June 5, 1957
2967
COMMUNICATIONS T O T H E EDITOR THE METHYL GROUP OF METHIONINE AS A SOURCE OF Czs IN ERGOSTEROL'
the residue, representing the ring system. I n the second case this relationship is reversed, as exSir: pected if the main incorporation of radioactivity It was reported recently that formate can serve were in Czs. In experiment 2, the ergosterol, obas a source of Czs in the biosynthesis of ergosterol2 tained from incubation of whole yeast with methioor of eburicoic acid.3 We have incubated cell-free nine-methyl-C14, although recrystallized to conyeast homogenates with labelled sodium bicar- stant specific activity, was subsequently found to bonate, formaldehyde, propionate-( 1 or 2)-C14 be impure. However, the dinitrophenylhydrazone and methionine-meth~1-C'~ and have found that of the a#-dimethyl-butyraldehyde was carefully more C14 is incorporated into the non-saponifiable purified before counting. Further degradation of lipids and the digitonin-precipitable sterols from this aldehyde shows that a predominant portion of methionine than from any of the other compounds. the total radioactivity is located in c28. When the Thus, in one experiment 20.270 of the methionine- correction factor suggested by Ehrensvard, et al.,6 m e t h ~ 1 - C 'radioactivity ~ was found in the non- for the combustion of C14HIsto BaC1403is applied, saponifiable fraction, as compared to 1.2% for the observed activity of is in acceptable agreea ~ e t a t e - 1 - C ' ~1.0% ) for NaHC03, 0.4% for form- ment with the figure calculated from the specific aldehyde and 0.5% for propionate. In compara- activity of the a,P-dimethyl-butyraldehyde. Cartive experiments with the same yeast homogenate bon 28 contains 20-30 times the radioactivity of which we ran after the paper of Danielson and the other carbon atoms in the molecule. Such a Bloch2 came to our attention, methionine-methyl- randomization of radioactivity was also observed C14 gave four times as much radioactivity in the by Dauben, et al., in the incorporation of C14 from digitonin-precipitable sterol fraction as sodium formic acid into the analogous position in eburicoic formate-C14. acid. To determine whether the radioactivity is conThe major incorporation of CI4 into CB of ergoscentrated in CB, samples of ergosterol from incuba- terol in our experiments may be accounted for by tions with acetate-l-C14 and methionine-meth~1-C'~ the well-known oxidation of the methyl group of were ozonized according to Hanahan and SVakil.4 methionine to formate in biological systems.e Comparison of the figures obtained with ergosterol However, transmethylation from sulfur to carbon, made from acetate and from methionine by a yeast which has not been observed up to now, cannot be homogenate (Table I, Expt. 1) shows that in the excluded since we find that aminopterin decreases the incorporation of C14 into ergosterol from forTABLE I mate-C14 but not from methionine-meth~1-C'~. DEGRADATION O F ERGOSTEROL-C14 Other experiments indicate that squalene, but not Source of '2x4, counts/mmole zymosterol, is converted to ergosterol in yeast carbon/min. homogenates. Acetate-1MetbionineExpt.
1"
ZC
Substance
Ergosterol Steam volatile fraction' Residue from steam distillation Ergosterol acetate a,@-Dimethylbutyraldehyde* Acetone' ( C Z ~ , Z ~ , U ) Cza
Cl'
methyl-Cl4
5,540 6,220
3,843 11,480
7,545
655 946
...
(5) G. Ehrensvard, Q. Reio, E. Saluste and R. Stjernholm, J. Biol. Chem., 189, 93 (1951). (6) J. S. Fruton and S. Simmonds, "General Biochemistry," John Wiley and Sons, Inc., New York, N. Y., 1953,p. 714.
WORCESTER FOUNDATIOX FOR GEORGE J. ALEXANDER ALLEN M. GOLD EXPERIMENTAL BIOLOGY SHREWSBURY, MASSACHUSETTS ERWINSCHWENK RECEIVED MARCH16, 1957
...
