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The non-enzymic formation of ribose oxime upon cleavage of adenine nucleotides and purine nucleosides by spores of Bacillus cereus var. terminalis in the presence of NH2OH
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OF
BIOCHEMISTRY
AND
BIOPHYSICS
86, 131-140 (19%)
The Non-enzymic Formation of Ribose Oxime upon Cleavage of Adenine Nucleotides and Pwrine Nucleosides by Spores of Bacillus cereus var. terminalis in the Presence of NH,OH Bernard J. Krask and George E. Fulk From Fort Detrick, Frederick, Maryland Received
March
30, 1959
INTRODUCTION
Recent studies (1) have shown that extracts from spores of BaciZZus contain a nucleoside phosphorylase which mediates in the reaction between inosine or adenosine and orthophosphate to produce ribose l-phosphate. Because adenosine or inosine alone or together with amino acids or glucose stimulates the rapid germination of spores of certain Bacillus species (a-5), the above results suggested the probable means by which these nucleosides contributed to germination. Unexplained observations on adenosine utilization by spores in the presence of NH20H, however, suggested that other pathways for nucleoside utilization were present and prompted an investigation of the nature of the reactions. The formation of an unidentified product similar to a hydroxamic acid on incubation of homogenates of B. cereus var. terminalis spores with NHzOH and adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine j-phosphate (AMP), or adenosine has been reported (6). The product formed a colored complex with iron in the method of Lipmann and Tuttle (7) for the determination of hydroxamic acids and was considered to be identical in the case of the reactions with either the nucleotides or adenosine. Product formation was optimal with adenosine and decreased as the number of phosphate groups on the nucleotides increased. Because B. cereus var. terminalis spores catalyze the synthesis of glutamohydroxamic acid from glutamine and NHgOH through glutamotransferase mediation (B), it was suggested that the unidentified product was formed by a similar amide transferase requiring an unknown subst’rate and using adenosine as a cofactor. The present report demonstrates that adenosine provides the substrate for the reaction with NH,OH and that the product, identified as ribose oxime, is the result of the following reactions: cereus var. terminalis
131
132
KRASK AND FULK (a) adenosine (b) ribose
---f adenine
+ NHtOH
+ ribose
+ ribose oxime
+ Hz0
Reaction (a) is catalyzed by a purine ribosidase recently reported in spores of this organism (5, 8, 9), while reaction (b) occurs non-enzymically (10). The reactions of the adenine nucleotides with NHSOH are shown to be dependent on the enzymic release of orthophosphate (Pi) and ribose prior to the reaction with NHzOH to form the pentose oxime. METHODS Partially clean suspensions of B. cereus var. terminalis spores were obtained from Dr. H. 0. Halvorson of the University of Illinois. The spores were freed of residual vegetative cells and autolyeed material by methods previously described (1). The clean preparations were lyophilized and stored in vacua over CaClp at 4°C. Spore homogenates were prepared by Mickle (11) disintegration in water at 4°C. until stained films showed the disrupted preparations were free of intact spores. One or two drops of 2-octanol were added to the Mickle cups to minimize foaming during disruption. The particulate debris fraction obtained by centrifugation at 25,000 X g for 45 min. at 4°C. was resuspended in water and was used in all experiments, its concentration being expressed in milligrams equivalent to intact lyophilized spores. Reaction mixtures were incubated at 47°C. in contrast to our previous studies at 37°C. (6). Ribose oxime was determined as a colored iron complex by a modification of the methods for estimating hydroxamic acids (7, 12) employing an increased concentration of iron. Since the color intensity of the iron complex was not as stable as that formed with the hydroxamic acids, a standard procedure was followed for all determinations. Reactions were stopped by transfer of 2.0-ml. aliquots to an acetoneDry Ice bath. On completion of an experiment, a series of six to eight aliquots was placed in an ice bath to thaw, and 0.5 ml. of 15ye trichloroacetic acid and 0.5 ml. of 2.5 N HCI was added to precipitate the protein. The aliquots were removed from the ice bath, and 0.5 ml. of 14.2% Fe& in 0.01 N HCl was added to develop the characteristic orange-brown or amber color of the iron complex. The solutions were clarified by centrifugation, and the color intensities of the supernatants were measured in a Klett-Summerson photoelectric calorimeter with a No. 54 filter. In each case, color determinations were made 20 min. from the time of addition of FeC13 . Maximum color intensity was obtained under these conditions. The concentration of the oxime is expressed as the color intensity of the iron complex and is given in Klett units/a.0 ml. of reaction mixture. The values presented in the results are the difference between a complete system and a control incubated with NHtOH, tris(hydroxymethyl)aminomethane (Tris) buffer, and the debris fraction in the absence of substrate. Substrate controls contributed no color in the absence of NHSOH but were included as chromatography controls. Reducing-sugar determinations were made according to the method of Nelson (13) on aliquots from reaction mixtures incubated without NHtOH. Pi was estimated in the presence and absence of NHzOH by the method of King (14). Chromatography methods are described in the Results section. Synthetic ribose oxime was prepared essentially by the method of Wohl (10).
