduced nucleotides were recovered. A summary of the yields of DPNJ4C and TPNJ4C obtained in several different preparations is shown in Table I. The proportions of the
two nucleotides and their location (in the cells or the medium) could be and were altered by varying the concentration of nicotinamide in the medium and the time at which the cells were harvested. The biosynthetic preparation of DPN14C and TPNJ4C described above complements an existing method for the preparation of larger quantities of DPNJ4C (6) that is based upon the exchange of nicotinamide-14C into the pyridine nucleotides through the use of spleen or bran DPNase (7,8). The DPNase method is best suited for the preparation of milligram quantities of the nucleotides, but the S. fragilis method can be most conveniently used for the biosynthesis of the nucleotides in microgram amounts. A very great advantage of the S. fragilis method is the incorporation of the labeled precursor into the nucleotides without a decrease in specific activity. The DPNase method can result in from two- to eightfold losses in specific activity depending on the yield of products desired. Yields obtained with the S. fragilis method are on the order of 1.5-2 times those obtained with the DPNase method. ACKNOWLEDGMENT The authors are grateful to Dr. Herman Phaff for helpful advice and for furnishing the strain of S. fragilis, C&l, used in this work. REFERENCES 1. BONAVITA, V., NARROD, S. A., AND KAPLAN, N. O., J. Biol. Chem. 236,936 (1961).
SACCHAROMYCES 2. BRAY, G. A., Anal.
Biochem. 1, 279 (1960). 3. CHAYEIN, S., DAOANI, M., JOHNSON, L., SAMLI, M., AND BATTAILE, J., Biochim. Biophys. Acta 100, 351 (1965). 4. OKA, Y., J. B&hem. (Japan) 41, 89 (1954). 23, 143 (1960). 5. ROSE, A. H., J. Gen. Microbial. 6. SILVERSTEIN, E., AND BOYER, P. D., in “Bio-
FRAGILIS
105
C&I
chemical Preparations” (A. Maehly, ed.), Vol. 11, p. 89. Wiley, New York (1964). 7. ZATMAN, L. J., KAPLAN, N. O., AND COLOWICK, S. P., J. Biol. Chem. 200, 197 (1960). 8. COLOWICK,
S. P.,
AND KAPLAN,
N.
O.,
in
“Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. IV, p. 848. Wiley, New York (1957).
OF
BIOCHEMISTRY
Nicotinamide
AND
BIOPHYSICS
Metabolism
118,
97-105
in a Nicotinamide-Requiring
Saccharomyces STERLING Department
CHAYKIN,
of Biochemistry
(1967)
fragilis
LANE KIKG,
and Biophysics,
Yeast,
G5:”
AND
RITA SCHINDLER
University of California,
Davis,
California
95616
Received June 10, 1966 Saccharomyces fragilis C&,1 was grown on Difco vitamin-free yeast base that had been supplemented with inositol, biotin, calcium pantothenate, and nicotinamide. It would not grow when nicotinamide was omitted from the medium. The metabolic fate of nicotinamide in this organism has been traced using nicotinamide-7-W. Nicotinamide was rapidly taken up by the cells and converted to DPN. No intermediates were detected. The appearance of TPN in the cells lagged behind that of DPN. During the period of maximum formation of TPN, the level of DPN in the cells decreased in what appeared to be a direct relationship to the increase in the level of TPN. Both DPN and TPN leaked out of the cells when one or more factors present in the vitamin-free yeast base were exhausted. The singular fate of nicotinamide in S. fragilis C 351offered a simple means for the preparation of DPN labeled with W in its nicotinamide moiety. A procedure has been worked out that employs S. fragilis C 351for the synthesis of small quantities of DPNW of high specific activity. TPN-W was an important by-product of the method. The nucleotides were isolated and separated by DEAE-cellulose chromatography.
The study of the metabolism of the pyridine nucleotides in mammals, in vivo, is a technically difficult procedure. The problems range from poor incorporation of labeled precursors into these compounds, to experimentally uncontrollable factors such as diet, hormone levels, and the potential of the different cell types present in an organism for individuality in their metabolic processes. Interpretation of data is oft’en impossible to make in the face of such a number of interacting variables. Thus at the time this investigation was begun, there was clearly a need for a model system in which the generalities of pyridine nucleo-
tide metabolism could be studied. Yeast have a number of attributes suited to such a model: They can be grown as a homogeneous cell type in a defined environment. The use of a nicotinamide-requiring strain would insure good incorporation of labeled precursors into the pyridine nucleotides. Furthermore, the principle organelles of mammalian cells have their counterparts in yeast. In particular, the study of the reactivities of the pyridine nucleotides in mitochondria might be possible if yeast were used as the experimental material. This presentation is a first report of t’he metabolism of the pyridine nucleotides in yeast.
