April 20; 19.53
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2010
Discussion It is apparent that the 3,5-substituted nucleo-
the antimetabolite whether the substituent on the 5-carbon is nucleophilic or electrophilic. The results agree with the observation of Woolley sides are less effective as antimetabolites than are the nucleosides in which only one of these structural and PringlelO who have demonstrated that as the changes exists. For example, 3-methyluridine or structural difference between metabolite and ana5-chlorouridine have an inhibition index of approxi- log increases, the degree of inhibition usually d e mately 0.5 when cytidine provides the pyrimidine creases. However, over the range of substrate requirement. When both structural changes are concentration tested, the doubly substituted numade on the same molecule, 3-methyl-5-chlorouri- cleosides retain their ability to inhibit in a compedine, the inhibition index against cytidine is in- titive manner. creased by a factor of about 3. A similar increase (10) D. W. Woolley and A. Pringle, J. B i d . Chcm., 194,729 (1952). in the inhibition index obtained with the doubly DEPARTMENT OF BIOCHEMISTRY AND NUTRITION substituted nucleosides is observed whether uridine, SCHOOL OF MEDICINE, AND cytidine, or uracil provide the pyrimidine require- LABORATORIES OF THE ALLAN HANCOCK FOUNDATION ment, It is of interest to note that a methyl group UNIVERSITY OF SOUTHERN CALIFORNIA in the %position of uridine decreases the activity of Los ANGELES,CALIFORNIA
COMMUNICATIONS T O T H E E D I T O R IONIC INHIBITION OF GROWTH IN LACTOBACILLUS LEICHMANNII 313 AND ITS REVERSAL WITH VITAMIN Biz
Sir: Vitamin B12,as a growth factor for Lactobacillus Leichmunnii 313, can be replaced by thymidine’ or
out the “reversible range” supports full growth a t slightly increasing (5-10 y per 5 ml.) levels; above the “reversible range,” the maximum growth response obtained with thymidine is the same as with excess vitamin BIZ(see Table I).
other desoxyribosides.2 It has been suggested’~~ that vitamin Bl2 might function as a catalyst (coenzyme) in the formation of desoxyribosides. The experimental data presented here seem to offer some indirect evidence for the existence of a vitamin B12-enzyme. We found that slightly hypertonic concentrations of various inorganic salts inhibit the growth of L. Leichmannii 313, in a basal medium4 supplemented with just sufficient (0.1 my per 5 ml.) vitamin Blz to allow full growth (in the absence of the salts). This inhibition can be reversed with an added excess of vitamin Bl2. When the salt concentration is increased, the vitamin B12 requirement sharply increases. Through a narrow salt concentration range, which we will term the “reversible range” (e.g., in the case of NaCl from 1.1 to 1.7%), the inhibition can be fully reversed by increasing the vitamin BIZlevel from the initial 0.1 my up to about 25 my (per 5 ml.); above this range, only partial reversal can be obtained during a standard, 16 hour, incubation period. Thymidine, throughTO, (1) W.Shive, J. M. Ravel and R . E. Eakin, THISJOURNAL, 2614 (1948). (2) E. Kitay, W. S. McNutt and E. E. Snell, J. B i d . Chcm., 177, 993 (l9$9). (3) E. Kitay, W. S. McNutt and E. E. Snell, J. Boct., 69, 727 (1950). (4) Per 100 ml.: acid-hydrolyzed casein, 0.5 g.; L-cysteine hydrochloride, 10 mg.; DL-tryptophan, 20 mg.; L-asparagine, 10 mg.; DL-alanine, 20 mg.; adenine sulfate, 1 mg.; guanine hydrochloride, 1 mg.;uracil, 1 mg.; xanthine, 1 mg.; thiamin hydrochloride, 100 y ; pyridoxine, 200 y ; pyridoxamine, 60 y ; pyridoxal, 60 y ; calcium pantothenate, 100 7 ; niacin, 200 y ; PABA, 20 y ; biotin, 0.2 7 ; folic acid, 0.4 y ; riboflavin, 100 y ; ascorbic acid, 0.2 g.; dextrose, 2.0 g.; tween 80, 100 mg.; ialts A, 1 ml.; aalts B, 1 ml.; sodium ncetate, 0.5 g. Incubntion, 16 hours at 3 7 O . Five ml. in each tube,
TABLEI Salt
Concentration % A44
Ir b
( B i ~ ) i / ~Thymidined max.
