Studies on the mode of action of vitamin K

Studies on the mode of action of vitamin K

STUDIES ON THE MODE OF ACTION OF VITAMIN K* ROBERT E. OLSON Department of Biochemistry,St. Louis UniversitySchoolof~Medicine, St. Louis, Missouri INTR...

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STUDIES ON THE MODE OF ACTION OF VITAMIN K* ROBERT E. OLSON Department of Biochemistry,St. Louis UniversitySchoolof~Medicine, St. Louis, Missouri INTRODUCTION Tim regulation of cell metabolism involves an interplay of numerous controls operating at three general levels: (a) modification of the activity of existing enzymes; (b) modification of the permeability and/or function of the cell and organelle membranes; and (c) modification of the number and/or concentration of cellular enzymes via the genetic mechanisms of induction and repression. An attempt to explain the action of the fat-soluble vitamins in terms of cofaetor formation has been uniformly unsuccessful. Nonetheless, it has clearly been established that, in certain fat-soluble vitamin deficiency diseases, the concentration of proteins or enzymes in the blood or various tissues may change markedly. It has long been recognized that liver xanthine oxidase in both rabbits tl) and rats ¢2) is elevated by vitamin E deficiency, as is a group of enzymes associated with lysosomes.~3) The sulfate-activating enzymes needed for the formation of 3'-phosphoadenosine-5'-phosphosulfate have been reported to be decreased markedly in vitamin A deficiency in the rat. c4) In vitamin D deficiency, a carrier protein for calcium transport in the intestinal mucosa appears markedly reduced in activity. The effect of vitamin K deficiency in lowering the concentration of prothrombin and related dotting proteins in plasma is perhaps the best known of the relationships between fatsoluble vitamin nutrition and protein concentration. Martius and his colleagues ¢5) attempted to explain the effect of vitamin K deficiency upon prothrombin formation in terms of a general failure of protein synthesis due to defective generation of ATP. He argued that the short half-life of prothrombin (8 hr) would make it particularly vulnerable to a decrease in the rate of ATP generation. The idea that uncoupling of oxidative phosphorylation occurs in vitamin K deficient or dicumarol-treated rats and chicks has not been confirmed,c6.7) Studies in our own laboratory have shown no fall in ATP concentration in the livers of vitamin K-deficient chicks. ¢s) * Supported in part by a grant-in-aid from the U.S.P.H.S. AM-09992from the National Institute of Arthritis and Metabolic Disease, Bethesda 14, Md. 181



Attempts to demonstrate the presence of vitamin K in the prothrombin molecule have also been fruitless. Likewise, reactivation of clotting by adding vitamin K in vitro to blood from vitamin K-deficient animals has not been observed. Similar data exist for systems affected by other fat-soluble vitamins. It occurred to us that these changes in the concentration of enzymes and biologically active proteins (the clotting proteins are actually proenzymes) induced by the fat-soluble vitamins might reflect the control of protein synthesis exercised by these lipids at the genetic level; i.e. at the level of DNAdependent RNA synthesis. We have therefore undertaken to test the hypothesis that the fat-soluble vitamins act as effector molecules in the model proposed by Jacob and Monod. ¢9) The experiments to be reported deal with a study of the possible action of vitamin K at the genetic level in stimulating prothrombin synthesis. This vitamin was chosen for study because of the ease with which the deficiency can be produced in young chicks, the rapidity of the response to vitamin K, and the simplicity of the one-stage assay for plasma prothrombin. A preliminary report of these experiments has been made. °°) MATERIALS AND METHODS One-day-old chicks were raised on a vitamin K-deficient diet containing soy protein ¢~J for 14 days, at which time they weighed roughly 120 g and showed prolonged clotting times as measured by the method of Lee and W h i t e O2) and one stage prothrombin times as measured by the method of Quick and Grossman. ¢13) This method actually measures the activity of 3 vitamin K-dependent clotting factors as indicated in Fig. 1 which shows the interrelationships of the clotting proteins. Recent studies¢x4) suggest that the clotting factors are all proenzymes which undergo stepwise conversion to their respective enzymes by partial proteolysis. Through the cascade effect presented in Fig. 1 a small stimulus gains first amplification which first leads to polymerization of the fibrinogen core peptide to produce fibrin strands. In the one stage assay thromboplastin (desiccated rabbit brain) converts proconvertin to its accelerator form which initiates the extrinsic side of the system. The three K-dependent factors which may limit the rate of clotting in this assay are proconvertin (Factor VII), Stuart Factor (X) and prothrombin itself. The experimental design is shown in Fig. 2. Vitamin K deficiency was produced in two ways. Vitamin K deficiency was produced in chicks by feeding a vitamin K-deficient diet slightly modified from Griminger. tx~) In some experiments a conditioned vitamin K deficiency was induced by administration of dicumarol. The drug was administered in the diet at the level of 0.05 per cent for a period of 48 hr and the animals bled by heart puncture and clotting and prothrombin times were determined. Curative


