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The occurrence of nicotinic acid mononucleotide in yeast
ARCHIVES
OF
BIOCHEMLSTRY
AND
82, 83-88 (1959)
BIOPHYSICS
The Occurrence of Nicotinic Acid Mononucleotide in Yeast’ Robert W.
Wheat
From the Department of Biochemistry, School of Medicine, ~~~vers~~y~ D,urh~m, North ~~ro~~n~
Duke
Received October 13, 1958
The importance of nicotinic acid mononucleotide as an intermediate in the synthesis of diphosphopyridine nucleotide (DPW) has been recently demonstrated by Preiss and Handler (l-3). This demonstration was contingent upon incubation of erythrocytes with nicotinic acid-C4. The present report indicates that nicotinic acid Inononucleotide can be isolated from a yeast preparation without prior exposure of the yeast to nicotinic acid. METHODS AND MATERIALS
Yeast Preparation Dry 20-40 yeast was purchased from Standard Brands Inc., New York City. In a personal coxnmunication, Mr. Robert F. Light of Standard Brands Inc., described Fleischmann’s Dried Yeast Type 20-40 as a strain of Saccharom~ces cerevisiae which is grown aerobically on the usual molasses wort with no added nicotinic acid, harvested, and air-dried at a temperature ranging from 38 t,o 93”C., to a moisture level of approximately 7yo,.
Chromatogmphg Ion-exchange chromatography using Dowex I-formate (X-2,2~-~) was carried out essentially by the procedures of Hurlbert et al. (4). Solvent systems for paper chromatography were 95yc ethanol-l M ammonium acetate (7.5:3) (5) and n-butanol saturated with 2.5 M ammonia (6).
Charcoal Adsorptions and elutions were carried out by batch techniques on Biichner funnels, following the elution procedure of Pontis et al. (7), using 20 vol./g. charcoal each of wash fluids and eluant. Darco KB, purchased from the Atlas Powder Co., New York City was washed with acid before use.
Spectra Spectra were taken with a Beckman DK recording spectrophotometer. Absorption peaks were also checked in a model DU Beckman speetrophotometer. Spectro_I_ -. 1 This work was supported by a Duke University Research Grant and by Research Grant E-1659 from the National Institute of Allergy and Infectious Diseases. 83
84
WHEAT
photometric titration of chromophoric pK groups by difference spectra was carried out according to the method of Hakala and Schwert (8).
Phosphate was determined by the method of Gomori (9). Pentose was estimated by comparison with 5’-adenylic acid as a standard, using a modified Mejbaum (10) orcinol reagent containing cont. HCl and a heating period of 45 min. Ammonia was determined by nesslerization (11) after distillation. The short procedure of Heppel and Hilmoe (12) was followed for preparation of 5’-nucleotidase. Pyridinium eompounds were detected in solution by the KCPj method of Colowick et al. (13), and on chromatograms by ultraviolet fluorescence after exposure to methyl ethyl ketone and ammonia according to the method of Kodicek and Reddi (6).