... ... 0.025” 1 . 8 *.. 1.0 1.4 0.239 0.239 2.0 ,256 .256 1.5 1.5 2.5 .291 6.0 1.7 .291 .325 (25. O)f (2.5)’ .325 1.9 ,217 1.8 ,217 0.50 1.62 KCl 1.8 .252 ,252 1.58 1.88 2.0 2.13 .285 .285 7.5 (2.2)’ ,318 (25If 2.37 .318 .224 0.45 1.2 .224 NHiCl 2.5 1.4 .262 .262 2.30 5.0 1.6 .299 .299 1.8 .336 (20)’ ,336 1.6 .276 .092 0.12 1.8 KzSO4 1.8 1.8 .309 ,103 0.40 2.0 ,365 ,115 0.60 ,132 1.20 2.3 .406 2.6 ,447 ,149 5.0 .166 2.9 .498 (15)’ 0.14 1.0 .196 MgC12..049 1.2 ,236 0.30 6Hz0 ,059 .273 1.15 1.4 ,069 .073 CaCll ,292 0.8 3.8 1.2 ,109 .436 (25)’ Gram moles per liter. * Ionic strength, I( = ‘/~Zcv*, where c = gram ions per liter; v = valence, for each ion. my’per 5 ml.; amount of additional (in excess of 0.1) vitamin BIZneeded for half maximum growth. y per 5 ml.; required for half maximum growth in vitamin Blz-free media. (We are indebted to Dr. W. Shive for a small sample of this substance.) e m y g 5 ml. basal medium It concentration above (vitamin BIS standard curve). “reversible range” ; only partial growth obtained. None NaCl
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2020
VOl. 75
Initial cholesterol fractions showed the presence of small quantities of similar C14-labeled components but there was only a slight change in activity of the cholesterol after purification by precipitation of the digitonide, dibr~mination~ and recrystallization. The observed changes in C14 content were log BIZ)^/^ ma=. = a blp not accompanied by comparable changes in total This equation seems to apply well within the cholesterol present in the tissuess but they were "reversible range'' and the values of the constants sufficient to indicate changes in the C1*-contentof a and b, respectively, are not too far apart for most other sterols. of the salts examined. Schwenk and N. T. Werthessen, Arch. Biochcm. Biophys., It is possible to arrive theoretically to a similar 40,(5)334E.(1952). functional relationship between (Bit) I/* max. and ( 6 ) . K. Guggenheim and R . E. Olson, J . Nutrition, 48, 345 (1952). from simple kinetic equations, if we make two R. R. BECKRR basic assumptions : jjrst, vitamin Blz combines H. B. BURCH with a protein apoenzyme (Ea) to give the enzyme DEPARTMENT OF CHEMISTRY L. L. SALOMON UNIVERSITY T. A. VENKITASUBRAMANIAN Ea $ BlzEa; second, the available COLUMBIA B1zEa:B12 YORK C. G. KING concentration of Ea is controlled by the ionic KEW YORK27, SEW RECEIVED MARCH 23, 1953 strength of the salt solutions in accordance with Cohn' s ' salting-out '' equation for proteins ?.6 These two assumptions allow the derivation of a theoretical equation which has the same form as the ENZYMATIC PHOSPHORYLATION OF NUCLEOSIDES experimental formula. The derivation itself, toBY PHOSPHATE TRANSFER gether with a critical appraisal of such interpreta- Sir: tion of our data, will be presented elsewhere. We have found a phosphatase preparation, ob( 5 ) E. J. Cohn, Pkysiol. Rev.,5, 349 (1935), A n n . Rev Btockcm., 4, tained by the fractionation with ammonium sulfate 93 (1935). of Merck malt diastase, which is able to phosphory( 6 ) M. Ingram, Pvoc. Roy. Soc., Scr. B , 134, 181 (1951). late ribose and desoxyribose nucleosides in the presTHEARMOUR LABORATORIES THOMAS J. BARDOS CHICAGO HARRYL. GORDON ence of sodium phenylphosphate. The reaction is 9, ILLINOIS dependent on the concentrations of both nucleoRECEIVED MARCH18, 1953 side and phenylphosphate. The PH activity curves for transphosphorylation and dephosphoryASCORBIC ACID DEFICIENCY AND CHOLESTEROL