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The utilization of inosine, adenosine and ribose by spores of Bacillus cereus var. terminalis
ARCHIVES
OF
BIOCHEMISTRY
AND
79, 86-90 (1959)
BIOPHYSICS
The Utilization of Inosine, Adenosine and Ribose by Spores of Bacillus cereus var. terminalis Bernard J. Krask and George E. Fulk From the United States Army, Chemical Corps, Fort Detrick, Frederick, Maryland Received April 28, 1958 INTRODUCTION
Inosine or adenosine alone or together with amino acids or glucose stimulate the rapid germination of spores of certain Bcu3ZZu.s species (14). The means by which these nucleosides contribute to germination are however unknown. The hydrolytic cleavage of inosine or adenosine to ribose and a purine base has been demonstrated, but the products of the cleavage individually or together would not stimulate germination (4-6). The disappearance of ribose as a reducing sugar following complete cleavage of adenosine by intact spores or extracts has also been reported, but neither phosphorylation nor oxidation of the pentose could be demonstrated (5). In this report evidence is presented which suggests that extracts of spores of Bacillus cereus var. terminalis contain the following enzymes (or enzyme systems): (a) a nucleoside phosphorylase (7) which mediates in the reaction between adenosine or inosine and orthophosphate to produce ribose l-phosphate (R-l-P); (5) a ribokinase (8) which catalyzes the reaction between adenosine triphosphate (ATP) and ribose in the presence of Mg++ to produce ribose 5-phosphate (R-5-P), and (c) a phosphoribomutase (9) which converts R-l-P to R-5-P. EXPERIMENTAL
AND RESULTS
Partially clean spore’preparations were obtained from Dr. H. 0. Halvorson of the University of Illinois. The spores were freed of residual debris by overlaying suspensions on sucrose solutions with a concentration gradient of 1040% sucrose and centrifuging at 1050 X 9 for 1 hr. at 4°C. The spore sediments were washed 6-8 times with distilled water, lyophilized, and stored in vacua over CaClz at 4°C. Spore homogenates were prepared by Mickle (10) disintegration for 15 min. at 4°C. Stained films showed the homogenates were free of intact spores. Pentose and pentose phosphate were demonstrated by one-dimensional ascending chromatography on Whatman No. 1 paper. Aliquots of reaction 86
UTILIZATION
OF
INOSINE,
ADENOSINE
ASD
RIBOSE
87
mixtures were centrifuged at 25,000 X g for 20 min. at 4°C. prior to application and were developed simultaneously in acetone&O% acetic acid (1: 1) at room temperature and methanol-NH40H-Hz0 (6: 1:s) at 4°C. Benzidine-acetic acid (1 I), phloroglucinol (12), and aniline-phthalate (13) were used as spray reagents. Pentose phosphate formation and orthophosphate liberation were also followed at the same time by paper chromatography in the two solvent systems using a molybdate-perchloric acid (14) spray reagent and ultraviolet development (15). Aliquots of reaction mixtures for reducing sugar determinations were treated according t,o the method of Nelson (16). In preliminary studies it was found that spore homogenat8es released ribose and orthophosphate from adenosine triphosphate (ATP). The debris fraction from homogenates separated by centrifugation at 25,000 X g at 4°C. for 45 min. also released ribose and orthophosphate from adenosine diphosphate, adenosine 5-phosphate, and, most significantly, from R-5-P. This fraction split adenosine and inosine to ribose and a purine base. All the activities were destroyed by heating for 15 min. at 65°C. with the exception of the hydrolytic nucleosidase which was stable at 100°C. for I hr. The breakdown of R-5-P to ribose and orthophosphate by debris fractions from spores suggested that the inability of previous workers to demonstrate phosphorylation of ribose following adenosine or inosine cleavage was due to phosphatase activity. A marked decrease in phosphatase activity against R-5-P in extracts prepared from homogenates at 25,000 X g was demonstrated by incubating the extract from 12 mg. of spores with R-5-P (25 Imoles), MgClz (24 pmoles), tris(hydroxymethyl)aminomethane (Tris) buffer (40 pmoles, pH 7.5) and chloromycetin (100 pg.) in a l.O-ml. volume for up to 4 hr. at 37°C. Hydrolytic nucleosidase activity toward adenosine or inosine was completely absent or negligible when 5 pmoles of either nucleoside was substituted for R-5-P in the above reaction mixture. Replacement of R-5-P with inosine or adenosine (5 pmoles) and Tris buffer with potassium phosphate buffer (20 pmoles, pH 7.5) resulted in a product> which on chromatography occupied the same position as R-5-P in the two solvent systems previously mentioned. The phloroglucinol spray identified the reaction product as an aldopentose, and the molybdate spray showed it to be phosphorylated. Since R-l-P was unavailable for comparison, advantage was taken of the fact that R-l-P is extremely acid labile (17). When the pentose phosphate product and a standard of R-5-P were eluted from chromatograms with water and subjected to hydrolysis in 0.5 N HCl at lOO”C., both were found to be stable to acid hydrolysis for 20 min. as determined by chromatography. The reaction product and the standard R-5-P were degraded to approximately the same extent after acid hydrolysis for 40 min. This stability to hydrolysis eliminated R-l -I’ as the reaction product.
