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COMMUNICATIONS TO THE EDITOR
It is of interest that two distinct and metabolically separated steps of a biosynthetic sequence are catalyzed by one enzyme and are under the control of a common genetic unit. (7) Predoctoral Fellow of the National Science Foundation.
DIVISIONOF BIOCHEMISTRY RICHARD w'. MILLER' DEPARTMENT OF BIOLOGY LEWISN. LWKENS' MASSACHUSETTS INSTITUTE OF TECHNOLOGY CA4M13RIDGE39, MASSACHUSETTS JOHN M. BUCHANAS RECEIVED FEBRUARY 16, 1957
SYNTHESIS OF DIPHOSPHOPYRIDINE NUCLEOTIDE FROM NICOTINIC ACID BY HUMAN ERYTHROCYTES I N VITRO1
Sir: In 1943,2 in vitro synthesis of pyridine nucleotides from nicotinic acid (NA)3by human erythrocytes was demonstrated under conditions wherein no comparable synthesis was obtained with nicotinamide (NAm). Erythrocytes were shown to be freely permeable to both compounds; however, the microbiological assay employed was non-specific and the synthesized material might equally well have been nicotinamide mononucleotide (NMN), DPN, T P N ?or nicotinamide riboside. Later i t was observed that, in the presence of very high concentrations of NAm, pyridine nucleotides were synthesized of which 75-95y0 was NMN and the remainder DPN.4 Preiss and Handler5 have shown NMK formation in this system to occur by condensation of NAm with 1-pyrophosphoryl ribose-5phosphate. However, since extremely high and non-physiological concentrations of NAm were required for this reaction and since no DPN-pyrophosphorylase has been detected in human erythroc y t e ~ ,NMN ~ , ~ may not be an intermediate in DPN synthesis in the human erythrocyte. In consequence, it appeared desirable to reinvestigate the reported synthesis of pyridine nucleotides from NA by erythrocytes and establish the nature of the synthesized material. Table I shows that a t low concentration of NA there was appreciable synthesis of pyridine nucleotide, all of which was accounted for as DPN by the alcohol dehydrogenase assay, whereas hThn a t similar concentration did not elevate the pyridine nucleotide level significantly. Only a t higher concentrations was NAm an effective precursor for DPN synthesis. A t a concentration sufficiently great to permit significant DPN synthesis, N M N accumulated in almost equal quantity, while a t still higher NA4m concentration, NMN synthesis was dominant. I n contrast, NMN synthesis has not been observed a t any concentration of NA. (1) These studies were supported in part by contract AT-(40-1)-289 between Duke University and the United States Atomic Energy Commission and by Grant RG-91 from t h e National Institutes of Health. (2) P. Handler and H. I. Kohn, J . B i d . Chem., 150, 447 (1943). (3) These abbreviations a r e used: N A , nicotinic acid; NAm, nicotinamide; GAm, glutamine: N M N , nicotinamide mononucleotide; D P N . diphosphopyridine niicleotide; T P N , triphosphopyridine nucleotide: T R I S , trishydroxymethylaminomethane. (4) I. G . Leder and P. Handler, J . B i d . Chcm., 189, 889 (1951). ( 5 ) J. Preiss a n d P. Handler, Abstracts of the 130th National Meetng of t h e American Chemical Society (1956), p. 44c; J . B i d . C h r n . , in press. (0) A. hfalkin and 0 1'. I>enstedt, C'anudian J . Riorhem. Pkysiol., 34, 121 (1956).