FORMATION
OF
EXPERIMENTAL
Purine Ribosidase Mediation
RIBOSE
133
OXIME
RESULTS
in the Adenosine-NHzOH
Reaction
Further study on the characteristics of the reaction between adenosine and NHzOH indicated a marked similarity to purine ribosidase activity of B. cereus spores (5,8,9). Formation of the iron complex occurred on incubation of adenosine and NHzOH with either intact resting or germinated spores; this activity was limited to the particulate debris fraction resulting from centrifugation of disrupted spore homogenates. Boiling of the aqueous suspensions of whole homogenates or the debris fraction for 1 hr. resulted in only a 25 % loss of activity. The reaction was independent of added metals, orthophosphate, and arsenate over a pH range of 5.5-8.0, and product formation was found to be dependent on stoichiometric rather than catalytic concentrations of adenosine. These similarities suggested that purine ribosidase was concerned with the adenosine-NHzOH reaction. Evidence for purine ribosidase mediation was obtained when purine and pyrimidine nucleosides were substituted for adenosine (Table I). The iron complex was formed only on incubation of the purine nucleosides with the debris fraction and NHZOH. The release of approximately theoretical concentrations of reducing sugar identified as ribose from adenosine, inosine, and guanosine in the absence of NHtOH paralleled the formation TABLE
The Dependence
I
of Iron-Complex Formation on the Release of Ribose from Purine Nucleosides
Reaction mixtures contained, per 2.0 ml.: substrate, 5 pmoles; spore debris fraction, 10 mg.; (=t) NHzOH, 200rmoles, Tris buffer, pH 7.5,200 pmoles. Time of incubation 2 hr. at 47°C. Products Substrate
With NHzOH Iron complex
per 2.0 ml.
of reaction
Without NHzOH Ribose pmoles
Adenosine Inosine Guanosine Xanthosine Cytidine Uridine Ribose Adenine Hypoxanthine Cytosine
28 30 23 4 0 0 30 0 0 0
5.44 5.56 4.94 0.60 0.19 0.15 5.44 0 0.15 0
134
KRASK
AND
FULK
of the iron complex and indicated a relationship between the extent of cleavage of the nucleosides and the color intensity of the complex. The relatively slower release of ribose from xanthosine was accompanied by a corresponding decrease in the color intensity of the complex. The relationship between iron complex formation and the release of ribose became apparent when the products of the cleavage of the nucleosides were tested individually (Table I). The iron complex was formed only from ribose and NHZOH, and its color intensity was equal to that obtained on incubation with equivalent concentrations of either adenosine or inosine. That ribose as such was available for reaction with NHSOH was suggested by the complete recovery of the sugar after incubation in the absence of NHtOH. Preincubation experiments further established the role of purine ribosidase and ribose in the reactions between the nucleosides and NH,OH (Fig. 1). The initial rate of iron complex formation after addition of NHzOH to debris fraction preincubated with adenosine was five times that attained on addition of adenosine and NHzOH to debris fraction preincubated without substrate. That the rate curves were a measure of the same reactions
MINUTES
FIG. 1. The effect of preincubation of debris fraction with and without adenosine on the rate of the adenosine-NHzOH reaction. Final reaction mixtures contained, per 2.0 ml.: adenosine, 10 pmoles; spore debris fraction, 12 mg.; NHsOH, 200 pmoles; Tris buffer, pH 7.5,200 pmoles. (0) Debris fraction preincubated 2 hr. at 47°C. with adenosine and buffer; NHzOH added and mixture reincubated at 47°C. (0) Debris fraction preincubated with buffer; adenosine and NHzOH added and mixture reincubated at 47°C.
FORMATION
OF
RIBOSE
OXIME
ADENOSINE
or RIBOSE
(Mlcromoles
per 2 0 ml)
135
FIG. 2. The effect of adenosine and ribose concentration on iron-complex formation. Reaction mixtures contained, per 2.0 ml; (0) adenosine or (0) ribose; spore debris fraction, 25 mg.; NHsOH, 200 pmoles; Tris buffer, pH 7.5, 200 pmoles. Time of incubation 2 hr. at 47°C.