1,044 165 .. 110 C*ad ... 3,210 a The two samples of ergosterol in Expt. 1 are not directly comparable, since much more C14 was used in the case of acetate. Isolated as dinitrophenylhydrazone. All samples counted after combustion to BaC08, corrected t o infinite thickness. Combustion of CHI, reportedly gives BaCOs of low specific activity.6
...
first case the steam-volatile fraction, a, /%dimethylbutyraldehyde, contains less radioactivity than (1) This investigation was supported by the U. S. Public Health Service Grant No. C321, an Institutional Grant from the American Cancer Society, T h e Schering Corporation, BloomBeld. New Jersey and the Jane Coffin Childs Memorial Fund. (2) H. Danielson and K. Bloch, THIS J O U R N A L , 19, 500 (1957). (3) W.G.Dauben, G. J. Fonken and G. A. Boswell, ibid., '79, 1000 (1967). (4) D. G. Hanahan and S. J. Wakil, ibid., '76, 273 (1953).
DIFFUSION OF
0 ' 8
AND OF PROTIUM IN DZO-HZO MIXTURES'
sir: We wish to report some interesting and unexpected results obtained in this Laboratory in the course of studies on diffusion in liquid systems.2 The data were obtained for 25 =t0.02' by means of the diaphragm cell technique.2 Deuterium analyses were made pycnometrically, and those for 0l8 by the gradient tube density m e t h ~ d ,after ~ first converting the samples of D2018-H2018to H1Ol8. This last was accomplished by vaporizing (1) These investigations were supported in part by the O 5 c e of Ordnance Research. (2) A. W.Adamson and R. R. Irani, Abstracts 130th Mtg., Amer. Chem. Soc., Atlantic City, N. J., September, 1956. (3) A. Hvidt, G. Johansen, K. Linderstrom-Lang and F. Vaslow, Compl. rend. Irav. Lab. Carlsberg, Ser. Chim., 99, No. 9.
COXNUNICATIONS TO THE EDITOR
2968
a few cc. of the mixture to be analyzed, and passing the vapor over hot copper wire; the resulting Cu0l8 was then converted to water and Cu by reaction with hydrogen gas. Since the 0 I 8 data yielded essentially self-diffusion coefficients, these are plotted in the figure as points (reproducibility, 2%). The protium diffusion data were treated as giving integral diffusion coefficients, and the values are given as lines drawn between the average compositions of the upper and lower compartments of the diffusion cell (reproducibility, f 4%).
Vol. 79
SITE OF VANADIUM INHIBITION OF CHOLESTEROL BIOSYNTHESIS
Sir : Vanadium compounds have been shown to inhibit the incorporation of radioacetate into cholesterol by rat and rabbit liver in vitro and in V ~ V O . ' . ~ The following compounds have been generally accepted as intermediates in the biosynthesis of cholesterol from acetate : acetoacetate, B-hydroxy@-methyl glutarate (HMG),4 @-methyl crotonate (BMC),6 squalene,6 lanosterol7 and zymosterol.' Recently P,G-dihydroxy-/3-methylvaleric acid (mevalonic acid)g has been proposed as an intermediate because of its extremely efficient rate of conversion to cholesterol. This communication demonstrates 2.8 that the inhibition by vanadium occurs between the six and five carbon intermediates. 4 2.4 U Rat liver slices were incubated for two hours in % phosphate buffer' containing 10 mg. of sodium ace? tate-1-CI4 (1.0 mc.,/mRI.) or 1 mg. of mevalonic 5 2.0 a ~ i d - 2 - C ' ~(0.005 mc./mR>I.).l0 Aliquots of the !n same batch of slices were used for each experis ment. Vanadium in a final concentration of x 1.6 AI was added to one flask as diamnionium oxytarQ tratovanadate.!O An equimolar amount of tartrate was added to the control flask. Following 12 incubation of the flasks containing acetate sub1 strate, 30 mg. HMG, 40 mg. BMC, 35 mg. squalene, 0 20 40 60 80 100 and 2 mg. of cholesterol were added to each flask as carrier. After saponification in 7 0 7 , ethanol 3' %Dz0. and 5y0 potassium hydroxide under nitrogen for Fig. 1.