51 57
Oct. 20, 195-4
NOTES
TABLE I1 THE AGING OF A DILUTEDSOLUTION OF ENZYME A Dilutionagedat3-4'. I n kineticruns: temp.,25.12=!c0.02°, [substratelo = 3.34 X 10-3 M , [phosphate] = 0.012 M
is furnished by these compounds for the biosynthesis of cellulose, thus permitting some insight as to the role played by these supplements as well as possibly elucidating upon the mechanism whereby the additional cellulose is produced. In this note we wish to report the results of experiments in which A . xylinum was cultured in a glucose-enriched medium plus one of each of the following as the sole labeled ingredient: ethanol1-Cl4, ethanol-2-CI4, sodium acetate-l-Cl4 and sodium acetate-2-CL4. Analyses of the culture products are presented.
pH
Added Mg++,M
Versene,
.If
Activity after dilution 0 hr 21 hr. 62 hr.
7.4 0.016 100 100 100" 7.4 0 15 50 76 8.2 0 0.014 18 79 91 a T h e absolute activity in this standard run with M g f f present was found to have dropped only 1% from that observed a t 0 hours.
It will be most interesting to note in further study whether any measurable change in state of the enzyme (molecular weight, ionic charge, light-scattering properties) accompanies this change in its response to Mg++ activation. Experimental The apparatus and techniques employed in the kinetic experiments were those previously Recrystallized acetylcholine chloride as substrate and triply distilled water were used throughout. T h e enzyme preparation B sent to Bethesda for the comparison of Table I was characterized by a specific activity (Ap value) of 6000 mg. acetylcholine hydrolyzed/hr./mg. of protein per ml. The Ap value of the stock solution of A was 11,000 and its content of ammonium sulfate was 5% by weight, with PH adjusted to 7. Results identical t o those given for immediate dilution of stock enzyme A were obtained on a similar but older Bethesda preparation assayed a t an Ap of 34,000. In the aging experiments of Table 11, the dilution of stock solution A (7.8 mg. protein per ml.) which was allowed t o stand a t 3-4' was a 1:25 dilution in water with a final ammonium sulfate content of 0.2%. A 1.0-ml. aliquot of the aging 1:25 dilution was made t o 500 ml. with water just prior t o kinetic experiments, and 0.50-ml. aliquots withdrawn for the individual runs. U. S. NAVALMEDICALRESEARCH INSTITUTE BETHESDA,MARYLAND, AND DEPARTMENT OF NEUROLOGY COLUMBIA UNIVERSITYCOLLEGEOF PHYSICIANS AND SURGEONS NEW YORK,NEW YORK
Experimental Culture Conditions.-Except for the nutrient medin, the methods of sterilization of medium, inoculation and incubation were the same as those used with ~ - g l u c o s e - l - C ' ~ . In ~ this study the culture medium consisted of 1.0% unlabeled D-glucose; 0.3% KHtP04; evperiments 1 t o 4 differed in the use of 0.5% Difco yeast extract, and experiments 5 t o 7 differed in the use of 2.0% Difco yeast extract. Unlabeled sodium acetate, 0.6%, was added to experiments 3 and 4, and 0.1% t o experiments 5 to 7, while none was added to experiments 1 and 2. Unlabeled ethanol, 0.76% was added to all experiments except 3 and 4. The total microcuries of C14-labeled ethanol or sodium acetate added t o each experiment appear in Table I . Culture Products.-The C14 yields in the CO? formed by each culture were determined according to the procedures described earlier,4 results appearing in Table I . The CI4 contents of the celluloses (purified by the procedure described earlier4) nere determined in two ways. The CI4 in the celluloses from cultures 1 to 4 , inclusive, was determined from a count of BaC03 obtained from the cellulose by a wet ashing p r o c e d ~ r e using ,~ Suclear Instrument and Chemical Corporation's Q-gas Flow Counter, Model D16A, in conjunction with Model 163 Scaler. Following the usual purification procedures the celluloses from cultures 5 to 7 inclusive were hydrolyzed, and the D-glucose purified by the method of Whistler and DursoB and by several recrystallizations before assay for C14,using a vibrating reed electrometer which is more sensitive than the measuring instrument utilized in experiments 1 to Fj, Table I.