88
KRASK
AND
FULK
These findings indicated that in the presence of orthophosphate, inosine and adenosine are split to R-l-P and a purine base by nucleoside phosphorylase and that the R-l-P is subsequently converted to R-5-P by phosphoribomutase. A similar breakdown of adenosine to R-5-P was first observed with red blood cell hemolyzates (18) and a purified nucleoside phosphorylase was subsequently isolated (19,20). Evidence for the formation of R-l-P by spore extracts was obtained indirectly by substituting arsenate for orthophosphate and determining reducing sugar according to the following reaction (7,19) : Inosine or adenosine + arsenate -+ R-l-arsenate
+ purine
1Ha0
ribose
Table I shows that the ribose liberated by the enzymic arsenolysis of inosine is 20 times that released from inosine incubated in the absence of arsenate and that the arsenate-stimulated reaction is heat labile. The ribose produced in the absence of arsenate is most probably due to incomplete separation of the heat-stable hydrolytic nucleosidase by centrifugation of the homogenates. Ribose liberation from adenosine does not occur in the absence of arsenate, and the initial slower rate of reaction of adenosine arsenolysis suggests that adenosine must first be deaminated to inosine. Adenosine deaminase has been demonstrated in spore extracts (4), and it has been suggested that adenosine is converted to inosine prior to its utiliTABLE I of Inosine and Adenosine by Extracts from Bacillus cereus var. terminalis Spores Reaction mixtures contained per ml.: Inosine or adenosine, 5 crmoles; extract equivalent to 12 mg. intact spores; Tris buffer, pH 7.5, 16 rmoles. Mixtures with NalHAs04.7Hz0 contained 12.5 pmoles/ml. Extract was inactivated by heating at 65°C. for 15 min. Reaction mixtures were incubated at 37°C. Arsenolysis
Micromoles Experimental
Inosine + Inosine + Inosine Inosine + Adenosine Adenosine Adenosine
of riboselml.
arsenate arsenate + heated extract heated extract + arsenate + arsenate + heated extract
of reaction mixture
Hours
conditions 0
0.5
1
2
3
0.28 0.05 0 0 0 0 0
3.00 0.09 0.05 0.05 0.80 0 0
3.75 0.14 0.09 0.13 1.90 0 0
4.37 0.29 0.16 0.19 3.70 0 0
4.57 0.34 0.23 0.23 4.45 0 0
UTILIZATION
OF
INOSINE,
ADENOSINE
AND
RIBOSE
89
zation for spore germination. The specificity of nucleoside phosphorylase for inosine and guanosine (7, 19, 21) further supports this suggestion. The formation of R-5-P from inosine and adenosine indicated that R-5-I’ metabolism might be important in germinating spores and that spores may contain an alternate mechanism for R-5-P synthesis. R-5-P synthesis by a ribokinase was demonstrated by paper chromatography after incubation of extract (from 15 mg. spores), ribose (4 pmoles), ATP (10 kmoles), MgCI, (20 pmoles), and potassium phosphate buffer (10 pmoles, pH 7.5) in a volume of 1.0 ml. at 37°C. for up to 4 hr. Aliquots spotted at 1-hr. intervals for 4 hr. in the two solvent systems showed an increase with time in the color intensity of the spot corresponding to R-5-P. Simultaneous reducing sugar determinations showed that 0.54 and 1.00 pmoles of the ribose disappeared from the reaction mixture at 2 and 4 hr., respectively. Mg++ and ATP were necessary for the disappearance of reducing sugar and R-5-t’ formation. The ribokinase activity was lost by heating extracts at 65°C. for 15 min. R-5-P formation from ATP breakdown in the absence of ribose was negligible. DISCUSSION