was indicated by their essentially parallel paths after 20 min. of reaction with NHzOH and by the extent of iron complex formation in both reactions. The necessity for the release of ribose to form the iron complex was indicated by taking an aliquot from the reaction mixture preincubated with adenosine immediately before the addition of NHzOH; 10.29 pmoles ribose was recovered from preincubation with 10 pmoles adenosine. The rate curves were thus essentially a measure of the rat.e of reaction between ribose and NHzOH. The effect of the concentration of adenosine and ribose on iron complex formation is shown in Fig. 2. Equivalent concentrations of the substrates up to 20 pmoles gave rise to approximately equivalent concentrations of the iron complex. The Non-enxymic Formation of Ribose Oxime The above results suggested the formation of ribose oxime. Wohl (10) reported the chemical synthesis of sugar oximes in alcoholic solutions of
136
KRASK
AND
FULK
NHzOH under mild conditions of reaction and noted that arabinose oxime was formed at a more rapid rate than glucose oxime. When pentose and hexose sugars (10 pmoles) and ribitol (50 pmoles) were incubated with and without NHzOH (200 pmoles) in the absence of debris fraction for 2 hr. at 47”C., the iron complex was formed only in the presence of the reducing sugars and NH20H. The color intensities obtained in the nonenzymic reactions were; ribose, 36; xylose, 56; arabinose, 74; glucose, 10; and ribitol, 0. The reactions occurred without any significant change in the color intensities when acetate buffer at pH 5.5 or phosphate buffer at pH 7.5 were substituted for Tris buffer at pH 7.5. On the basis of these observations, synthetic ribose oxime was prepared (lo), and its ability to form a complex with iron was tested. Figure 3 compares the color intensity of the iron complex formed after incubation of ribose oxime with and without inactivated debris fraction to that formed after incubation of ribose and NHzOH with active and inactive prepara100
- 80 -z 0 r-4 ;a “, =c3 60
RIBOSE
OR RIBOSE
(Micromoles
FIG. 3. Iron-complex
OXIME
per 2 0 ml)
formation with ribose oxime and the product of the riboseNHzOH reaction. Reaction mixtures contained, per 2.0 ml.: ribose with active (0) or autoclaved (a) spore debris fraction, 15 mg.; NHtOH, 200 Hmoles; Tris buffer, pH 7.5, 200 pmoles. Ribose oxime with (A) or without (A) autoclaved spore debris fraction, 15 mg.; Tris buffer, pH 7.5, 200 pmoles. Time of incubation 2 hr. at 47°C.
FORMATION
OF
RIBOSE
OXIME
137
tions. The iron complex with the synthetic oxime was formed independently of the debris fraction, and its color intensity in the presence of inactivated debris was roughly equivalent to that obtained in the reactions between ribose and NHZOH. Although not included in the figure, the absence of change in the color intensity of the iron complex formed after incubation of ribose oxime with active debris fraction for 2 hr. demonstrated that ribose oxime was not altered by the spore preparation. The equivalence in the color intensities obtained after incubation of ribose and NHzOH with both active and inactive debris fractions established the nonenzymic nature of ribose oxime formation by spores. That ribose was not utilized to form some other component which might react similarily with NHzOH was demonstrated by the recovery of 5.20, 9.97, 15.39, and 19.50 pmoles ribose after incubation of active debris fraction with 5, 10, 15, and 20 pmoles ribose without NHzOH. The reason for the increased color intensity when ribose oxime was incubated with the inactivated debris fraction is not apparent at present. A similar effect was observed when ribose and NHzOH were incubated with either active or inactivated debris fractions. The Identijkation
of Ribose Oxime
Ribose oxime was identified by one-dimensional ascending chromatography on Whatman No. 1 paper. Reaction mixtures containing debris fraction and the purine nucleosides or ribose with and without NH,OH were incubated for 2 hr. at 47°C. Aliquots were centrifuged at 25,000 X g for 1 hr. at 4”C., and the supernatants were developed simultaneously in pyridine-butanol-water (6:4: 3) and in the upper phase of ethyl acetatepyridine-water (2: 1:2). The synthetic ribose oxime and the product of the ribose-NHtOH reaction in the absence of spores were used as standards. The oximes were located by spraying with a hydroxamic acid reagent (15), which resulted in their appearance as orange spots against a yellow background. Ribose was demonstrated with benzidine-acetic acid (16) and aniline-hydrogen phthalate (17) as spray reagents. Spots staining with a marked intensity with the hydroxamic acid reagent appeared only with aliquots taken from reaction mixtures which formed the iron complex, and their position corresponded to that of the oxime standards. Although the Rf values varied in the experiments, the positions of the unknown oxime and the standard were always identical. In freshly prepared solvents the Rf values of ribose oxime and ribose were 0.75 and 0.64, respectively, in pyridinebutanol-water and 0.73 and 0.63 in ethyl acetate-pyridinewater. Several peculiarities were observed with respect to the staining response of ribose oxime. When the oxime standards and reaction mixtures forming the iron complex were developed in the pyridine solvents and sprayed with