1 This investigation was supported in part by Grants AM03464 and AM05741 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service. 2 A brief account of this work was presented at the Ninth Symposium on Advances in Tracer Methodology that was held in San Francisco on October 23, 1964.
fragilis CSS~was maintained for as long as a year at 4” under mineral oil on slants containing 1% Difco yeast extract, 2% peptone, 2yo dextrose, and 2% agar. Cells were transferred to and grown overnight at 37” on a liquid medium before experimental use. The liquid medium contained Difco vitamin free yeast base, 16.7 gm/liter
MATERIALS Saccharomyces
97
AND METHODS
98
CHAYKIN,
KING,
(prepared in a lo-times concentrated form and filtered before dilution), which had been supplemented with inositol, 4.8 mg/liter; biotin, 4.8 fig/liter; calcium pantothenate, 4.8 mg/liter; and nicotinamide 32 rg/liter. Nicotinamide was the growth-limiting factor in the liquid medium. Cells grown overnight on this medium were in stationary phase and nicotinamide-starved. Experimental flasks were inoculated with a onetenth volume of such a culture. All cultures were shaken vigorously in order to maintain aerobic growth conditions. Unless specified otherwise, cultures were incubated at 37”. Growth was estimated by following changes in optical density at 600 rnp. When optical density readings exceeded 0.500 the samples were diluted with 0.9% NaCl until the optical density readings fell below 0.500. Samples of culture taken as part of the kinet)ic studies were treated as follows. Five-ml samples were placed in cold 12-ml centrifuge tubes and quickly cooled by swirling the tubes in a mixture of ice and water. The cells were recovered by centrifugation for 2 minutes at 1500 g. Appropriate aliquots of the supernatant fluid were taken for counting of radioactive materials or paper chromatographic analysis, or both. The remaining supernatant fluid was removed by decantation. The cells were resuspended in 1.5 ml of cold 0.9% NaCl and again recovered by centrifugation. After removal of the supernatant fluid, the cells were suspended in 0.4 ml of 0.01 M phosphate buffer, pH 7.5, and the centrifuge tube containing the cells was placed in a boiling water bath for 3 minutes. The tubes were quickly cooled again and frozen overnight. Seven minutes elapsed from the time the culture sample was taken until the cells were heat-treated. The radioactive metabolites of nicotinamide7J4C were separated and identified by descending paper chromatography. Samples (0.3 ml) of the cell-free culture medium or 0.2.ml samples of the cell extracts were applied to 1.5 X 22-inch strips of Whatman No. 1 filter paper in preparation for paper chromatographic analysis. Appropriate chromatographic markers were applied to the chromatograms to aid in the identification of radioactive materials. They included DPN, TPN, nicot,inamide, and nicotinic acid. Each chromatogram was run in n-butanol saturated with water (1) and then re-chromatographed in an ethanol: ammonium acetate solvent system (5 parts of 95(% ethanol + 2 parts of 1 M ammonium acetate; adjusted to pH 7.5 with concentrated NHdOH). The chromatogram was developed in the first solvent for 18 hours. The first solvent was used to separate nicotinamide and nicotinic acid from the pyridine nucleotides. Their RF values were 0.69,
AND
SCHINDLER
0.11, and 0.00, respectively. After the determination of radioactivity associated with the various radioactive components separated by the first solvent had been completed, the chromatograms were developed in the second solvent, for 5(r60 hours. Serrated edges were cut on the lower ends of the chromatograms in preparation for the second chromatographic development. This procedure was used in order to prevent skewing of the materials being chromatographed by uneven dripping of the solvent. Nicotinamide and nicotinic acid ran off the chromatograms during the second chromatographic procedure; the pyridine nucleotides, DPN and TPN, had RF values of 0.50 and 0.25, respectively. The nicotinamide-7-l% and nicotinic acid-7-l% used in these studies were purchased from New England Nuclear Corp.; their specific activit’ies were 42.1 mC/mmole and 10 mC/mmole, respectively. Radioactive substances in aqueous solution were counted on a liquid scintillation counter in the solvent developed by Bray (2). Radioactive materials on paper chromatograms were quantitated with a Vanguard paper strip counter with integrator attachment. When cells were grown as a source of small quantities of DPN-1% and TPN-I%, 500 ml of the liquid medium described above was used. The culture medium was cooled to 0” before the cells were collected by centrifugation for 15 minutes at 17,000 g. The cells (1.3-1.5 gm, wet weight) were suspended in 10 ml of 0.01 M phosphate buffer, pH 7.5, heated in a boiling water bath for 3 minutes, and cooled quickly. Cell debris was removed by centrifugation; the pH of t,he extract was 7.0. The pyridine nucleotides were isolated by chromatography of the extract on DEAE-cellulose using a linear gradient of ammonium bicarbonate (3) RESULTS