The utilization of inosine or adenosine to form R-5-P suggests that the function of these nucleosides in stimulating spore germination is to provide utilizable phosphate esters with the minimum investment of energy by the organism. R-5-P formation from inosine or adenosine does not require ATP and provides a source of triose phosphates for ATP regeneration. Spores are reported to be devoid of hexokinase (22) and may well be depleted of ATP. Inosine or adenosine utilization in such a manner in germination would parallel the reported effect of these nucleosides in restoring viability to stored red blood cells through resynthesis of phosphate esters and ATP (23, 24). The utilization of inosine or adenosine to form R-5-P and t,he synthesis of R-5-P by ribokinase suggests that carbohydrate metabolism via a pentose phosphate pathway occurs in spores requiring nucleosides for germination. The recent report that spores of B. cerew var. terminalis germinate readily in the presence of R-5-P (25) adds significance to our rexultjs and speculation. SUMMARY
Extracts from spores of B. cereus var. terminalis contain the following enzymes: (a) a nucleoside phosphorylase which mediates in the reaction between adenosine or inosine and orthophosphate to produce ribose-lphosphate; (b) a phosphoribomutase which converts ribose-l-phosphate to ribosed-phosphate and (c) a ribokinase which catalyzes the formation of ribose-5-phosphate from ribose and ATP in the presence of Mg++. The
90
KFtASK AND
FULK
experimental evidence suggests deamination of adenosine to inosine prior to phosphorolysis. Spore homogenates and debris fractions from separated homogenates release ribose and orthophosphate from adenosine triphosphosphate, adenosine diphosphate, adenosine monophosphate and ribosed-phosphate. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
HILLS, G. M., Biochem. J. 46, 363 (1949). POWELL, J. F., J. Gen. Microbial. 6, 993 (1951). CHURCH, B. D., HALVORSON, H., AND Halvorson, H. O., J. Bacterial. 68,393 (1954). POWELL, J. F., AND HUNTER, J. R., Biochem. J. 62, 381 (1956). LAWRENCE, N. L., J. Bacterial. 70, 577 (1955). LAWRENCE, N. L., J. Bacterial. 70, 583 (1955). KALCKAR, H. M., J. Biol. Chem. 167, 429 (1947). SABLE, H. Z., Proc. Sot. Exptl. Biol. Med. 76, 215 (1950). ABRAMS, A., AND KLENOW, H., Federation Proc. 10, 153 (1951). Sot. 68, 10 (1948). MICKLE, H., J. Roy. M~CTOSCO~. HORROCKS, R. H., Nature 164, 444 (1949). DISCHE, Z., AND BORENFREUND, E., Biochim. et Biophys. Acta 67, 239 (1957). PARTRIDGE, S. M., Nature 164, 443 (1949). HANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107 (1949). BANDURSKI, R. S., AND AXELROD, B., J. Biol. Chem. 193, 405 (1951). 16. NELSON, N., J. Biol. Chem. 166, 375 (1944). 17. KALCKAR, H. M., J. Biol. Chem. 167, 477 (1947). 18. DISCHE, Z., in “Phosphorus Metabolism” (McElroy, D. W., and Glass, B., eds.), Vol. I, p. 171. Johns Hopkins Press, Baltimore, Md., 1951. 19. GABRIO, B. W., AND HUENNEKENS, F. M., Biochim. et Biophp. Acta 18,585 (1955). 20. Tsuboi, K. K., AND HUDSON, P. B., J. BioZ. Chem. 224, 879 (1957). 21. HEPPEL, L. A., AND HILMOE, R. J., J. Biol. Chem. 198, 683 (1952). 22. CHURCH, B. D., AND HALVORSON, H., J. BacterioZ. 73, 470 (1957). 23. GABRIO, B. W., DONOHUE, D. M., AND FINCH, C. A., J. Clin. Invest. 34,1509 (1955). 24. RUBINSTEIN, D., AND DENSTEDT, 0. F. Can. .J. Biochem. Physiol. 34, 927 (1956). 25. HALVORSON, H., AND CHURCH, B. D., J. Appl. Bacterial. 20, 359 (1957).