VOI. 79
Indeed, in most experiments in which DPN synthesis was observed from NA, the NMN of the erythrocyte, which usually accounts for about 50% of the total pyridine nucleotides, disappeared. With both substrates, virtually all of the total nucleotide synthesized, as measured by the fluorimetric assay, as accountable as NMN and/or DPN. TABLE I PYRIDINE NUCLEOTIDE FORMATION FROM SICOTINIC ACID AND NICOTINAMIDE BY HUMAN ERYTHROCYTES The reaction mixture contained: 50 pmoles phosphate pII 7.4, 22.5 mg. glucose, defibrinated whole blood 3.0 ml Na4, S A m , and glutamine in the amounts indicated. Total volume W L S 3.5 ml ; incubation time 22 hours. I
Additions pmoles
@moles
NA
0.3
NA
0.3 1.0 1.0 10.0 10.0 100 .0 0.3 1.3 1.0 1.0 30.0 10.0 100.0 300 . 0
NA
NA NA NA h7A NAm NAm IGAm NAm NAm NAm NAm NAm
ATotal Pyridine nucleotidea @moles
ADPNb pmoles
A N A4 ?: pmoles
0.045 .201 .OB0 .222 ,061 126 .OR3
0.041 .211 ,049 .195 .0*;9 ,123 ,139
... ...
,016
.ooo
.01,5
.000
... ...
.I119
. 000
...
GAni 20 GXm 20
GAm 20
G.Sm 20 GrZm 20
GAm 20 GAm 20
... ...
... ...
,
oon
...
,111
,
(1413
...
,036 ,323 .385
... 0.208 .010
1.13
,
. . I
O(10
,068 ,5587
C
Systems lacking NAm and NA contained 0.103 pmole. This value was subtracted from the observed value, yielding the increment shown. Increment over the control value of 0.089 pmole. e Assayed with alcohol dehydrogenase after aliquot was treated with D P N pyrophosphorylase and ATP. N M S was calculated as the increment in DPK due to this treatment. The control contained 0.093 pmole S h f N . a
It is evident from these data that free NAni cannot be an intermediate in DPN synthesis from NA, suggesting that amidation may occur after nicotinic acid is converted to some presently unknown nucleotide derivative. Several explanations might be offered to account for DPN synthesis a t high YAin concentration, but further work is necessary to establish the mechanism of this process. Table I1 shows that DPN synthesis from NA is dependent on phosphate, glucose, and ammonia which may be supplied as glutamine. Asparagine TABLE I1 REQUIREMENTS FOR DPN SYNTHESIS BY ERYTHROCYTES The complete reaction mixture contained 10 pmoles Ka4, 10 pmoles GAm, 50 pmoles phosphate pH 7.4, 22.5 mg. glucose, 20 pmoles Mg++,defibrinated blood 3.0 ml., 0.9% NaCl t o 4.74 rnl., incubation time 21 hours. Omissions
None
NA TRIS instead of phosphate Glucow Mgi-+ GAm NH4+instead of C7.1ni ..isparagiiie instead ul GXni
Final D P N , pmole
ADPN, pmole
0.285 ,137 ,153 ,180 ,294 ,182 276 .199
0.148 ,000 ,016 ,043 ,157 ,045 ,139 ,062
,March 20, 19.57
COMMUNICATIONS TO THE EDITOR
showed almost no activity as the amide donor. Under the conditions of the experiment shown in Table 11, glutamine supply limits DPN synthesis. Thus, in a separate experiment under similar conditions, with NA held constant a t 10 pmoles per vessel, DPN synthesis in the presence of 0, 4, 10 and 20 pmoles of glutamine was 0.052, 0.104, 0.172 and 0.274 pmole, respectively. Further investigations are in progress seeking t o elucidate the mechanism of pyridine nucleotide synthesis from nicotinic acid and its amide.
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(7) Predoctoral Fellow of the National Institute9 of Health.
DEPARTMENT OF BIOCHEMISTRY DUKEUKIVERSITY SCHOOL OF MEDICINE J. PREISS’ DURHAM, NORTHCAROLINA PHILIPHANDLER 11, 1957 RECEIVED FEBRUARY pJ
THE ISOTROPIC LENGTH OF POLYMER NETWORKS
01.