AND
DISCUSSION
Figure 1 shows the time course of the growth of S. fragilis and the rate of the uptake of nicotinamide upon which that growth depends. The rate of disappearance of nicotinamide from the medium was only slightly affected by the temperature at which the cells were incubated. However, there was a considerable temperature effect on the length of the lag period before the cells began to grow rapidly. Since the cells entered the rapid growth phase fast’er when grown at 37”, that temperature was adopted for routine use. Figure 2 presents data that support the contention that nicotinamide is the growth-
SACCHAROMYCES
FRAGILIS
99
Cm
8 t w’ HOURS
FIG. 1. The effect of temperature on growth and nicot,inamide-7-l% uptake. The concentration of nicotinamide in the medium was 15Opg/liter. All other conditions were as described in the text. n ando; 24”;M andA, 37”;(---) nicotinamide-T-14C uptake; (-) I&.
limiting nutrient in the culture medium. The optical density at 600 rnp was more t,han tripled when the nicotinamide concentration was quadrupled. The final optical density was essentially the same when either nicotinamide or nicotinic acid was used to satisfy t,he growth requirement. The lag phase was slightly short’er when nicotinic acid was used as growth factor. When the distribution of radioactivity between the cells and the medium was examined over a 24-hour period, an interesting phenomenon came to light (Fig. 3). Cells crown at, low concentrations of nicotinamide-7-14C or nicotinic acid-7-14C took up the radioactive material and retained most of it throughout the 24-hour duration of the experiment’. The distribution of radioactivity at higher concentrations of nicotinamide and nicot,inic acid had a complex time course. Radioactivity first moved into the cells and then back into the medium again. The radioactivit,y in t,he cells was restricted t,o a relatively few compounds. The time course of the appearance of radioactivity
in the various
compounds
in cells
grown on nicotinamide-7-14C is shown in Figs. 4 and 5. When cells grown on nicotinic acid were examined the spectrum of compounds
was
qualitat’ively
identical
to the
2
0
6
12
16
24
HOURS FIG. 2. Growth at high and low concentrations of nicot,inamide and nicotinic acid. The low concentration was 33 pg/liter and the high concentration was 130 pg/liter. (- - -) Nicotinic acid; (-) nicotinamide.
patterns obtained with cells grown on nicotinamide. Since the only differences noted were small quantitative ones, the experiments in which nicotinic acid was used are not presented in detail. The order of appearance of radioactive compounds in cells grown at both high and low concentrations of either nicotinamide-7J4C or nicotinic acid-7J4C was nicotinamide, DPN, nicotinic acid, and TPN. No other radioactive materials were observed. Nicotinamide and nicotinic acid had a transient existence in cells grown at both high and low concent#rations of the growth-limiting nutrient. The levels of the pyridine nucleotides, on the other hand, were quite stable in nicobinamide-limited
cells,
but,
fluctuated
in
cells grown at high concentrations of nicotinamide. Although it is not obvious from a ronsideration of Figs. 4 and 5 that DPN - -~~.~-.-~~
100
CHAYKIN,
KING,
AND SCHINDLER
IO
6.
A. t 5
‘5- z,a*-, -O‘----$----CELLS,.
, 0
I
I
6
12
I6
24
/---“” I 6
1 I2
-
I 18
HOURS
FIQ. 3 Distribution of radioactivity between the cells and the medium. These data were collected in the same experiment as the data shown in Fig. 2. A and B were at the high and low concentrations of nicotinamide and C and D at the high and low concentrations of nicotinic acid, respectively.
1
TPN
--------
--__________
---a-_
0
DPN
8\c.-8------0
6
I 12
--
--
-I 18
NICOTINIC ----m
ACID
I 24
HOURS
FIG. 4. Radioactive compounds found in cells grown at a low concentration of nicotinamide. The data were collected in the same experiment as shown in Fig. 3B. The radioactive compounds were derived from the cells found in a &ml sample of the culture at the times specified in the figure.