138
KRASK
AND
FULK
either benzidine-acetic acid or aniline-hydrogen phthalate to identify the reducing sugars, the position corresponding to ribose oxime was also stained by these reagents. Since ketoximes are converted to their respective ketones by acid hydrolysis (18), our observations suggested the ribose oxime was hydrolyzed in situ to the aldo sugar when the papers were sprayed with the acidic reagents and heated at 105°C. for 5 min. This apparent acid lability was also indicated by the absence of an oxime spot when secbutanol-formic acid-water (75: 15: 10) was used as a solvent and the hydroxamic acid reagent was used for color development. In the acidic solvent only a single benzidine-staining spot corresponding to ribose was found with either the oxime standards or aliquots from reaction mixtures forming the iron complex. A similar observation was made with acetone30 % acetic acid (1: 1). Application of higher concentrations of the oxime standard to the latter solvent, however, produced a single spot which was stained by both the hydroxamic acid and reducing sugar reagents. Its position was identical to that of ribose. The assumptions made to explain the staining response of ribose oxime to benzidine were supported by the results of the hydrolysis experiments used to identify the synthetic oxime. The oxime was hydrolyzed with HCl, and the extent of hydrolysis was determined by the decrease in iron complex formation and the appearance of ribose on chromatograms. Tubes containing 40 pmoles oxime in 2.0 ml. water or HCl in a final concentration of 0.01, 0.05, 0.10, 0.50, and 1.00 M were incubated at 37°C. for 1 hr. As an additional control an oxime-water solution was held at room temperature. The acid mixtures were neutralized with 1.0 ml. NaOH, and the controls were brought to 3.0 ml. with water. Iron-complex determinations were made on 2.0-ml. aliquots. The color intensities were 99 for both controls and 60, 9, 5, 6, and 11, respectively, for the acid mixtures. Ribose was released from all the mixtures with the exception of the oxime-water controls. The marked lability of ribose oxime to acid suggests that our interpretation of the benzidine-acetic acid reaction with ribose oxime is correct. Ribose Oxime Formation from Adenine Nucleotides Recently we have shown that the debris fraction contains heat-labile enzymes which release Pi from ATP, ADP, and AMP (1). Our present findings implied that ribose oxime was the product of the breakdown of the nucleotides in the presence of NHZOH. When iron-complex formation and the release of Pi on incubation of the debris fraction with the nucleotides and NH&H was compared with the release of ribose and Pi in the absence of NH&H, the relationship between iron-complex formation and ribose release was again apparent (Table II). That ribose was released from ADP
139
FORMATION OF RIBOSE OXIME TABLE Iron-Complex
II
Formation upon Cleavage of Adenine Nucleotides Adenosine in the Presence of NHzOH
and
Reaction mixtures contained, per 2.0 ml.: substrate, 10 pmoles; spore debris fraction, 10 mg.; (=I=) PJHZOH, 200 pmoles; Tris buffer, pH 7.5, 200 rmoles. Time of incubation 2 hr. at 47°C. Figures in brackets represent reactions with spore debris fraction heated 20 min. at 65°C. Products With
Substrate
ATP ADP AMP Adenosine
Iron
complex
Rlett
units
0 (0)
16
(0)
34 (0) 59 (59)
per 2.0 ml. of reaction
NHzOH
Without Ribose
Pi
pmoles
0.59 3.14 5.70
NHlOH
(0) (0) (0)
/.Lmoles
0.03 3.22 5.63 8.58
(0) (0) (0) (8.34)
Pi
Jmoles
1.20 (0) 3.60 (0) 5.87 (0)
and AMP in the presence of NHzOH was indicated by the equivalent concentrations of Pi recovered on incubation with or without NHzOH and by the inability of heated preparations to give rise to the iron complex. Confirmation of ribose oxime formation from ADP and AMP was made by chromatography. The failure to elicit the color complex or recover ribose on incubation with ATP suggested an insufficient rate of ADP formation at 47°C. NH20H also appeared to inhibit the ATPase activity. Incubation of ATP with the debris fraction and NHsOH at 37°C. for 24 hr. gave rise to the iron complex in solution, and ribose oxime was demonstrated on chromatograms. SUMMARY
The non-enzymic formation of ribose oxime on incubation of adenine nucleotides and purine nucleosides with NHzOH and spores of Bacillus cereus var. terminalis is described. Ribose oxime formation is contingent upon the enzymic release of ribose from the nucleotides and nucleosides. REFERENCES 1. KRASK, B. J., AND FULK, G. E., Arch. Bioch,em. Biophys. 79, 86 (1959). 2. HILLS, G. M., Biochem. J. 46, 363 (1949). 3. POWELL, J. F., J. Gen. llficrobiol. 6, 993 (1951). 4. CHURCH, B. D., HALVORSOS, H., AND HALVORSOX, H. O., J. Bacterial.
68, 393
(1954). 5. POWELL, J. F., ASD HUNTER, J. It., Biochem. J. 62, 381 (1956). 6. KRASK, B. J., in “Spores” (Halvorson, H. O., ed.) Publ. No. 5, p. 135. American Institute of Biological Sciences, Washington, D. C., 1957. 7. LIPJIANN, F., ASD TUTTLE, L. C., J. Biol. Chem. 169.21 (1945).