Fig. 1.-Plot of ratio of isotropic length after cross-linking
Li t o initial length LOagainst the square root of the fraction Sir: A general theory of the elastic properties of of the units crosslinked p ’ l z . polymer networks was developed in a recent paper’ linking density decreases deviations from linearity and this theory was applied t o the cross-linking of occur and Li/Lo appears to approach unity. Achighly oriented chains. Whereas for a network cording to equation (38) of ref. 1, Li/Lo should formed in the usual way by cross-linking chain vary directly as p ’ l P for chains with perfect axial molecules in random arrangement the isotropic orientation, and for an infinitesimal amount of length Li of the network (;.e., its length under no cross-linking Li should shrink to zero. This bestress) must obviously be independent of the de- havior is indicated by the linear portion of the gree of cross-linking, it was shown t h a t for a net- curve and its extrapolation to the origin. Since work formed by the random cross-linking of highly the chains prior t o network formation are neither oriented chains Li should increase directly as the completely nor perfectly oriented, deviations from square root of the fraction p of the units cross- linearity would be expected a t low cross-linking linked. Although i t has been reported that the densities where L i should tend to remain constant cross linking of stretched rubber results in an in- as observed. The slope of the linear portion of the crease in its isotropic (zero stress) length,*v3 curve is fifteen while theoretically it is estimated to adequate data are not available to test the afore- be about ten. It appears that “racked rubber” mentioned deduction. We wish to report the re- can serve as a good model for the physical behavior sults of studies of the isotropic length of natural of the fibrous proteins. rubber networks formed from chains in a highly Further details of the experimental methods, a oriented state. These results give strong support more thorough discussion of these results as well to the theoretical conclusions. as a comparison of the isotropic melting temperaThe highly oriented state of the rubber, prior t o ture and swelling behavior of different type netcross-linking is obtained by modification of the works will appear in a forthcoming paper.6 “racking process” originally described by F e ~ c h t e r . ~ (6) D . E. Roberts and L. Mandelkern, in preparation. The wide angle X-ray pattern6 indicates that the DOKALD E. ROBERTS BUREAU OF STANDARDS specimen is in a highly oriented state and the ratio NATIONAL 25, D. C. LEOMANDELKERN of the extended length to retracted length is about WASHINGTON BAKERLABORATORY OF CHEMISTRY eleven. The samples were cross-linked by subject- CORNELL UNIVERSITY PAULJ. FLORY ing them to y-ray irradiation from a Co60 source. ITHACA, N. ‘17. The efficiency of cross-linking in the highly oriented RECEIVED JANUARY 28, 1957 racked’ rubber was found to be twice that for unoriented rubber. HORMONES AND RELATED COMPOUNDS. I n Fig. 1 the ratio of Ll to the initial length Lo ADRENAL V. FLUORINATED &METHYL STEROIDS is plotted against p a l z . A fiftyfold range in crosslinking is encompassed by these experiments and S i r : We recently have reported’ the preparation of a the isotropic length increases by a factor of two and a half. At the higher degrees of cross-linking the number of 6-methylated analogs of adrenal hordata are well represented by a straight line which mones which show unusual potentiation of glucoextrapolates to the origin. However, a s the cross- corticoid activity with no sodium-retaining properties. The group of Sa-fluoro- and 21-fluoro-6(1) P.J. Flory, THISJOURNAL, 78, 5222 (1956). methyl steroids reported herein represents a con(2) R. D. Andrews, E. E. Hanson and A. V. Tobolsky, J . A p p l . Phrs., 17, 352 (1946). tinuation of this work. Compound 111 described (3) J. P. Berry, J. Scanlan and W. F. Watson, Trans. Faraday SOC. below is by far the most potent glucocorticoid re62, 1137 (1956). ported to date. (4) H . Feuchter, Karrtschrrk, Dec.. p. 6 (1925); pp. 8, 28 (1928). ( 5 ) C. C. Davis and J. T. Blake, “The Chemistry and Technology of Rubber,” Reinhold Publishing Corporation, New York, N . Y.. 1937, p. 78.
(1) G. B. Spero, J. L. Thompson, R. J. Magerlein, A. R. Hanze, H. C. Murray, 0. K. Sebek and J. A. Hogg, TEIS JOURNAL,78, 6213 (195G).