Five hundred grams Fleischmann’s dry 20-40 yeast was mixed with 2000 ml. distilled water in a daring blendor and stirred into an equal volume of hot 95% ethanol. The mixture (maintained at 65-75°C. during the addition) was cooled, and after 2 days at 0-5°C. was filtered with the aid of Celite. The filtrate of pH 6-7 was passed through a 5-g. cake of Darco KB on a Biichner funnel to effect partial removal of flavines (7). Alcohol was then removed by evaporation under reduced pressure to one half the original filtrate volume. ~ucleotides were adsorbed onto Dareo KB at pH 3 or below by successive filtration through two 25-g. cakes of the charcoal on 15”cm. Biichner funnels. The filter cakes were washed with 500 ml. (20 vol./g. charcoal) each of water and 0.01 M ethylenediaminetetraacetic acid at pH 7 (7). Adsorbed materials were then eluted from the charcoal with 500 ml. of 50% ethanol. The combined alcohol eluates were diluted with water, neutralized to pH 7, and passed through a 3 X 14 cm. Dowex 1-formate column of about 100 ml. bed volume. The column was washed with 11. water and then eluted batchwise with 1 1. each (IO-column bed volumes) of 0.25, 1.0, 2.0, 2.5, and 3.0 II1 formic acid (3). Later work indicated B-column bed volumes of each eluant were satisfactory. The eluates were concentrated by the charcoal procedure as above, and aliquots of each fraction were assayed for pyridinium compounds by measuring absorption at 315 and 325 rnp after treatment with 1 M KCN (3, 13, 14). Only the 0.25 and 1.0 M formic acid fractions contained KCN-reactive material, and the 0.25 M formic acid fraction, which was assumed to contain DPN (4, 3), was not examined further. The 1.0 nf formic acid fraction contained material which exhibited after several minutes a maximal absorption in 1.0 M KCN near 315 rnp. This material was rechromatographed twice on 1 X 20 cm. columns of Dowex I-formate, using linear gradients with 500 ml. eluant, and collecting 5-ml. fractions. The first gradient was varied between 0.25 and 1.0 M formic acid. The material in tubes 37-44 (Fig. 1A) containing most of the KCN-reactive n~aterial was again freed of
NICOTINIC
ACID
TUBE
FIG. 1. Uowex
l-form&e
85
MONONUCLEOTIDE
NUMBER
chr~~m~tograph~
of the nucleotide.
3 See t,ext for
de-
tails.
formic acid by t#hecharcoal procedure as above, using 10 g. charcoal. About 80% of this material was rechromatogrsphed on Dowex 1-formate, using 2%linear gradient of water vs. 0.2 ill ammonium formate at pH 4.65. The results are shown in Fig. 1B. The material in tubes 36-39 was pooled, concent,rated as above on 2.5 g. charcoal on a 7.5~cm. Biichner funnel, eluted, neutralized with ammonium hydroxide, reduced in volume to 5 ml. under reduced pressure, and filtered. Absorption spectra of the nucleotide are shown in Fig. 2. In water and in 0.1 &f acid and alkali the compound exhibited a major absorption peak at 265.5-266 rnp which, in 1.0 ,!V KCN, shifted to 315 mE;1,indicating it to be a pyridinium derivat,ive of nicotinic acid (13,3, 14). Maximal absorption in KCN occurred several minutes after mixing. Further analyses a,re shown in Table I. Phosphate was determined before and after treatment of aliquots with 5’-nucleotidase or after wet ashing with sulfuric acid and hydrogen peroxide. No free inorganic phosphate was found prior to treatment. Total nitrogen was determined as ammonia after wet ashing an aliquot t,reated with Dowex 50 (H form) for removal of cations. Ratios of unity were found between total phosphate, 5’-phosphate, pentose, and nitrogen. In addition to the positional Dowex 1 chromat’ography (3, 4) and t’he 315-my absorption peak in 1 &Z KCN, the following further evidence was obtained which indicates that the isolated compound is ni~oti~i~ acid mono-
86
WHEAT 0.80071
FIG. 2. Absorption spectrum of nuoleotide in 0.1 N HCl (a), 0.1 N NaOH (A), and 20 min. after addition to 1 M KCN (X). TABLE I Phosphate and Pentose and Nitrogen Analysis of Nicotinic Acid MononucEeotidoa P total @noles~ml.
13.7 f
0.8”
S-P wlmles/mE.
13.4 f
0.46
Pentose
Nitrogen
pmoles/mZ.
I”toles/nd.