Foreign Body Hagernan Factor ( X L I ) S

(Factor IX)~--~""- z /


~'~""'~q /


c 9/ /


{ \ \

Proaccelerin. (Factor V) ~,~,

/ /

\ Tissue Juice ~ (Thromboplastin)



~'~Ac 10 /

~ Prothrombinase (Ae 5) Prothrornbin / (Factor ~} ~ / (M. w. 6s, ooo) _ ~ # : ~ " ~ T h r o r n b i n (Ac 2) (M. W. 35.000) Fibrinogen,~ / (Factor I) ~ /

(M. w. 340,000 )"-,,,~,~.

: ~ F i b r i n (M. W. 330,000n) FIG. I. Mechanism for the coagulation of the blood (modified from Breckenridge and Ratnoff~.~14) The intrinsic scheme is pictured to the left beginningwith the action of an intravascular "foreign body" upon Factor XII and culminating in the conversion of fibrinogen to fibrin. The extrinsic scheme activated during injury is pictured to the right. These two systems converge at Factor X which leads to a final common pathway. The cascade is based upon sequential conversion of proenzymes to enzymes which are protcolytic in nature and remove a peptide in the next proenzyme to activate it. The molecular weights of the proenzymes and corresponding enzymes are indicated where known. Vitamin K is concerned with the synthesis of one procnzyme in the intrinsic scheme, above the branch point (Factor IX), one proenzyme in the extrinsic scheme above the branch point (Factor VII) and two in the final common pathway (Factors II and X).




doses of vitamins K a and K , were administered respectively to chicks and rats to establish normal responses to vitamin K in each species. Two methyl-l, 4 naphthoquinone (vitamin K3) was administered to chicks in doses of 2 g per 100 g body weight by intubation of the crop and the clotting and prothrombin times were measured on blood obtained by heart puncture 6 hr after the administration of the vitamin. In rats in which the conditioned deficiency was induced by dicumarol, vitamin K , was administered in doses of 1 mg (approximately 300 gg per 100 g of rat) the prothrombin and clotting times were also measured 6 hr after administration of the vitamins. Deficiency Period

Therapy Period


Deficiency in chicks | x = 14 Dicumarol (0.05%} in rats x-.- Z

/ J

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-~( Days"


K3 (chicks) KI (iats}


.~'AI_ U_ C 14








6 Hours

Actinornyc in I


Puromycin Schedule

FIo. 2. The experimental design and procedure for the current experiments. A vitamin K homologne was given at zero time. Hours and days to the left of time indicate events preceding the administration and the vitamin and hours to the right indicate events following the administration of the vitamin. Actinomycin was administered in one dose intraperitoneally in propylene glycol at either --4 or --2 hr. Puromycin was administered intraperitoneally in phosphate buffer pH 7.0 in 4 equally divided doses at the times indicated. The isotopic substrates were administered to selected chicks to validate the effect of the inhibitors. Various inhibitors of R N A and protein synthesis were administered in order to determine whether they would alter the normal response to vitamin K in deficient chicks and rats. Actinomycin D in propylene glycol was administered intraperitoneally in doses ranging from 30--800/~g per 100 g of body weight. Specific doses employed in given experiments are indicated for both chicks and rats. Puromycin was administered intraperitoneally at 2-hr intervals in experiments designated to block synthesis at the ribosomal level. The dose of puromycin ranged from 5-40 mg per 100 g of rat in 4 divided doses. To evaluate the effect of actinomycin upon the synthesis of hepatic R N A in these birds, 5/~curies of adenine-8-*4C (9.3 mcuries/mmole) was administered twice to chicks 6 hr and 4 hr before sacrifice. In the groups to be described, actinomycin D in various doses was given 8 hr before sacrifice and