140 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
=SK
AND
FULK
LAWRENCE, N. L., J. Bacterial. 70,577 (1955). LAWRENCE, N. L., J. Bacterial. 70,583 (1955). WOHL, A., Ber. 26,730 (1893). MICKLE, H., J. Roy. Microsop. Sot. 68,lO (1948). GROSSOWICZ, N., WAINFAN, E., BOREK, E., AND WAELSCH, H., J. Biol. Chem. 187, 111 (1950). NELSON, N., J. Biol. Chem. 163, 375 (1944). KING, E. J., Biochem. J. 26,292 (1932). STADTMAN, E. R., AND BARKER, H. A., J. BioZ. Chem. 164,769 (1950). HORROCKS, R. H., Nature 164, 444 (1949). PARTRIDGE, S. M., Nature 164,443 (1949). JOHNSON, R. W., AND STIEGLITZ, J., J. Am. Chem. Xoc. 66,1904 (1934). PARTRIDGE, S. M., Biochem. J. 42,238 (1948).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
86, 131-140 (19%)
The Non-enzymic Formation of Ribose Oxime upon Cleavage of Adenine Nucleotides and Pwrine Nucleosides by Spores of Bacillus cereus var. terminalis in the Presence of NH,OH Bernard J. Krask and George E. Fulk From Fort Detrick, Frederick, Maryland Received
March
30, 1959
INTRODUCTION
Recent studies (1) have shown that extracts from spores of BaciZZus contain a nucleoside phosphorylase which mediates in the reaction between inosine or adenosine and orthophosphate to produce ribose l-phosphate. Because adenosine or inosine alone or together with amino acids or glucose stimulates the rapid germination of spores of certain Bacillus species (a-5), the above results suggested the probable means by which these nucleosides contributed to germination. Unexplained observations on adenosine utilization by spores in the presence of NH20H, however, suggested that other pathways for nucleoside utilization were present and prompted an investigation of the nature of the reactions. The formation of an unidentified product similar to a hydroxamic acid on incubation of homogenates of B. cereus var. terminalis spores with NHzOH and adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine j-phosphate (AMP), or adenosine has been reported (6). The product formed a colored complex with iron in the method of Lipmann and Tuttle (7) for the determination of hydroxamic acids and was considered to be identical in the case of the reactions with either the nucleotides or adenosine. Product formation was optimal with adenosine and decreased as the number of phosphate groups on the nucleotides increased. Because B. cereus var. terminalis spores catalyze the synthesis of glutamohydroxamic acid from glutamine and NHgOH through glutamotransferase mediation (B), it was suggested that the unidentified product was formed by a similar amide transferase requiring an unknown subst’rate and using adenosine as a cofactor. The present report demonstrates that adenosine provides the substrate for the reaction with NH,OH and that the product, identified as ribose oxime, is the result of the following reactions: cereus var. terminalis
131
132
KRASK AND FULK (a) adenosine (b) ribose
---f adenine
+ NHtOH
+ ribose
+ ribose oxime
+ Hz0
Reaction (a) is catalyzed by a purine ribosidase recently reported in spores of this organism (5, 8, 9), while reaction (b) occurs non-enzymically (10). The reactions of the adenine nucleotides with NHSOH are shown to be dependent on the enzymic release of orthophosphate (Pi) and ribose prior to the reaction with NHzOH to form the pentose oxime. METHODS Partially clean suspensions of B. cereus var. terminalis spores were obtained from Dr. H. 0. Halvorson of the University of Illinois. The spores were freed of residual vegetative cells and autolyeed material by methods previously described (1). The clean preparations were lyophilized and stored in vacua over CaClp at 4°C. Spore homogenates were prepared by Mickle (11) disintegration in water at 4°C. until stained films showed the disrupted preparations were free of intact spores. One or two drops of 2-octanol were added to the Mickle cups to minimize foaming during disruption. The particulate debris fraction obtained by centrifugation at 25,000 X g for 45 min. at 4°C. was resuspended in water and was used in all experiments, its concentration being expressed in milligrams equivalent to intact lyophilized spores. Reaction mixtures were incubated at 47°C. in contrast to our previous studies at 37°C. (6). Ribose oxime was determined as a colored iron complex by a modification of the methods for estimating hydroxamic acids (7, 12) employing an increased concentration of iron. Since the color intensity of the iron complex was not as stable as that formed with the hydroxamic acids, a standard procedure was followed for all determinations. Reactions were stopped by transfer of 2.0-ml. aliquots to an acetoneDry Ice bath. On completion of an experiment, a series of six to eight aliquots was placed in an ice bath to thaw, and 0.5 ml. of 15ye trichloroacetic acid and 0.5 ml. of 2.5 N HCI was added to precipitate the protein. The aliquots were removed from the ice bath, and 0.5 ml. of 14.2% Fe& in 0.01 N HCl was added to develop the characteristic orange-brown or amber color of the iron complex. The solutions were clarified by centrifugation, and the color intensities of the supernatants were measured in a Klett-Summerson photoelectric calorimeter with a No. 54 filter. In each case, color determinations were made 20 min. from the time of addition of FeC13 . Maximum color intensity was obtained under these conditions. The concentration of the oxime is expressed as the color intensity of the iron complex and is given in Klett units/a.0 ml. of reaction mixture. The values presented in the results are the difference between a complete system and a control incubated with NHtOH, tris(hydroxymethyl)aminomethane (Tris) buffer, and the debris fraction in the absence of substrate. Substrate controls contributed no color in the absence of NHSOH but were included as chromatography controls. Reducing-sugar determinations were made according to the method of Nelson (13) on aliquots from reaction mixtures incubated without NHtOH. Pi was estimated in the presence and absence of NHzOH by the method of King (14). Chromatography methods are described in the Results section. Synthetic ribose oxime was prepared essentially by the method of Wohl (10).
