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 , ~ of L o n g s ~ o r t hwithin ,~ those of II'ang, et ~ l . and 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).
June 5, 1957
COMMUNICATIONS TO THE EDITOR
2969
TABLE I EFFECTOF VANADIUM O N THE INCORPORATION OF ACETATE1-ci4 AND MEVALONIC ACID-2-Ci4INTO CHOLESTEROL Each flask contained 20 ml. phosphate buffer,’ CI4labeled substrate, 1 g. liver slices, and tartrate (control) or e diammonium oxytartratovanadate (vanadium) in a final concentration of 10-3M. Gas phase was 95% 0 ~ 5 %COZ. Substrate
Radioactivity recoveredo Ex: periConCompound isolated ment trol Vanadium
10 mg. sodium P-Hydroxy-p1 2,908 4,820 acetate-l-CI4 methyl glutaric 2 3,138 6,839 (1.0 mc./ acid mM.) 10 mg. sodium 6-Methylcro1 3,510 482 2 3,920 986 acetate-l-Ci4 tonic acid (1.0 mc./ mM.) 10 mg. sodium Squalene 1 280 92 acetate-1-C’4 2 303 94 (1.0 mc./ mM.) 10 mg. sodium Cholesterol 1 41,248 9,446 acetate-l-CI4 2 51,500 11,575 (1.0 me./ mM.) 1 mg. meva- Cholesterol 1 340 52 388 26 lonic acid-22 C’4 (0.005 mc./mM.) Recovered radioactivity is expressed as counts per minute per infinitely thick layer.
0 20
o 4G
0 30
PI“)
Fig. 1.-Adsorption
isotherms of krypton bromide.
on
sodium
the prescription d In dlI/d In 0 = y ( = 1 for Kr) leads to the isotherm in p(1
- e)/el+Y
=
+
- p ( x - 2~9.8) const. (1)
where /3 = l / k T , and x and w are the energies of adsorption and of lateral interaction as employed by Fowler and G ~ g g e n h e i m . ~As a result of important but compensating differences, our equation with y = 1 is similar to that of Rushbrooke.* With y = 0 it reduces to the Fowler “crude” t h e ~ r y and , ~ with w = 0 in addition, to the Langmuir result. In Fig. 2 we show how the data of Fig. 1 are rectilinearized according to the prescription of Eq. (1). Evidently the intercepts and slopes may be used
substrate,l’ no inhibition by vanadium was found between squalene and cholesterol. These data are interpreted as demonstrating that vanadium inhibits cholesterol biosynthesis between HMG and BMC. Vanadium also inhibits the conversion of mevalonic acid to cholesterol. l 2 (11) F. Dituri, F. A. Cobey. J. V. B. Warms and S. Gurin, J. B i d Chcm., 221, 181 (1956). (12) Supported in part by U.S.P.H.S. Grant H-1947C-2. (13) U.S.P.H.S. Research Fellow of the National Heart Institute. (14) Established Investigator of the American Heart Associatlon.
DEPARTMENT OF INTERNAL MEDICINE DANIELL. A Z A R X O F F ~ ~ UNIVERSITY O F KANSAS MEDICAL CENTER KANSASCITY, KANSAS GEORGEL. CURRAN“ M A Y 6, 1957 RECEIVED
TWO-DIMENSIONAL TRANSITIONS IN ADSORBED MONOLAYERS
Sir: Because of the obvious interest’s2 in theoreticallysimple adsorption systems which exhibit transitions, we are moved to make this preliminary report on our measurements for krypton adsorbed on sodium bromide, Fig. 1. Localized adsorption with nearest-neighbor interactions which, in addition to their usual effects, alter the lateral frequency W I I of the adsorbed molecule with increasing coverage 8 according to (1) S. Ross and G . E. Boyd, “New Observations on Two-Dimensional Condensation Phenomena,” M D D C Report, 864 (1948); S. Ross, THIS JOURNAL 70, 3830 (1948); S.Ross and H. Clark, ibid., 76, 6081 (1953); 76, 4291, 4297 (1954); S. Ross and W. Winkler, i b i d . , 7 8 , 21337 (1854);J . Coli. Sci., 10, 319; 330 (1955). (2) H. Edelhoch and H . S. Taylor, J . P h y s . Chcm., 68, 344 (1954).
e Fig. Z.-Adsorption isotherms of krypton on sodium bromide, plotted according to Eq. (l), y = 1.
to evaluate x and w,both of which are in reasonable agreement with independent theoretical calculations. The authors wish to thank the Institute of Geophysics, University of California a t Los Angeles, (3) R. H . Fowler and E. A. Guggenheim, “Statistical Thsrmodynamics,” Cambridge University Press, 1939,p. 1007. (4) G.S. Rushbrooke. Proc. Camb. Phil. SOL., 3 4 , 424 (1938).