vitamin K, 6 hr before sacrifice. The liverswere quickly removed, chilled, and homogenized with cold 0.5 M perchloric acid, and the homogenate was centrifuged at 800 x g for 30 rain. Adenosine-Y-phosphate was isolated from the supematant by the method of Edmonds and Le Page.(is) The R N A in the washed residue was differentiallyhydrolyzed by the method of Schmidt and Thannhauser (t6) and the resulting adenosine-T- and adenosine-Yphosphates were isolated on Dowex-I formate. The isolatedadenine nucleotides were dissolved in hyamine and counted with diplenyloxazolein toluene with a Packard scintillationspectrometer. This procedure eliminates from consideration terminal adenosine residues incorporated by exchange into sRNA. Furthermore, the specificactivitiesof the two isomeric adenylates were found to be identical in these experiments, indicating that they were derived from the core of newly biosynthesized R N A . Since.adenosine-Yphosphate isolated from the supernatant is in isotopic equilibrium with cell ATP, the specificactivityobserved can be regarded as that of the triphosphate precursor of RNA-bound adenylate. The ratio of specificactivityof product to that of precursor is then employed as a measure of the relativeincorporation of A T P into R N A in the presence and absence of the vitamin. In order to measure the efficacyof the inhibition of protein synthesis by puromycin, 10 #curies of L. phenylalanine-U:4C hydrochloride was injected I hr prior to sacrifice. The liverwas homogenized with 9 vol. of I0 per cent trichloraceticacid and the precipitatescentrifuged. A n aliquot of the supernatant was counted for unincorporated amino acid by drying, dissolving in hyamine and counting in toluene with PPO. The precipitatecontaining the protein precipitatewas dissolved in 10 ml of trichloraceticacid, heated for 15 rain at 90 degrees in a waterbath, centrifuged, and the supernatant discarded. The precipitatewas washed twice with trichloraceticacid, dissolved in I0 ml of 0.4 N N a O H and reprecipitated with trichloraceticacid. The washing was repeated and the protein was then washed 3 times with acetone and dried under nitrogen. The protein was ground into a fine powder and a weighed portion counted by suspending in 20 ml of gel-scintilIator(Cab-o-sel). The specificactivityof the injected L. phenylalanine-U:4C was 2 mlcuries/ mmole. The extent of inhibition of protein synthesis was determined by the per cent of total homogenate activityappearing in the protein precipitate. In the absence of puromycin approximately 90 per cent of the activity in liverhomogenate was found in the protein precipitate. RESULTS

The response of young vitamin K-deficient chicks to an oral dose of 2 ~g of vitamin K 3 is shown in Fig. 3. The histograms present the per cent of 20 to 40 chicks in each group showing the clotting and prothrombin times expressed in arbitrary logarithmic units observed for each treatment. It may



E. O L S O N

be seen that all deficient birds had prothrombin times in excess of 240 sec and 80 per cent had clotting times in excess of 10 min. After 2/ag of vitamin K 3, all birds showed prothrombin times below 240 see and clotting times below 30 rain, respectively. The average prothrombin time was 90 sec and the average clotting time 4 min. The administration of actinomycin D in doses of 800/~g/100 g to chicks 4 hr in advance of vitamin K s established the effect of the vitamin. This effect of actinomycin D at this dosage was not


I00 :~LUS2.~J K3





v -I-



I00 'PLUS 2pg Ks


~J n 50



~i 6





x~ 3o



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FIG. 3. Effect of vitamin K3 and vitamin K3 plus actinomycin D upon the clotting and prothrombin times of vitamin K-deficient chicks. The histograms represent distribution curves for both prothrombin and clotting times under the conditions specified. The times are plotted on a logarithmic scale. The dose of vitamin K3 was 2gg/100 g chick and the dose of actinomycin was 800 pg/lO0 g chick given intraperitoneally; 20--40chicks were used in each group. Actinomycin was given 4 hr before vitamin K3 and all chicks were bled by heart puncture 6 hr after vitamin K3.