FORMATION
OF
EXPERIMENTAL
Purine Ribosidase Mediation
RIBOSE
133
OXIME
RESULTS
in the Adenosine-NHzOH
Reaction
Further study on the characteristics of the reaction between adenosine and NHzOH indicated a marked similarity to purine ribosidase activity of B. cereus spores (5,8,9). Formation of the iron complex occurred on incubation of adenosine and NHzOH with either intact resting or germinated spores; this activity was limited to the particulate debris fraction resulting from centrifugation of disrupted spore homogenates. Boiling of the aqueous suspensions of whole homogenates or the debris fraction for 1 hr. resulted in only a 25 % loss of activity. The reaction was independent of added metals, orthophosphate, and arsenate over a pH range of 5.5-8.0, and product formation was found to be dependent on stoichiometric rather than catalytic concentrations of adenosine. These similarities suggested that purine ribosidase was concerned with the adenosine-NHzOH reaction. Evidence for purine ribosidase mediation was obtained when purine and pyrimidine nucleosides were substituted for adenosine (Table I). The iron complex was formed only on incubation of the purine nucleosides with the debris fraction and NHZOH. The release of approximately theoretical concentrations of reducing sugar identified as ribose from adenosine, inosine, and guanosine in the absence of NHtOH paralleled the formation TABLE
The Dependence
I
of Iron-Complex Formation on the Release of Ribose from Purine Nucleosides
Reaction mixtures contained, per 2.0 ml.: substrate, 5 pmoles; spore debris fraction, 10 mg.; (=t) NHzOH, 200rmoles, Tris buffer, pH 7.5,200 pmoles. Time of incubation 2 hr. at 47°C. Products Substrate
With NHzOH Iron complex
per 2.0 ml.
of reaction
Without NHzOH Ribose pmoles
Adenosine Inosine Guanosine Xanthosine Cytidine Uridine Ribose Adenine Hypoxanthine Cytosine
28 30 23 4 0 0 30 0 0 0
5.44 5.56 4.94 0.60 0.19 0.15 5.44 0 0.15 0
134
KRASK
AND
FULK
of the iron complex and indicated a relationship between the extent of cleavage of the nucleosides and the color intensity of the complex. The relatively slower release of ribose from xanthosine was accompanied by a corresponding decrease in the color intensity of the complex. The relationship between iron complex formation and the release of ribose became apparent when the products of the cleavage of the nucleosides were tested individually (Table I). The iron complex was formed only from ribose and NHZOH, and its color intensity was equal to that obtained on incubation with equivalent concentrations of either adenosine or inosine. That ribose as such was available for reaction with NHSOH was suggested by the complete recovery of the sugar after incubation in the absence of NHtOH. Preincubation experiments further established the role of purine ribosidase and ribose in the reactions between the nucleosides and NH,OH (Fig. 1). The initial rate of iron complex formation after addition of NHzOH to debris fraction preincubated with adenosine was five times that attained on addition of adenosine and NHzOH to debris fraction preincubated without substrate. That the rate curves were a measure of the same reactions
MINUTES
FIG. 1. The effect of preincubation of debris fraction with and without adenosine on the rate of the adenosine-NHzOH reaction. Final reaction mixtures contained, per 2.0 ml.: adenosine, 10 pmoles; spore debris fraction, 12 mg.; NHsOH, 200 pmoles; Tris buffer, pH 7.5,200 pmoles. (0) Debris fraction preincubated 2 hr. at 47°C. with adenosine and buffer; NHzOH added and mixture reincubated at 47°C. (0) Debris fraction preincubated with buffer; adenosine and NHzOH added and mixture reincubated at 47°C.
FORMATION
OF
RIBOSE
OXIME
ADENOSINE
or RIBOSE
(Mlcromoles
per 2 0 ml)
135
FIG. 2. The effect of adenosine and ribose concentration on iron-complex formation. Reaction mixtures contained, per 2.0 ml; (0) adenosine or (0) ribose; spore debris fraction, 25 mg.; NHsOH, 200 pmoles; Tris buffer, pH 7.5, 200 pmoles. Time of incubation 2 hr. at 47°C.