SACCHAROMYCES
FRAGILIS
Gas,
101
FIG. 5. Radioactive compounds found in cells grown at a high concentration of nicotinamide. The data were collected in the same experiment as shown in Fig. 3A. The radioactive compounds were derived from the cells found in a 5-ml sample of the culture at the times specified in the figure. and TPN had a precursor-product relationship, presentation of the data in an alternate form makes that relationship clearly visible (Fig. 6). Increases in the amounts of TPN in the cells were directly compensated for by equivalent decreases in the amounts of DPN present. The relative amounts of DPN and TPN in the cells at the conclusion of the experiment were a function of the original nicotinamide concentration in the medium. Cells grown on nicotinamide-deficient media produced more TPN than DPN; those grown on nicotinamide-rich media contained more DPN than TPN. The shift in radioactivity from the cells to the medium (Fig. 3A), which occurred as the cells grown at high concentrations of nicotinamide entered the stationary phase (Fig. 2), appeared to be most closely related to the decrease in the cellular pyridine nucleotide levels. It was therefore not surprising to find that the radioactivity that
began to appear in the medium after the sixth hour of incubation was associated with DPN and TPN. The time course of the appearance of the nucleotides is shown in Fig. 7. The identity of the pyridine nucleotides in both the cells and the medium was established by enzymic conversion to their reduced forms and identification of the reduced forms by chromatography on DEAE-cellulose (3). The DPN and TPN that appeared in the medium did not seem to be subject to degradation. When nicotinic acid was used as the growth factor it was the only radioactive material present in the medium during the first 2 hours of incubation of the cells. When nicotinamide was used as the growth factor both nicotinamide and nicotinic acid appeared in the medium (Fig. 7). Approximately 60% of the growth factor was in the form of nicotinic acid in the first sample taken (30 minutes) in the experiment shown
CHAYKIN,
KING,
TPN
.----------”
I
6
I
12
AND SCHINDLER
4
I
I6
24
I
I
I
6
12
16
2I
HOURS
HOURS
FIG. 6. Percentage of total cellular pyridine nucleotide present as DPN and TPN. (A) Derived from data presented in Fig. 4; (B) derived from data presented in Fig. 5.
HOURS FIG. 7. Radioactive compounds found in the culture medium when cells were grown at a high concentration of nicotinamide. The radioactivity is that found in a 5-ml sample of the culture medium at the times specified in the figure. (- - -) Nicotinamide; (-. . .-) nicotinic acid.
in Fig. 7. This observation is compatible with the high levels of nicotinamide deamidase activity known to be present in yeast (4). The experimental evidence presented above leads to the following picture of
nicotinamide metabolism in S. fro&s C&51. This strain of yeast can deamidate nicotinamide and cause nicotinic acid to appear in the medium. The enzyme does not appear to be an extracellular one since the cell-free medium lacks deamidase activity. The ap-
SACCHAROMYCES
25
50
FRAGILIS
C&
103
75 FRACTION
NUMBER
FIG. 8. The chromatograpbic isolation of DPN-i4C and TPN-i4C. The cell extract was applied to a 1.5 X 25-cm DEAF-cellulose (bicarbonate) column. The column was developed with a linear gradient of ammonium bicarbonate (O-O.4 M; 1500 ml). Five-ml fractions were collected. (-) Absorbance at 260 rnp; (- - -) radioactivity; peak I, nicotinamide; peak II, DPN; peak IV, TPN; peaks III and V, unidentified. pearance of the acid in the medium can be explained by either leakage from the cells or by a deamidase associated with the cell Both nicotinamide and nicotinic membrane.
acid appear inside the cells when C& is grown on either form of the growth factor. DPN is the first nucleotide form of the growth factor which is found in the cells; TPN appears to be derived from DPN. None of the possible intermediates in the synthesis of DPN was observed. The reduced nucleotides were not found either. The absence of these compounds may have been due to metabolism, both synthetic and oxidative, during the 7-minute period required to harvest, wash, and kill the cells. A more rapid harvesting procedure will have to be used if the biosynthetic intermediates and reduced nucleotides are to be detected. Cells grown under conditions in which a factor(s) present in the vitamin-free yeast base was limiting, leaked nucleotides into the medium. The vitamin-free yeast base was shown to be involved in the leak-
age phenomenon in the following way. A medium similar to the one used in Fig. 3A was supplemented with four. times the concentrations of the vitamin supplements (exclusive of nicotinamide). The leakage was the same as that shown in Fig. 3A. Tripling of the concentration of the vitamin-free yeast base reduced the leakage of the pyridine nucleotides. The distribution of radioactivity between the cells and the medium was very much like that shown in Fig. 3D. The observation of the leakage of nucleotides from yeast is not without precedent. Rose (5) found that yeast grown on a biotin-deficient medium leaked desamido DPN into the medium. Since the pyridine nucleotides were the unique end products of the met’abolism of nicotinamide in S. fragilis C& it seemed possible that this organism might be used for the biosynthesis of DPNJ4C and TPN14C from nicotinamide-14C. The system appeared to offer a means for the preparation of small quantities of the nucleotides,
104
CHAYKIN,
KING,
AND SCHINDLER
TABLE NUCLEOTIDE Tie
32 32 120
Stationary
I YIELDSO To Fbdioactivity
of harvest
TPN
phase
Intracellular Extracellular Intracellular Extracellular Intracellular Extracellular Total
Just before stationary phase Stationary phase
(1The amounts of radioactivity originally were recovered in DPN and TPN.