seen in normal animals nor animals pretreated with vitamin Ka. Figure 4 shows the prothrombin and clotting times of such chicks with and without actinomycin. It would appear that under the conditions of these experiments, the effect of actinomycin D upon preexisting prothrombin concentration cannot be detected with the methods employed. When 800 #g/100 g of actinomycin D was administered to vitamin Kdeficient chicks 2 hr before the administration of vitamin K 3 (2 pg/100 g) the action of the vitamin was greatly reduced but not abolished. The doseresponse curves for actinomycin D given 2 and 4 hr before vitamin K a are


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30NTROL: Img 1(31 koj. DIET


~, ~

CI_OTTINO TIMIr IN M|NLffE$ FIO. 4. Effect of vitamin K3 and vitamin K3 plus actinomycin D upon the clotting and prothrombin times of control chicks. These chicks were fed the basal diet plus 1 mg of vitamin K3 per kg of diet and showed normal clotting and prothrombin times. i o o ACTINOMYCIN 4 ° PRIOR K 3 • ACTIN


u N

~A m_z ~m


,6o ' ~ o 5bo ~o OOSE OF ACTI.O.',CIN IN pg/,OOg. Fie. 5. Dose-response curve for the inhibition of prothrombin synthesis by actinomycin when administered at different times before vitamin K3 in vitamin K-deficient chicks. The percentage of chicks given 2 pg vitamin K3/100 g chick showing no change from their deficient state (prothrombin times > 2 4 0 sec) is plotted on the ordinate. Legend: O , actinomycin at 4 ° before vitamin K3; tq, actinomycin at 2 ° before vitamin K3. 187

0 a. 20-

I -m

i 1.0-


o w Q.







FIG. 6. The relationship between R N A synthesis and clotting defect in vitamin K-deficient chicks given 2 pg/lO0 g chick postoperatively preceded 2 hr earlier by various doses of actinomycin D. R N A was labeled with adeninc-8-14C and clotting defect plotted as the percentage of chicks showing no change from deficient prothrombin time 6 hr after the administration of vitamin K3. ~IAZED

I00- ~'LUS I mg. K I




l¢1 ~

o i-z c.)









I00 PLUS I mg. KI

I mg. K I I00 PLUS PLUS 60 ,l~J. ~,CTINOMYClN



~, ~b ~o


~, ib~o


FIG. 7. Effect of vitamin K l and vitamin K1 plus actinomycin D upon the clotting and prothrombin times of adult rats previously administered dicumarol. The animals had received 0.05 per cent of dietary dicumarol for 48 hr prior to J~he experiment. The conventions are the same as in Fig. 3; the vitamin K1 was administered in sesame oil per os and the actinomycin was given intraperitoneally in propylene glycol 4 h r prior to the Kl. 188



oA w~E --Z

. ~40" Zl--









DOSE OF ACTINOMYClNIN #(J/IOOtJ. F]O. 8. Do~-~sponse curve for the inhibition of prothrombin synth~s by actinomycin in rats previously given dicumarol. The conventions are the same as Fig. 5. The inhibitor was administered 4 hr prior to vitamin K].

Ib u


.~_ ~ I=-






"r= ~


plus Dicumarol

.s'o ¢.o Dose of Vitamin K3 in pg./100g.


FIo. 9. Dose-response curve for the action of vitamin K3 in the vitamin Kdeficient chick in the presence and absence of prior dicumarol. The percentage of chicks showing a response (prothrombin times less than 240 sec) is plotted against the dosage of vitamin Ks in #g/100 g of bird. Legend: response in vitamin K-deficient chicks; O response in vitamin K-deficient chicks who had received 3 mg dicumarol/100 g 16 hr prior to the vitamin,




PLUS rm'::l 2)JqK3 ~


~o ' 85 ' 240

~o '

d5 '







~ ~b ~

i ~ ~b ~0 I S IO 3 0 CLOTTING TIME IN MINUTES FIe. 10. Effect of vitamin K3 and vitamin K3 plus puromycin upon the clotting and prothrombin times of vitamin K-deficient chicks. The conventions are the same as in Fig. 3. The dose o f vitamin K3 was 2/tg/100 g chick and the dose o f puromycin was 20 mg total given intraperitoneally in 4-5 mg doses at 2-hr intervals commencing 2 hr prior to the oral administration of the vitamin.