--Variation of the diffusion coefficient with deu- one hour, the non-saponifiable fraction was exterium enrichment in water mixtures: present work, 0 01* tracted with petroleum ether and from it squalene as tracer, - H~O-DZO;Longsworth, 0 , HzO-DzO; Wang, and cholesterol were isolated by means of an et al., OI8 as tracer, A H~O-DZO, H 3 as tracer. alumina column.6 The radioactivity of squalene was determined as the hexahydrochloride and Considering first the region of low D20 content, cholesterol as the digitonide. The saponifiable there has been a considerable fluctuation in litera- fraction was acidified to pH 2 with concentrated ture values, but our results are in agreement with hydrochloric acid and extracted continuously with those of II'ang, et ~ l . and , ~ of L o n g s ~ o r t hwithin ,~ ether for 24 hours. The ether was evaporated to experimental error. The point of interest, how- dryness and HMG4 and BMC5 isolated and their ever, is that while the 0l8 difl'usion coefficients radioactivity determined. Derivatives of each varied with composition almost exactly as the in- compound retained the calculated amount of verse of the viscosity, the protium diffusion coeffi- radioactivity. cients showed a marked minimum which, if corThe radioactivity (Table I) of B N C . squalene rected for the viscosity effect, fell a t 50% DzO and cholesterol from acetate as substrate was decontent. pressed by vanadium in comparison to the control. The explanation for these findings must be that The increased radioactivity of HbIG in the vanasomewhat different mechanisms are available for dium inhibited reaction might be expected since it oxygen than for protium diffusion. For the is known that inhibition a t any step in a sequence former, there is the possibility of oxygen exchange will produce an accumulation of the intermediate between clusters, and for the latter, there is a just below the inhibition with increased trapping of variety of ways for a similar exchange to occur, the radioactive molecules. The radioactivity of since either long or short protium bonds and either cholesterol obtained with mevalonic acid as suba four or a five coordinated transition state may be strate was also depressed by vanadium. When bioinvolved. I n any event, the results cannot be ex- synthetic CI4-labeled squalene was employed as plained in terms of the usual assumption that dif(1) G. L. Curran, J. Bioi. Chem.. 210, 765 (1954). fusion in water occurs only by motion of individual (2) G. L. Curran and R. L. Costello, J. E x $ . M e d . , 103, 49 ( 1 Y A ) . molecules as units. (3) G. L, Curran, J. Bioi. Chem., 191, 775 (1951).
*
4
i
v
( 3 ) J. L. Rabinowitz and S. Gurin, ibid., 208, 307 (1951). (5) J. L. Rahinowitz and S. Gurin, THIS JOURNAL 76, 51FS (1954). (6) R. G. Langdon and K. Bloch, J . Bioi. Chem., 200, 12'3 ( 1 9 . 3 ) and 200, 135 (1953). DEPARTMEST OF CHEMISTRY ARTHURW. XDAMSON ( 7 ) R. B. Clayton and K. Bloch, ibid.. 218, 319 (1956). (8) J. D. Johnson and K. Bloch, THISJOURNAL, 79, 1145 (1957) OF SOUTHERN CALIFORNIA UNIVERSITY (9) P . A. Tavormina, hi. H. Gibbs and J. W. Huff, i b i d . . 78, 4498 RIYADR. IRANI Los ANGELES 7 , CALIFORNIA (1956). RECEIVED MARCH23, 1957 (10) Generously supplied b y XIerck Sharp and Dohme
(4) J. H. Wang, C. V. Robinson and I. S. Edelman, THISJ O U R N A L , 76, 466 (1953). (5) L G. Longsworth, J . Phys. Chein., 68, 770 (1954).