13.5 f
1.P
12.1
0 See text for details. b SymboIs * indicate average value of two or more determinations.
nucleotide. Paper chromatograms of the nucleotide exhibited a single ultraviolet-absorbing spot which gave a negative test for /?-amide pyridinium compounds upon exposure to methyl ethyl ketone and ammonia (6). An acid group (other than phosphate) with a pK near pH 2.4 was indicated by a difference spectrum titration in the region of 250-300 rnp. This is in in the range of 2.1 for the carboxyl of nicotinic acid and trigonelline (Nmethylnicotinic acid), as found by Hakala and Schwert (8). Hydrolysis in 0.1 N HCl for 45 min. at 100” [known to cleave the pyridinium riboside bond without further conversion of ni~otinamide to nicotinic acid and ammonia (15)] resulted in loss of 8U-90% of the 315-m& KCN reactivity with the concomitant appearance of a new ultraviolet-absorbing spot after paper chromatography. The new spot exhibited Rf values of 0.67 and 0.28 which were similar to Rf values of nicotinic acid (0.66 and 0.28) as compared to
87
NICOTINIC ACID MONONUCLEOTIDE
nicotinamide (0.73 and 0.70) in neutral ethanol-ammonium acetate and in ~-butanol-water-ammonia, respectively. COMMENT
The data obtained indicate the compound to be nicotinie acid mononucleotide, with an apparent molar absorbancy index in 1 M KCN of about 4.8 X 103. On this basis, 500 g. of the 20-40 yeast preparation contained about 100 pmoles of the nucleotide. The natural occurrence of nieotinic acid ribotide in biological material raises the question as to whether it is a product of synthesis or degradation. Systems which deamidate nicotinamide ribotide or DPN ha,ve not been reported, although the deamidation of free nicotinamide is known (16). However, the nicotinic acid analog of DPN (deamido DPN) has been prepared enzymically
by exchange
of nicoti~lic
acid with DPN
in the presence
of beef liver DPNase (17)) and chemically by degradation of acetylpyridine DPN
(14). It is possible,
in view of the work of Preiss and Handler
(3)) that
the present observation and the recent report of deamido DPN isolation from PeniciZZium chrysogenum (17) indicate the accumulation of normal int,ermediates
in the synthesis
of DPN.
ACKNOWLEDGMENTS The author would like to express appreciation to Dr. Handler and Dr. Preiss for making certain information available prior to publication and for their helpful suggestions during the course of this work. It is a pleasure to acknowledge the technical assistance of Mrs. Elaine B. Krick. SUMMARY
A nucleotide was isolated from water-alcohol extracts of yeast by charcoal adsorption-elution and anion-exchange chromatography. The compound was identified as nicotinic acid mononucleotide. 1. PREISS, J., ARD HANDLER, P.,J. Am. Chem. Xoc. 79, 1514 (1957). 2. PREISS, J., AND HANDLER, P., J. Am. Chem. Sot. 79,4246 (1957). 3. PREISS, J., AND HANDLER, P.,J.Biol. Chem.233,488 (1958). 4. HURLBERT, R.B., SCHMITZ,H.,RRUMM, A.F., ANDPOTTER, V.R.,J.BioZ. 209,23
Chem.
(1954).
5. PALADINI, A. C., AND LELOIR, L. F., ~~0chem.J. 61,426 (1952). 6. KODICEK,E., AND REDDI,K. K.,.Nature 168,475 (1951). 7. PONTIS, H. G., CABIB, E., AND LELOIR, L. F., Biochim. et Biophys. Acta 26, 146 (1957). 8. HAKALA, M. T., AND SCHWERT,G. W., Biochim. et Riophys. Acta 16, 489 (1955). 9. GOMORI, G., .I. Lab. Clin. Med. 27, 955 (1941). 10. MEJBAIJM, W., 2. physiol. Chew 268, 117 (1939).
88 11. 12. 13. 14.
WHEAT
VANSELOW, A. P., Ind. Eng. Chem., Anal. Ed. 12, 516 (1940). HEPPEL, L. A., AND HILMOE, R. J., J. Biol. Chem. 188, 665 (1951). COLOWICK, S. P., KAPLAN, N. O., AND CIOTTI, M., J. Biol. Chem. 191,447 (1951). LAMBORG, M., STOLZENBACK,F. E., AND KAPLAN, N.O., J.BioZ. Chenz. 231, 685
(1958). 15. SCHLENK, F., Arch. Biochem. 3, 93 (1943). 16. ELLINGER, P., AND KADER, A., Biochem. J. 44, 77 (1949) ; OKA, Y., J. Biochem. (Tokyo) 4l,89 (1954); HUGHES, II. E., AND WILLIAMSON, D. H., Rio&hem. J. 66, 851 (1955). 17. S~RI,~PI-~RESCENZI,~., AND BALLIO, A.,~Vutu~el~,l2~ (1957).