I0~ ¢J ~o


~.~ 60 ~.)IM. 0D O:E

n ~ ~ p - 20


~o 2'o ~




'~ 80 (~





tb zb




FIG. 11. Dose--response curve for the inhibition of prothrombin synthesis by puromycin in vitamin K-deficient chicks (left) and the corresponding degree in the inhibition of total protein synthesis in the liver (right). The percentage of total radioactivity of the liver homogenate found in trichloracetic acid insoluble fraction was used as a relative measure of protein synthesis. The phenylalanine-U-14C in an amount o f 10 pcuries per 120 g of chick was given 1 hr prior to sacrifice.


19 1

shown in Fig. 5. The chick appears to be considerably more resistant to actinomycin than the rat. This impression was confirmed by studying the effect of various doses of actinomycin upon RNA synthesis in the chick. The effect of actinomycin D upon RNA synthesis and dotting in groups of chicks is shown in Fig. 6. The clotting defect induced by actinomycin D is expressed in terms of per cent of chicks showing prothrombin times in excess of 240 sec; i.e. no change from the control. It is clear from these data OPERON 0






s SS


"OIX~lOOi~ PI ~





i/ I



RIP. nK ,~ RF~ Kn + n=O,.~1 ~ Kn On= Fie. 12. General scheme for the regulation of protein synthesis (modified from Jacob and Monod).(9) Legend: GR = regulatory gene; O = operator; S G b SG2 = structural genes; ml, m2, mr = messenger RNAs, transcribed from S U b SG2, GR, D N A by base pairing; aa = mixture of aminoacyl sRNAs available for protein synthesis; P1, Pz = proteins made by ml, mz; R P = represser protein made by mr; R P : K n = represser allosterically altered by combination with vitamin K to become a nonrepressor; P P . K n • Dn = P P . Kn, a nonrepresser altered allosterically by dicumarol (D) to become a represser.

that doses of actinomycin which inhibit RNA synthesis also inhibited prothrombin synthesis in a parallel manner. This relationship is even more striking in chicks given actinomycin 4 hr before vitamin K. Studies of this same phenomena in rats treated with dicumarol give similar results. Figure 6 shows the prolonged clotting and prothrombin times in rats given dietary dicumarol at the 0.05 per cent level for 48 hr. This clotting defect could be corrected in 6 hr by administration of 1 mg of vitamin KI to each rat by mouth. Administration of actinomycin D in doses from 30-240 ~g/100 g of body weight 4 hr before vitamin K~ modified the action of the