OF
BIOCHEMISTRY
AND
79, 86-90 (1959)
BIOPHYSICS
The Utilization of Inosine, Adenosine and Ribose by Spores of Bacillus cereus var. terminalis Bernard J. Krask and George E. Fulk From the United States Army, Chemical Corps, Fort Detrick, Frederick, Maryland Received April 28, 1958 INTRODUCTION
Inosine or adenosine alone or together with amino acids or glucose stimulate the rapid germination of spores of certain Bcu3ZZu.s species (14). The means by which these nucleosides contribute to germination are however unknown. The hydrolytic cleavage of inosine or adenosine to ribose and a purine base has been demonstrated, but the products of the cleavage individually or together would not stimulate germination (4-6). The disappearance of ribose as a reducing sugar following complete cleavage of adenosine by intact spores or extracts has also been reported, but neither phosphorylation nor oxidation of the pentose could be demonstrated (5). In this report evidence is presented which suggests that extracts of spores of Bacillus cereus var. terminalis contain the following enzymes (or enzyme systems): (a) a nucleoside phosphorylase (7) which mediates in the reaction between adenosine or inosine and orthophosphate to produce ribose l-phosphate (R-l-P); (5) a ribokinase (8) which catalyzes the reaction between adenosine triphosphate (ATP) and ribose in the presence of Mg++ to produce ribose 5-phosphate (R-5-P), and (c) a phosphoribomutase (9) which converts R-l-P to R-5-P. EXPERIMENTAL
AND RESULTS
Partially clean spore’preparations were obtained from Dr. H. 0. Halvorson of the University of Illinois. The spores were freed of residual debris by overlaying suspensions on sucrose solutions with a concentration gradient of 1040% sucrose and centrifuging at 1050 X 9 for 1 hr. at 4°C. The spore sediments were washed 6-8 times with distilled water, lyophilized, and stored in vacua over CaClz at 4°C. Spore homogenates were prepared by Mickle (10) disintegration for 15 min. at 4°C. Stained films showed the homogenates were free of intact spores. Pentose and pentose phosphate were demonstrated by one-dimensional ascending chromatography on Whatman No. 1 paper. Aliquots of reaction 86
UTILIZATION
OF
INOSINE,
ADENOSINE
ASD
RIBOSE
87
mixtures were centrifuged at 25,000 X g for 20 min. at 4°C. prior to application and were developed simultaneously in acetone&O% acetic acid (1: 1) at room temperature and methanol-NH40H-Hz0 (6: 1:s) at 4°C. Benzidine-acetic acid (1 I), phloroglucinol (12), and aniline-phthalate (13) were used as spray reagents. Pentose phosphate formation and orthophosphate liberation were also followed at the same time by paper chromatography in the two solvent systems using a molybdate-perchloric acid (14) spray reagent and ultraviolet development (15). Aliquots of reaction mixtures for reducing sugar determinations were treated according t,o the method of Nelson (16). In preliminary studies it was found that spore homogenat8es released ribose and orthophosphate from adenosine triphosphate (ATP). The debris fraction from homogenates separated by centrifugation at 25,000 X g at 4°C. for 45 min. also released ribose and orthophosphate from adenosine diphosphate, adenosine 5-phosphate, and, most significantly, from R-5-P. This fraction split adenosine and inosine to ribose and a purine base. All the activities were destroyed by heating for 15 min. at 65°C. with the exception of the hydrolytic nucleosidase which was stable at 100°C. for I hr. The breakdown of R-5-P to ribose and orthophosphate by debris fractions from spores suggested that the inability of previous workers to demonstrate phosphorylation of ribose following adenosine or inosine cleavage was due to phosphatase activity. A marked decrease in phosphatase activity against R-5-P in extracts prepared from homogenates at 25,000 X g was demonstrated by incubating the extract from 12 mg. of spores with R-5-P (25 Imoles), MgClz (24 pmoles), tris(hydroxymethyl)aminomethane (Tris) buffer (40 pmoles, pH 7.5) and chloromycetin (100 pg.) in a l.O-ml. volume for up to 4 hr. at 37°C. Hydrolytic nucleosidase activity toward adenosine or inosine was completely absent or negligible when 5 pmoles of either nucleoside was substituted for R-5-P in the above reaction mixture. Replacement of R-5-P with inosine or adenosine (5 pmoles) and Tris buffer with potassium phosphate buffer (20 pmoles, pH 7.5) resulted in a product> which on chromatography occupied the same position as R-5-P in the two solvent systems previously mentioned. The phloroglucinol spray identified the reaction product as an aldopentose, and the molybdate spray showed it to be phosphorylated. Since R-l-P was unavailable for comparison, advantage was taken of the fact that R-l-P is extremely acid labile (17). When the pentose phosphate product and a standard of R-5-P were eluted from chromatograms with water and subjected to hydrolysis in 0.5 N HCl at lOO”C., both were found to be stable to acid hydrolysis for 20 min. as determined by chromatography. The reaction product and the standard R-5-P were degraded to approximately the same extent after acid hydrolysis for 40 min. This stability to hydrolysis eliminated R-l -I’ as the reaction product.