OF
BIOCHEMLSTRY
AND
82, 83-88 (1959)
BIOPHYSICS
The Occurrence of Nicotinic Acid Mononucleotide in Yeast’ Robert W.
Wheat
From the Department of Biochemistry, School of Medicine, ~~~vers~~y~ D,urh~m, North ~~ro~~n~
Duke
Received October 13, 1958
The importance of nicotinic acid mononucleotide as an intermediate in the synthesis of diphosphopyridine nucleotide (DPW) has been recently demonstrated by Preiss and Handler (l-3). This demonstration was contingent upon incubation of erythrocytes with nicotinic acid-C4. The present report indicates that nicotinic acid Inononucleotide can be isolated from a yeast preparation without prior exposure of the yeast to nicotinic acid. METHODS AND MATERIALS
Yeast Preparation Dry 20-40 yeast was purchased from Standard Brands Inc., New York City. In a personal coxnmunication, Mr. Robert F. Light of Standard Brands Inc., described Fleischmann’s Dried Yeast Type 20-40 as a strain of Saccharom~ces cerevisiae which is grown aerobically on the usual molasses wort with no added nicotinic acid, harvested, and air-dried at a temperature ranging from 38 t,o 93”C., to a moisture level of approximately 7yo,.
Chromatogmphg Ion-exchange chromatography using Dowex I-formate (X-2,2~-~) was carried out essentially by the procedures of Hurlbert et al. (4). Solvent systems for paper chromatography were 95yc ethanol-l M ammonium acetate (7.5:3) (5) and n-butanol saturated with 2.5 M ammonia (6).
Charcoal Adsorptions and elutions were carried out by batch techniques on Biichner funnels, following the elution procedure of Pontis et al. (7), using 20 vol./g. charcoal each of wash fluids and eluant. Darco KB, purchased from the Atlas Powder Co., New York City was washed with acid before use.
Spectra Spectra were taken with a Beckman DK recording spectrophotometer. Absorption peaks were also checked in a model DU Beckman speetrophotometer. Spectro_I_ -. 1 This work was supported by a Duke University Research Grant and by Research Grant E-1659 from the National Institute of Allergy and Infectious Diseases. 83
84
WHEAT
photometric titration of chromophoric pK groups by difference spectra was carried out according to the method of Hakala and Schwert (8).
Phosphate was determined by the method of Gomori (9). Pentose was estimated by comparison with 5’-adenylic acid as a standard, using a modified Mejbaum (10) orcinol reagent containing cont. HCl and a heating period of 45 min. Ammonia was determined by nesslerization (11) after distillation. The short procedure of Heppel and Hilmoe (12) was followed for preparation of 5’-nucleotidase. Pyridinium eompounds were detected in solution by the KCPj method of Colowick et al. (13), and on chromatograms by ultraviolet fluorescence after exposure to methyl ethyl ketone and ammonia according to the method of Kodicek and Reddi (6).