was indicated by their essentially parallel paths after 20 min. of reaction with NHzOH and by the extent of iron complex formation in both reactions. The necessity for the release of ribose to form the iron complex was indicated by taking an aliquot from the reaction mixture preincubated with adenosine immediately before the addition of NHzOH; 10.29 pmoles ribose was recovered from preincubation with 10 pmoles adenosine. The rate curves were thus essentially a measure of the rat.e of reaction between ribose and NHzOH. The effect of the concentration of adenosine and ribose on iron complex formation is shown in Fig. 2. Equivalent concentrations of the substrates up to 20 pmoles gave rise to approximately equivalent concentrations of the iron complex. The Non-enxymic Formation of Ribose Oxime The above results suggested the formation of ribose oxime. Wohl (10) reported the chemical synthesis of sugar oximes in alcoholic solutions of
136
KRASK
AND
FULK
NHzOH under mild conditions of reaction and noted that arabinose oxime was formed at a more rapid rate than glucose oxime. When pentose and hexose sugars (10 pmoles) and ribitol (50 pmoles) were incubated with and without NHzOH (200 pmoles) in the absence of debris fraction for 2 hr. at 47”C., the iron complex was formed only in the presence of the reducing sugars and NH20H. The color intensities obtained in the nonenzymic reactions were; ribose, 36; xylose, 56; arabinose, 74; glucose, 10; and ribitol, 0. The reactions occurred without any significant change in the color intensities when acetate buffer at pH 5.5 or phosphate buffer at pH 7.5 were substituted for Tris buffer at pH 7.5. On the basis of these observations, synthetic ribose oxime was prepared (lo), and its ability to form a complex with iron was tested. Figure 3 compares the color intensity of the iron complex formed after incubation of ribose oxime with and without inactivated debris fraction to that formed after incubation of ribose and NHzOH with active and inactive prepara100
- 80 -z 0 r-4 ;a “, =c3 60
RIBOSE
OR RIBOSE
(Micromoles
FIG. 3. Iron-complex
OXIME
per 2 0 ml)
formation with ribose oxime and the product of the riboseNHzOH reaction. Reaction mixtures contained, per 2.0 ml.: ribose with active (0) or autoclaved (a) spore debris fraction, 15 mg.; NHtOH, 200 Hmoles; Tris buffer, pH 7.5, 200 pmoles. Ribose oxime with (A) or without (A) autoclaved spore debris fraction, 15 mg.; Tris buffer, pH 7.5, 200 pmoles. Time of incubation 2 hr. at 47°C.
FORMATION
OF
RIBOSE
OXIME
137
tions. The iron complex with the synthetic oxime was formed independently of the debris fraction, and its color intensity in the presence of inactivated debris was roughly equivalent to that obtained in the reactions between ribose and NHZOH. Although not included in the figure, the absence of change in the color intensity of the iron complex formed after incubation of ribose oxime with active debris fraction for 2 hr. demonstrated that ribose oxime was not altered by the spore preparation. The equivalence in the color intensities obtained after incubation of ribose and NHzOH with both active and inactive debris fractions established the nonenzymic nature of ribose oxime formation by spores. That ribose was not utilized to form some other component which might react similarily with NHzOH was demonstrated by the recovery of 5.20, 9.97, 15.39, and 19.50 pmoles ribose after incubation of active debris fraction with 5, 10, 15, and 20 pmoles ribose without NHzOH. The reason for the increased color intensity when ribose oxime was incubated with the inactivated debris fraction is not apparent at present. A similar effect was observed when ribose and NHzOH were incubated with either active or inactivated debris fractions. The Identijkation
of Ribose Oxime
Ribose oxime was identified by one-dimensional ascending chromatography on Whatman No. 1 paper. Reaction mixtures containing debris fraction and the purine nucleosides or ribose with and without NH,OH were incubated for 2 hr. at 47°C. Aliquots were centrifuged at 25,000 X g for 1 hr. at 4”C., and the supernatants were developed simultaneously in pyridine-butanol-water (6:4: 3) and in the upper phase of ethyl acetatepyridine-water (2: 1:2). The synthetic ribose oxime and the product of the ribose-NHtOH reaction in the absence of spores were used as standards. The oximes were located by spraying with a hydroxamic acid reagent (15), which resulted in their appearance as orange spots against a yellow background. Ribose was demonstrated with benzidine-acetic acid (16) and aniline-hydrogen phthalate (17) as spray reagents. Spots staining with a marked intensity with the hydroxamic acid reagent appeared only with aliquots taken from reaction mixtures which formed the iron complex, and their position corresponded to that of the oxime standards. Although the Rf values varied in the experiments, the positions of the unknown oxime and the standard were always identical. In freshly prepared solvents the Rf values of ribose oxime and ribose were 0.75 and 0.64, respectively, in pyridinebutanol-water and 0.73 and 0.63 in ethyl acetate-pyridinewater. Several peculiarities were observed with respect to the staining response of ribose oxime. When the oxime standards and reaction mixtures forming the iron complex were developed in the pyridine solvents and sprayed with
138
KRASK
AND
FULK