vitamin as shown in Figs. 7 and 8. Actinomycin in doses above 120 ~g/100 g of body weight completely blocked the procoagulation effect of vitamin K1. Below this level the response was dose dependent. If actinomycin was given after the vitamin or given to normal rats, no anticoagulant effect was noted in a period of 6-10 hr. Of particular interest in connection with the mode of competition between dicumarol and vitamin K was a series of experiments in which a doseresponse curve for vitamin K3 was determined in vitamin K-deficient chicks with and without prior dicumarol. As shown in Fig. 9, a curve typical for saturation kinetics was obtained in which the half-maximal dosage to 0.1 ~g/ 100 g bird. If 3 nag of dicumarol were administered 16 hr prior to the vitamin K a an S-shaped shift to the right occurred which raised the half-maximal dosage to 0.7/zg/100 g of bird. The relationship between these 2 curves is analogous to the oxygen dissociation curves of myoglobin and hemoglobin and v vs. S plots of other enzymes subject to aUosteric transition in the presence of a substrate binding to an other-than-active enzymatic site. (~7) The chicks given a total dose of 20 mg of puromycin on the schedule described also showed a marked decrease in clotting as shown in Fig. 10. The clotting defect paralleled the extent to which protein synthesis was depressed as measured by phenylalanine incorporation into liver protein (Fig. 11). DISCUSSION These experiments suggest that vitamin K acts to induce the synthesis of prothrombin (and related clotting proteins) at the genetic level; i.e. the level of DNA-dependent RNA synthesis, most likely by modifying the properties of a represser protein. These tentative conclusions are based on the assumption that the inhibitors of DNA-dependent RNA synthesis (actinomycin D) and protein synthesis at the ribosomal level (puromycin) are functioning in accordance with the actions demonstrated for them in vitro. (1s'19) Based on the model proposed by Jacob and Monod/9) it is postulated that in the absence of vitamin K, a regulatory gene represses the activity of the operon or operons concerned with the elaboration of the coagulation proenzymes which, in various species, may range from one to four. In higher mammals, four vitamin K-dependent proenzymes for clotting are known, viz: prothrombin (Factor II), proconvertin (Factor VII), plasma thromboplastin component (Factor IX) and Stuarts Factor (Factor X) (Fig. 1). As shown in Fig. 11, vitamin K, presumably in its alkylated form, combines with represser protein and changes its conformation. This alteration in structure results in a release or derepression of the operator so that messenger RNA for the clotting proenzymes can be synthesized, and lead, in turn, to the synthesis of the corresponding proteins in the ribosomal system. Despite the relative



resistance of chick liver chromatin to inhibition by actinomycin, there was a correspondence in our experiments between the degree of depression in liver RNA synthesis and the prevention of the action of vitamin K in deficient chicks. In addition, the inhibitory effect of actinomycin D was dependent upon the dose given in both chicks and dicumarolized rats. The dose-response curve of chicks to vitamin K3 after prior administration of dicumarol is of particular interest. The shift in the curve from that of a simple adsorption isotherm to a sigmoid shape after dicumarol as shown in Fig. 8 provides kinetic evidence for an allosteric interaction of dicumarol with a vitamin K-binding protein. It has been known for years that the interaction between vitamin K and dicumarol is consistent with neither a classical competitive nor non-competitive inhibition. (2°'21) Since the action of vitamin K1 is also antagonized by actinomycin D in the rat pretreated with dicumarol, it follows that the site of competition between these two substances must be at the site of transcription of information from DNA codons to RNA codons. Since repressor proteins are postulated to act at this level, the most reasonable working hypothesis is that vitamin K and dicumarol both modify the properties of the repressor protein by combining at allosteric sites. The active site of such a protein would, of course, be the site of combination with DNA. Vitamin K, in this model, is an allosteric inhibitor making the native protein less able to combine with DNA; contrariwise, the coumarin drugs are allosteric activators, making the native protein better able to combine with DNA and block transcription. That the repressor protein has sites for both anti- and procoagulants is also suggested by a recent report of hereditary coumarin drug resistance in a cohort of human subjects who have a normal sensitivity to vitamin K and a normal drug metabolism. (22) This hypothesis also explains the failure of vitamin K to act in hereditary hypothrombinemic states, (23) in which it may be assumed that the structural gene components of the operon are defective. Suggestions have appeared in the literature to the effect that vitamin K may act to cleave giant macromolecules into the several vitamin K-dependent proenzymes for coagulation, or that it may act to release prothrombin from the liver. These possibilities seem unlikely in view of the present experimental work which indicates that the vitamin exerts its influence prior to the completion of the prothrombin molecule. The action of puromycin, which acts as an antimetabolite to tyrosyl-S-RNA at the ribosomal level, in blocking the procoagulation effect of vitamin K in the chick indicates clearly that the prothrombin which appears in the circulation after vitamin K is newly synthesized. Further evidence in favor of neosynthesis come from the studies of Barnhart and Anderson. (24'25) By using a fluorescent antibody against dog prothrombin, these workers demonstrated that liver prothrombin is associated with the microsomes of the parench~rmal cells of dog liver and that dicumarol decreased the amount of prothrombin visible by this technique. Further-