Five hundred grams Fleischmann’s dry 20-40 yeast was mixed with 2000 ml. distilled water in a daring blendor and stirred into an equal volume of hot 95% ethanol. The mixture (maintained at 65-75°C. during the addition) was cooled, and after 2 days at 0-5°C. was filtered with the aid of Celite. The filtrate of pH 6-7 was passed through a 5-g. cake of Darco KB on a Biichner funnel to effect partial removal of flavines (7). Alcohol was then removed by evaporation under reduced pressure to one half the original filtrate volume. ~ucleotides were adsorbed onto Dareo KB at pH 3 or below by successive filtration through two 25-g. cakes of the charcoal on 15”cm. Biichner funnels. The filter cakes were washed with 500 ml. (20 vol./g. charcoal) each of water and 0.01 M ethylenediaminetetraacetic acid at pH 7 (7). Adsorbed materials were then eluted from the charcoal with 500 ml. of 50% ethanol. The combined alcohol eluates were diluted with water, neutralized to pH 7, and passed through a 3 X 14 cm. Dowex 1-formate column of about 100 ml. bed volume. The column was washed with 11. water and then eluted batchwise with 1 1. each (IO-column bed volumes) of 0.25, 1.0, 2.0, 2.5, and 3.0 II1 formic acid (3). Later work indicated B-column bed volumes of each eluant were satisfactory. The eluates were concentrated by the charcoal procedure as above, and aliquots of each fraction were assayed for pyridinium compounds by measuring absorption at 315 and 325 rnp after treatment with 1 M KCN (3, 13, 14). Only the 0.25 and 1.0 M formic acid fractions contained KCN-reactive material, and the 0.25 M formic acid fraction, which was assumed to contain DPN (4, 3), was not examined further. The 1.0 nf formic acid fraction contained material which exhibited after several minutes a maximal absorption in 1.0 M KCN near 315 rnp. This material was rechromatographed twice on 1 X 20 cm. columns of Dowex I-formate, using linear gradients with 500 ml. eluant, and collecting 5-ml. fractions. The first gradient was varied between 0.25 and 1.0 M formic acid. The material in tubes 37-44 (Fig. 1A) containing most of the KCN-reactive n~aterial was again freed of
NICOTINIC
ACID
TUBE
FIG. 1. Uowex
l-form&e
85
MONONUCLEOTIDE
NUMBER
chr~~m~tograph~
of the nucleotide.
3 See t,ext for
de-
tails.
formic acid by t#hecharcoal procedure as above, using 10 g. charcoal. About 80% of this material was rechromatogrsphed on Dowex 1-formate, using 2%linear gradient of water vs. 0.2 ill ammonium formate at pH 4.65. The results are shown in Fig. 1B. The material in tubes 36-39 was pooled, concent,rated as above on 2.5 g. charcoal on a 7.5~cm. Biichner funnel, eluted, neutralized with ammonium hydroxide, reduced in volume to 5 ml. under reduced pressure, and filtered. Absorption spectra of the nucleotide are shown in Fig. 2. In water and in 0.1 &f acid and alkali the compound exhibited a major absorption peak at 265.5-266 rnp which, in 1.0 ,!V KCN, shifted to 315 mE;1,indicating it to be a pyridinium derivat,ive of nicotinic acid (13,3, 14). Maximal absorption in KCN occurred several minutes after mixing. Further analyses a,re shown in Table I. Phosphate was determined before and after treatment of aliquots with 5’-nucleotidase or after wet ashing with sulfuric acid and hydrogen peroxide. No free inorganic phosphate was found prior to treatment. Total nitrogen was determined as ammonia after wet ashing an aliquot t,reated with Dowex 50 (H form) for removal of cations. Ratios of unity were found between total phosphate, 5’-phosphate, pentose, and nitrogen. In addition to the positional Dowex 1 chromat’ography (3, 4) and t’he 315-my absorption peak in 1 &Z KCN, the following further evidence was obtained which indicates that the isolated compound is ni~oti~i~ acid mono-
86
WHEAT 0.80071
FIG. 2. Absorption spectrum of nuoleotide in 0.1 N HCl (a), 0.1 N NaOH (A), and 20 min. after addition to 1 M KCN (X). TABLE I Phosphate and Pentose and Nitrogen Analysis of Nicotinic Acid MononucEeotidoa P total @noles~ml.
13.7 f
0.8”
S-P wlmles/mE.
13.4 f
0.46
Pentose
Nitrogen
pmoles/mZ.
I”toles/nd.