16 None 9 None 10 6 16
DPN
Total
26
42
None
None
26
35
None
None
28 14 42
38 20 58
placed in the culture medium as nicotinamide-7-14C which
at high specific activity and in good yield. Cells of S. frugilis C 361were therefore grown on a large scale. The nucleotides were extracted in much the same fashion as that used in the kinetic studies discussed above. (The detailed methodology is presented in Experimental Procedure.) The separation of the components of the cell extract is exemplified by the data presented in Fig. 8. The nucleotides recovered from such a column were homogeneous by radiochemical criteria. They were, however, contaminated with nonradioactive materials from yeast. Chemically pure DPN and TPN were obtained by freeing the fractions obtained from the first column from NHJIC03 by lyophilization, reducing the nucleotides by specific enzymic means, rechromatography of the reduced nucleotides and if necessary oxidation, and a third chromatographic procedure in order to isolate the nucleotides, once again, in their oxidized forms. The method, including the use of 1 X lo-cm DEAEcellulose columns with batchwise elution of the nucleotides, has been presented in an earlier publication (3). The method depends on the fact that the chromatographic mobilities of the nucleotides and not those of the impurities are altered by the specific enzymic reduction procedure. Thus during the second chromatographic separation the impurities were eluted before the reduced nucleotides were recovered. A summary of the yields of DPNJ4C and TPNJ4C obtained in several different preparations is shown in Table I. The proportions of the
two nucleotides and their location (in the cells or the medium) could be and were altered by varying the concentration of nicotinamide in the medium and the time at which the cells were harvested. The biosynthetic preparation of DPN14C and TPNJ4C described above complements an existing method for the preparation of larger quantities of DPNJ4C (6) that is based upon the exchange of nicotinamide-14C into the pyridine nucleotides through the use of spleen or bran DPNase (7,8). The DPNase method is best suited for the preparation of milligram quantities of the nucleotides, but the S. fragilis method can be most conveniently used for the biosynthesis of the nucleotides in microgram amounts. A very great advantage of the S. fragilis method is the incorporation of the labeled precursor into the nucleotides without a decrease in specific activity. The DPNase method can result in from two- to eightfold losses in specific activity depending on the yield of products desired. Yields obtained with the S. fragilis method are on the order of 1.5-2 times those obtained with the DPNase method. ACKNOWLEDGMENT The authors are grateful to Dr. Herman Phaff for helpful advice and for furnishing the strain of S. fragilis, C&l, used in this work. REFERENCES 1. BONAVITA, V., NARROD, S. A., AND KAPLAN, N. O., J. Biol. Chem. 236,936 (1961).
SACCHAROMYCES 2. BRAY, G. A., Anal.
Biochem. 1, 279 (1960). 3. CHAYEIN, S., DAOANI, M., JOHNSON, L., SAMLI, M., AND BATTAILE, J., Biochim. Biophys. Acta 100, 351 (1965). 4. OKA, Y., J. B&hem. (Japan) 41, 89 (1954). 23, 143 (1960). 5. ROSE, A. H., J. Gen. Microbial. 6. SILVERSTEIN, E., AND BOYER, P. D., in “Bio-
FRAGILIS
105
C&I
chemical Preparations” (A. Maehly, ed.), Vol. 11, p. 89. Wiley, New York (1964). 7. ZATMAN, L. J., KAPLAN, N. O., AND COLOWICK, S. P., J. Biol. Chem. 200, 197 (1960). 8. COLOWICK,
S. P.,
AND KAPLAN,
N.
O.,
in
“Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. IV, p. 848. Wiley, New York (1957).