either benzidine-acetic acid or aniline-hydrogen phthalate to identify the reducing sugars, the position corresponding to ribose oxime was also stained by these reagents. Since ketoximes are converted to their respective ketones by acid hydrolysis (18), our observations suggested the ribose oxime was hydrolyzed in situ to the aldo sugar when the papers were sprayed with the acidic reagents and heated at 105°C. for 5 min. This apparent acid lability was also indicated by the absence of an oxime spot when secbutanol-formic acid-water (75: 15: 10) was used as a solvent and the hydroxamic acid reagent was used for color development. In the acidic solvent only a single benzidine-staining spot corresponding to ribose was found with either the oxime standards or aliquots from reaction mixtures forming the iron complex. A similar observation was made with acetone30 % acetic acid (1: 1). Application of higher concentrations of the oxime standard to the latter solvent, however, produced a single spot which was stained by both the hydroxamic acid and reducing sugar reagents. Its position was identical to that of ribose. The assumptions made to explain the staining response of ribose oxime to benzidine were supported by the results of the hydrolysis experiments used to identify the synthetic oxime. The oxime was hydrolyzed with HCl, and the extent of hydrolysis was determined by the decrease in iron complex formation and the appearance of ribose on chromatograms. Tubes containing 40 pmoles oxime in 2.0 ml. water or HCl in a final concentration of 0.01, 0.05, 0.10, 0.50, and 1.00 M were incubated at 37°C. for 1 hr. As an additional control an oxime-water solution was held at room temperature. The acid mixtures were neutralized with 1.0 ml. NaOH, and the controls were brought to 3.0 ml. with water. Iron-complex determinations were made on 2.0-ml. aliquots. The color intensities were 99 for both controls and 60, 9, 5, 6, and 11, respectively, for the acid mixtures. Ribose was released from all the mixtures with the exception of the oxime-water controls. The marked lability of ribose oxime to acid suggests that our interpretation of the benzidine-acetic acid reaction with ribose oxime is correct. Ribose Oxime Formation from Adenine Nucleotides Recently we have shown that the debris fraction contains heat-labile enzymes which release Pi from ATP, ADP, and AMP (1). Our present findings implied that ribose oxime was the product of the breakdown of the nucleotides in the presence of NHZOH. When iron-complex formation and the release of Pi on incubation of the debris fraction with the nucleotides and NH&H was compared with the release of ribose and Pi in the absence of NH&H, the relationship between iron-complex formation and ribose release was again apparent (Table II). That ribose was released from ADP
139
FORMATION OF RIBOSE OXIME TABLE Iron-Complex
II
Formation upon Cleavage of Adenine Nucleotides Adenosine in the Presence of NHzOH
and
Reaction mixtures contained, per 2.0 ml.: substrate, 10 pmoles; spore debris fraction, 10 mg.; (=I=) PJHZOH, 200 pmoles; Tris buffer, pH 7.5, 200 rmoles. Time of incubation 2 hr. at 47°C. Figures in brackets represent reactions with spore debris fraction heated 20 min. at 65°C. Products With
Substrate
ATP ADP AMP Adenosine
Iron
complex
Rlett
units
0 (0)
16
(0)
34 (0) 59 (59)
per 2.0 ml. of reaction
NHzOH
Without Ribose
Pi
pmoles
0.59 3.14 5.70
NHlOH
(0) (0) (0)
/.Lmoles
0.03 3.22 5.63 8.58
(0) (0) (0) (8.34)
Pi
Jmoles
1.20 (0) 3.60 (0) 5.87 (0)
and AMP in the presence of NHzOH was indicated by the equivalent concentrations of Pi recovered on incubation with or without NHzOH and by the inability of heated preparations to give rise to the iron complex. Confirmation of ribose oxime formation from ADP and AMP was made by chromatography. The failure to elicit the color complex or recover ribose on incubation with ATP suggested an insufficient rate of ADP formation at 47°C. NH20H also appeared to inhibit the ATPase activity. Incubation of ATP with the debris fraction and NHsOH at 37°C. for 24 hr. gave rise to the iron complex in solution, and ribose oxime was demonstrated on chromatograms. SUMMARY
The non-enzymic formation of ribose oxime on incubation of adenine nucleotides and purine nucleosides with NHzOH and spores of Bacillus cereus var. terminalis is described. Ribose oxime formation is contingent upon the enzymic release of ribose from the nucleotides and nucleosides. REFERENCES 1. KRASK, B. J., AND FULK, G. E., Arch. Bioch,em. Biophys. 79, 86 (1959). 2. HILLS, G. M., Biochem. J. 46, 363 (1949). 3. POWELL, J. F., J. Gen. llficrobiol. 6, 993 (1951). 4. CHURCH, B. D., HALVORSOS, H., AND HALVORSOX, H. O., J. Bacterial.
68, 393
(1954). 5. POWELL, J. F., ASD HUNTER, J. It., Biochem. J. 62, 381 (1956). 6. KRASK, B. J., in “Spores” (Halvorson, H. O., ed.) Publ. No. 5, p. 135. American Institute of Biological Sciences, Washington, D. C., 1957. 7. LIPJIANN, F., ASD TUTTLE, L. C., J. Biol. Chem. 169.21 (1945).
140 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
=SK
AND
FULK
LAWRENCE, N. L., J. Bacterial. 70,577 (1955). LAWRENCE, N. L., J. Bacterial. 70,583 (1955). WOHL, A., Ber. 26,730 (1893). MICKLE, H., J. Roy. Microsop. Sot. 68,lO (1948). GROSSOWICZ, N., WAINFAN, E., BOREK, E., AND WAELSCH, H., J. Biol. Chem. 187, 111 (1950). NELSON, N., J. Biol. Chem. 163, 375 (1944). KING, E. J., Biochem. J. 26,292 (1932). STADTMAN, E. R., AND BARKER, H. A., J. BioZ. Chem. 164,769 (1950). HORROCKS, R. H., Nature 164, 444 (1949). PARTRIDGE, S. M., Nature 164,443 (1949). JOHNSON, R. W., AND STIEGLITZ, J., J. Am. Chem. Xoc. 66,1904 (1934). PARTRIDGE, S. M., Biochem. J. 42,238 (1948).