88
KRASK
AND
FULK
These findings indicated that in the presence of orthophosphate, inosine and adenosine are split to R-l-P and a purine base by nucleoside phosphorylase and that the R-l-P is subsequently converted to R-5-P by phosphoribomutase. A similar breakdown of adenosine to R-5-P was first observed with red blood cell hemolyzates (18) and a purified nucleoside phosphorylase was subsequently isolated (19,20). Evidence for the formation of R-l-P by spore extracts was obtained indirectly by substituting arsenate for orthophosphate and determining reducing sugar according to the following reaction (7,19) : Inosine or adenosine + arsenate -+ R-l-arsenate
+ purine
1Ha0
ribose
Table I shows that the ribose liberated by the enzymic arsenolysis of inosine is 20 times that released from inosine incubated in the absence of arsenate and that the arsenate-stimulated reaction is heat labile. The ribose produced in the absence of arsenate is most probably due to incomplete separation of the heat-stable hydrolytic nucleosidase by centrifugation of the homogenates. Ribose liberation from adenosine does not occur in the absence of arsenate, and the initial slower rate of reaction of adenosine arsenolysis suggests that adenosine must first be deaminated to inosine. Adenosine deaminase has been demonstrated in spore extracts (4), and it has been suggested that adenosine is converted to inosine prior to its utiliTABLE I of Inosine and Adenosine by Extracts from Bacillus cereus var. terminalis Spores Reaction mixtures contained per ml.: Inosine or adenosine, 5 crmoles; extract equivalent to 12 mg. intact spores; Tris buffer, pH 7.5, 16 rmoles. Mixtures with NalHAs04.7Hz0 contained 12.5 pmoles/ml. Extract was inactivated by heating at 65°C. for 15 min. Reaction mixtures were incubated at 37°C. Arsenolysis
Micromoles Experimental
Inosine + Inosine + Inosine Inosine + Adenosine Adenosine Adenosine
of riboselml.
arsenate arsenate + heated extract heated extract + arsenate + arsenate + heated extract
of reaction mixture
Hours
conditions 0
0.5
1
2
3
0.28 0.05 0 0 0 0 0
3.00 0.09 0.05 0.05 0.80 0 0
3.75 0.14 0.09 0.13 1.90 0 0
4.37 0.29 0.16 0.19 3.70 0 0
4.57 0.34 0.23 0.23 4.45 0 0
UTILIZATION
OF
INOSINE,
ADENOSINE
AND
RIBOSE
89
zation for spore germination. The specificity of nucleoside phosphorylase for inosine and guanosine (7, 19, 21) further supports this suggestion. The formation of R-5-P from inosine and adenosine indicated that R-5-I’ metabolism might be important in germinating spores and that spores may contain an alternate mechanism for R-5-P synthesis. R-5-P synthesis by a ribokinase was demonstrated by paper chromatography after incubation of extract (from 15 mg. spores), ribose (4 pmoles), ATP (10 kmoles), MgCI, (20 pmoles), and potassium phosphate buffer (10 pmoles, pH 7.5) in a volume of 1.0 ml. at 37°C. for up to 4 hr. Aliquots spotted at 1-hr. intervals for 4 hr. in the two solvent systems showed an increase with time in the color intensity of the spot corresponding to R-5-P. Simultaneous reducing sugar determinations showed that 0.54 and 1.00 pmoles of the ribose disappeared from the reaction mixture at 2 and 4 hr., respectively. Mg++ and ATP were necessary for the disappearance of reducing sugar and R-5-t’ formation. The ribokinase activity was lost by heating extracts at 65°C. for 15 min. R-5-P formation from ATP breakdown in the absence of ribose was negligible. DISCUSSION