13.5 f
1.P
12.1
0 See text for details. b SymboIs * indicate average value of two or more determinations.
nucleotide. Paper chromatograms of the nucleotide exhibited a single ultraviolet-absorbing spot which gave a negative test for /?-amide pyridinium compounds upon exposure to methyl ethyl ketone and ammonia (6). An acid group (other than phosphate) with a pK near pH 2.4 was indicated by a difference spectrum titration in the region of 250-300 rnp. This is in in the range of 2.1 for the carboxyl of nicotinic acid and trigonelline (Nmethylnicotinic acid), as found by Hakala and Schwert (8). Hydrolysis in 0.1 N HCl for 45 min. at 100” [known to cleave the pyridinium riboside bond without further conversion of ni~otinamide to nicotinic acid and ammonia (15)] resulted in loss of 8U-90% of the 315-m& KCN reactivity with the concomitant appearance of a new ultraviolet-absorbing spot after paper chromatography. The new spot exhibited Rf values of 0.67 and 0.28 which were similar to Rf values of nicotinic acid (0.66 and 0.28) as compared to
87
NICOTINIC ACID MONONUCLEOTIDE
nicotinamide (0.73 and 0.70) in neutral ethanol-ammonium acetate and in ~-butanol-water-ammonia, respectively. COMMENT
The data obtained indicate the compound to be nicotinie acid mononucleotide, with an apparent molar absorbancy index in 1 M KCN of about 4.8 X 103. On this basis, 500 g. of the 20-40 yeast preparation contained about 100 pmoles of the nucleotide. The natural occurrence of nieotinic acid ribotide in biological material raises the question as to whether it is a product of synthesis or degradation. Systems which deamidate nicotinamide ribotide or DPN ha,ve not been reported, although the deamidation of free nicotinamide is known (16). However, the nicotinic acid analog of DPN (deamido DPN) has been prepared enzymically
by exchange
of nicoti~lic
acid with DPN
in the presence
of beef liver DPNase (17)) and chemically by degradation of acetylpyridine DPN
(14). It is possible,
in view of the work of Preiss and Handler
(3)) that
the present observation and the recent report of deamido DPN isolation from PeniciZZium chrysogenum (17) indicate the accumulation of normal int,ermediates
in the synthesis
of DPN.
ACKNOWLEDGMENTS The author would like to express appreciation to Dr. Handler and Dr. Preiss for making certain information available prior to publication and for their helpful suggestions during the course of this work. It is a pleasure to acknowledge the technical assistance of Mrs. Elaine B. Krick. SUMMARY
A nucleotide was isolated from water-alcohol extracts of yeast by charcoal adsorption-elution and anion-exchange chromatography. The compound was identified as nicotinic acid mononucleotide. 1. PREISS, J., ARD HANDLER, P.,J. Am. Chem. Xoc. 79, 1514 (1957). 2. PREISS, J., AND HANDLER, P., J. Am. Chem. Sot. 79,4246 (1957). 3. PREISS, J., AND HANDLER, P.,J.Biol. Chem.233,488 (1958). 4. HURLBERT, R.B., SCHMITZ,H.,RRUMM, A.F., ANDPOTTER, V.R.,J.BioZ. 209,23
Chem.
(1954).
5. PALADINI, A. C., AND LELOIR, L. F., ~~0chem.J. 61,426 (1952). 6. KODICEK,E., AND REDDI,K. K.,.Nature 168,475 (1951). 7. PONTIS, H. G., CABIB, E., AND LELOIR, L. F., Biochim. et Biophys. Acta 26, 146 (1957). 8. HAKALA, M. T., AND SCHWERT,G. W., Biochim. et Riophys. Acta 16, 489 (1955). 9. GOMORI, G., .I. Lab. Clin. Med. 27, 955 (1941). 10. MEJBAIJM, W., 2. physiol. Chew 268, 117 (1939).
88 11. 12. 13. 14.
WHEAT
VANSELOW, A. P., Ind. Eng. Chem., Anal. Ed. 12, 516 (1940). HEPPEL, L. A., AND HILMOE, R. J., J. Biol. Chem. 188, 665 (1951). COLOWICK, S. P., KAPLAN, N. O., AND CIOTTI, M., J. Biol. Chem. 191,447 (1951). LAMBORG, M., STOLZENBACK,F. E., AND KAPLAN, N.O., J.BioZ. Chenz. 231, 685
(1958). 15. SCHLENK, F., Arch. Biochem. 3, 93 (1943). 16. ELLINGER, P., AND KADER, A., Biochem. J. 44, 77 (1949) ; OKA, Y., J. Biochem. (Tokyo) 4l,89 (1954); HUGHES, II. E., AND WILLIAMSON, D. H., Rio&hem. J. 66, 851 (1955). 17. S~RI,~PI-~RESCENZI,~., AND BALLIO, A.,~Vutu~el~,l2~ (1957).