Results From the data presented in Table I it can be seen that little of the C14 originally furnished in the Cellulose Production by Acetobacter xylinum from labeled acetate or ethanol was to be found in the Unlabeled Glucose and C14-Acetateand C14- Ethanol resultant cellulose. The possibility that the traces of activity found in the cellulose from the earlier BY GLENNA. GREATHOUSE, HAROLD G. SHIRK AND FRANCIS experiments (cultures 1-4 inclusive) represented W. MINOR non-cellulosic impurities was tested by using a more RECEIVEDMAY10, 1954 elaborate purification procedure and the vibrating The significant enhancement of cellulose yield by reed as the measuring instrument in experiments 5 , A . xylinum brought about by the addition of TABLE I ethanol to a medium otherwise suitable for pellicle Total productionlJ is indicative that this 2-carbon C'4 in Cli alcohol can be an important ingredient in bacterial Ex; CUIfound C14 found periture, in,COn, cellulose production: A similar response and Labeled micromicro- in,cellulose, ment substrate curies curies no. microcurieq effect have been observed when acetate is added Ethanol-1-C l 4 14 14 to the same type of m e d i ~ m . ~ Despite the in1 0.005 creased yield of bacterial cellulose from a glucose 2 Ethanol-2-CI4 14 13.5 ,04 medium containing either one of these 2-carbon Sodium acetate-l-Cl4 19 14.6 3 .12 compounds, the organism is incapable of producing 4 10.8 Sodium acetate-2-CI4 16 .18 cellulose when either the ethanol or acetate is Ethanol-l-CI4 33 21 None 5 supplied as the sole carbon source.1-2 The avail46 36 Sodium acetate-l-Cl4 ,0003 6 ability of labeled acetate and ethanol now makes 7 Sodium acetate-2-Cl4 79 68 ,006 i t possible to determine how much, if any, carbon (4) F. W . Minor, G. A . Greathouse, H . G. Shirk, A. A I , Schwartz (1) H. L. A. Tarr and H. Hibbert. Can. J . Reseauck, 4, 372 (1930). (2) S. Hestrin, M. Aschner and V. J. Mager, Natuue, 159, 64 (1947). (3) F. W. Minor, G. A. Greathouse, H. G. Shirk, A. M. Schwartz and M. Harris, THIS JOURNAL, 76, 5052 (1954).
and M. Harris, THISJ O U R N A L , 76, 1655 (1964). (5) A. Lindenbaurn, J. Schubert and W. D. Armstrong. A n a l . C h e m . , 20, 1120 (1948). (6) R. L. Whistler and D. F. Durso, THIS JOURNAL, 72, 677 (1950).
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VOl. ’76
KOTES
6 and 7. The reduction in the proportion of radioactivity found in the cellulose from these cultures shows that impurities must have been present in experiments 1 to 4, and accounts for the higher value recorded. As may be seen, the greater proportion of the CI4 in each culture can be accounted for in the COz evolved. The liquid culture products, exclusive of the COZ and cellulose, were radioactive and accounted for the remainder of the activity. These observations indicate that, while both the ethanol and acetate are extensively metabolized by A . xylinum, neither of these 2-carbon compounds, nor any fragments thereof, appeared in the final cellulose product. It is interesting to note in this same respect the report’ that this microorganism cannot synthesize cellulose from glycol, another 2-carbon compound. This information suggests that the use of any 2-carbon metabolic intermediate may be similarly restricted, and further investigation of the mechanism of cellulose synthesis by A . xylinum may well deal with this possibility. The results of the present experiments indicate conclusively that even in the presence of D-glucose, neither ethanol nor acetate contribute carbon to the cellulose synthesis. The enhanced yields of cellulose are therefore caused in some other way. Acknowledgment.-We wish to express our gratitude to the United States Atomic Energy Commission for the grant which supported this research. We are also indebted to Dr. B. M. Tolbert of the University of California for making available samples of ethanol-l-C14 and sodium acetate-2-C14 for some of these experiments, and to Dr. H. S. Isbell and his associates a t the National Bureau of Standards for the vibrating reed measurements. NATIONAL RESEARCH COUNCIL, AND HARRISRESEARCH LABORATORIES WASHINGTON, D. C.
Regularities in the Basicity of Some Tertiary Ethylenediamines, Trimethylenediamines and 2-Hy-
droxytrimethylenediamines BY ALEXANDERGERO RECEIVED MAY8, 1954
Pharmacological studies on certain tertiary amines called for an investigation of their basicities in aqueous solution. The amines were symdisubstituted ethanes X-CH2-CH2-X (I), propanes X-CH2-CH2-CH2-X (11), and 2-propanols X-CH2CHOH-CH2-X (111), the substituents X being diethylamino (A), pyrrolidino (B), piperidino (C) or morpholino (D) groups linked to the ends of the carbon chain by their nitrogen atoms. These number and letter symbols will be used throughout the present paper; thus I I I D is 1,3-dimorpholino-2hydroxypropane 0