lation have the same shape, with an optimum SYNTHESIS1 around PH 5.2. Both reactions are partially inSir: hibited by inorganic phosphate to the same extent. In continuing studies of chemical changes charThe organic phosphates formed were separated acteristic of or regulated by ascorbic acid2s3and by paper chromatography with aqueous isobutyric related metabolites,* we have recently observed a acid buffered with ammonium isobutyrate as the relationship to steroid metabolism that is of con- solvent.' Their RF values were identical with siderable interest. Although l-CI4-labeled ascor- those of the corresponding nucleotides. bic acid is not appreciably incorporated into cholesIn a large-scale experiment, 166 p moles of riboterol, the vitamin does exert a marked effect upon cytidine was incubated, in a total volume.of 4 ml., the conversion of acetate-l-CI4 to cholesterol and with 800 p moles of phenylphosphate and 8 mg. of other steroids in guinea pigs. Preliminary find- enzyme in 0.1 M acetate buffer of PH 5 for 87 ings showed that severely scorbutic guinea pigs, hours a t 30'. At this stage, 80% of the phosphate compared with normal animals fed ad Lib., incor- donor were split and 17 p moles of cytidylic acid porated 6 times as much C14from acetate-1-C14 (10.2% of the nucleoside) were formed. The cytiinto cholesterol isolated from adrenals. dylic acid fraction, isolated by ion-exchange chroGuinea pigs of comparable age (10-12 weeks) matographyI2 contained equimolar quantities of and size (350-400 g.), on a vitamin C- and choles- organic phosphorus and of nucleoside (determined terol-free chow diet showed the following values spectrophotometrically) and was completely de(3 animals per group) for specific activities in puri- phosphorylated by the 5-nucleotidase of rattlefied adrenal and liver cholesterol, respectively, four snake venom which, under the conditions used, hours after receiving the last of three intraperi- failed to attack commercial cytidylic acid consisttoneal injections of labeled sodium acetate (1 mg., ing, presumably, of a mixture of the 2'- and 3'2.68 X lo7 c.p.m./mg. each a t 9 hour intervals): nucleotides. This evidence tends to indicate that normal, fed ad lib., 100 and 80; mild scurvy (15-20 the 5'-nucleotide had been produced. days depletion), 170 and 75 (pair-fed controls, 150 All nucleosides tested could thus be phosphoryand 80); severe scurvy (21-28 days depletion), lated. Preliminary results, listed in Table I, 600 and 145 (pair-fed controls, 195 and 90). apparently show that, under identical conditions, (1) This work w a s supported in part by grants from the Nutrition desoxyribon~cleosides~ are phosphorylated with Foundation, Inc., and the Division of Research Grants, U. S. Puhlk greater ease than the corresponding ribosides. Health Service. Table I gives the amounts of vitamin BIZ required for half maximum growth a t various salt concentrations. The logarithm of the witemin E12 requirement a@eurs to be a linearjunction of the ionic strength of the salt solutions
+
+
~ 74, (2) L. L. Salomon, J. J Burns and C. 0.King, T E JOUBNAX., 5161 (1952). (3) J. J. Burns, H. B. Burch and C. G. King, 1. B i d . Chcm., 191, 601 (1961). (4) Hugh H. Horowitr and C. G. King, ibid., 900, 126 (19533.
(1) B. Magasanik. E. Vischer. R. Doniger. D. Elson and E. Chargaff, J . Biol. Chcm., 186, 37 (1950). (2) W. E. Cohn and E. VolHn, Nature, 157, 483 (1951). (3) Uracil desoxyriboside was obtained through the courtesy of Prof. A. R. Todd.