The utilization of inosine or adenosine to form R-5-P suggests that the function of these nucleosides in stimulating spore germination is to provide utilizable phosphate esters with the minimum investment of energy by the organism. R-5-P formation from inosine or adenosine does not require ATP and provides a source of triose phosphates for ATP regeneration. Spores are reported to be devoid of hexokinase (22) and may well be depleted of ATP. Inosine or adenosine utilization in such a manner in germination would parallel the reported effect of these nucleosides in restoring viability to stored red blood cells through resynthesis of phosphate esters and ATP (23, 24). The utilization of inosine or adenosine to form R-5-P and t,he synthesis of R-5-P by ribokinase suggests that carbohydrate metabolism via a pentose phosphate pathway occurs in spores requiring nucleosides for germination. The recent report that spores of B. cerew var. terminalis germinate readily in the presence of R-5-P (25) adds significance to our rexultjs and speculation. SUMMARY
Extracts from spores of B. cereus var. terminalis contain the following enzymes: (a) a nucleoside phosphorylase which mediates in the reaction between adenosine or inosine and orthophosphate to produce ribose-lphosphate; (b) a phosphoribomutase which converts ribose-l-phosphate to ribosed-phosphate and (c) a ribokinase which catalyzes the formation of ribose-5-phosphate from ribose and ATP in the presence of Mg++. The
90
KFtASK AND
FULK
experimental evidence suggests deamination of adenosine to inosine prior to phosphorolysis. Spore homogenates and debris fractions from separated homogenates release ribose and orthophosphate from adenosine triphosphosphate, adenosine diphosphate, adenosine monophosphate and ribosed-phosphate. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
HILLS, G. M., Biochem. J. 46, 363 (1949). POWELL, J. F., J. Gen. Microbial. 6, 993 (1951). CHURCH, B. D., HALVORSON, H., AND Halvorson, H. O., J. Bacterial. 68,393 (1954). POWELL, J. F., AND HUNTER, J. R., Biochem. J. 62, 381 (1956). LAWRENCE, N. L., J. Bacterial. 70, 577 (1955). LAWRENCE, N. L., J. Bacterial. 70, 583 (1955). KALCKAR, H. M., J. Biol. Chem. 167, 429 (1947). SABLE, H. Z., Proc. Sot. Exptl. Biol. Med. 76, 215 (1950). ABRAMS, A., AND KLENOW, H., Federation Proc. 10, 153 (1951). Sot. 68, 10 (1948). MICKLE, H., J. Roy. M~CTOSCO~. HORROCKS, R. H., Nature 164, 444 (1949). DISCHE, Z., AND BORENFREUND, E., Biochim. et Biophys. Acta 67, 239 (1957). PARTRIDGE, S. M., Nature 164, 443 (1949). HANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107 (1949). BANDURSKI, R. S., AND AXELROD, B., J. Biol. Chem. 193, 405 (1951). 16. NELSON, N., J. Biol. Chem. 166, 375 (1944). 17. KALCKAR, H. M., J. Biol. Chem. 167, 477 (1947). 18. DISCHE, Z., in “Phosphorus Metabolism” (McElroy, D. W., and Glass, B., eds.), Vol. I, p. 171. Johns Hopkins Press, Baltimore, Md., 1951. 19. GABRIO, B. W., AND HUENNEKENS, F. M., Biochim. et Biophp. Acta 18,585 (1955). 20. Tsuboi, K. K., AND HUDSON, P. B., J. BioZ. Chem. 224, 879 (1957). 21. HEPPEL, L. A., AND HILMOE, R. J., J. Biol. Chem. 198, 683 (1952). 22. CHURCH, B. D., AND HALVORSON, H., J. BacterioZ. 73, 470 (1957). 23. GABRIO, B. W., DONOHUE, D. M., AND FINCH, C. A., J. Clin. Invest. 34,1509 (1955). 24. RUBINSTEIN, D., AND DENSTEDT, 0. F. Can. .J. Biochem. Physiol. 34, 927 (1956). 25. HALVORSON, H., AND CHURCH, B. D., J. Appl. Bacterial. 20, 359 (1957).