more, they showed that vitamin KI caused a marked increase in the antiprothrombin fluorescence of microsomes in increased numbers of parenchymal cells prior to release of prothrombin into the blood. Finally, Goswami and Munro tz6) found that the prothrombin content of microsomes from rat liver was increased by incubation in vitro and that this increase was blocked by ribonuclease. It is tempting to generalize this hypothesis to the other fat-soluble vitamins. It is interesting that some genetic disorders in man mimic some of the manifestations of the fat-soluble vitamin deficiency diseases. For examples, one may list vitamin D-resistant rickets, the clotting disorders already alluded to, icthyosis, gestational disorders and muscle dystrophy. As regards the function of vitamin E, we have recentlytz7) observed that ethionine, a wellestablished inhibitor of protein synthesis, reduces the plasma creatine phosphokinase content of vitamin E-deficient rabbits in the prodromal phase of muscular dystrophy as effectively as a-tocopherol. This result suggests that vitamin E may function as a repressor of enzymes which become destructive or redundant in the deficiency state. The chemically related steroid hormones also appear to function, at least in part, at the genetic level to stimulate enzyme synthesis. Ecdysone, the insect maturation hormone, c2s) androgens, estrogens, and the adrenal cortical hormones all appear to act in this manner. Further study of the steroids and the fat-soluble vitamins is needed to determine if trace isoprenoid lipids, as a class, function biologically by controlling protein synthesis at the genetic level. SUMMARY The regulation of cell metabolism involves controls imposed at three general levels: (I) modification of the activity of existing enzymes, (2) modification of the permeability and function of cell and organelle membranes, and (3) the control of the synthesis of enzymes and other proteins of biological significance mediated by genetic mechanisms. Attempts to explain the action of the fat-soluble vitamins in terms of the first two mechanisms cited above have not been successful. In view of the fact, however, that the fat-soluble vitamins as a class do influence the concentrations of enzymes and other proteins in various organisms, it appeared possible that these changes reflected an action at the genetic level. It has been shown that actinomycin D inhibits vitamin K-induced prothrombin formation in chicks deficient in vitamin K. Doses of actinomycin which inhibited the normal response to vitamin K inhibited incorporation of adenine-8-14C into hepatic RNA. These results were consistent with the hypothesis that vitamin K acts by stimulating messenger RNA formation for the synthesis of clotting proteins. Puromycin was also shown to inhibit the response to vitamin K in proportion to its inhibitory effect on liver protein



synthesis. Since the coumarin drugs antagonize vitamin K, it seemed possible that the site of antagonism was a molecule which regulates this expression of genetic information. To test this hypothesis, rats were fed a diet containing 0.05 per cent dicumarol for 48 hr, at which time they showed prolonged clotting and one-stage prothrombin times. This dotting defect could be corrected in 6 hr by the administration of 1 mg of vitamin K t to each rat by mouth. The administration of actinomycin D in doses from 30-240 #g/100 g body weight 4 hr prior to the vitamin Kt either modified or prevented the action of the vitamin. I f actinomycin was given after the vitamin, or given to normal rats, no anticoagulant effect was noted. The dose-response curve of vitamin K-deficient chicks to vitamin K3 in the presence and absence of prior dicumarol was suggestive of an allosteric interaction. These data are consistent with the view t h a t the site of antagonism between vitamin K and the coumarin drugs is a molecule which controls DNA-dependent R N A replication. ACKNOWLEDGMENTS

The author thanks Clement Stone, Merck Institute for Therapeutic Research, for supplies of actinomycin D; Dr. Samuel Kushner of the Lederle Laboratories for supplies of puromycin; Dr Mary Edmonds for technical advice on the fractionation of adenine nucleotides; and Mrs. Lakeles Dorman, Miss Nancy Fleming, Miss Hanson Tsao, and Mr. Ben Dudley for technical assistance. REFERENCES 1.

J . S . DINNING,An elevated xanthine oxidase in livers of vitamin E-deficient rabbits, J. Biol. Chem. 202, 213-215 (1953).

2. R. E. OLSONand J. S. DrNNr~o, Enzyme abnormalities associated with dietary necrotic liver degeneration in rats, Ann. N. Y. Acad. Sci. 57, 889--895(1954). 3. I . D . Dv..~I, C. C. CALVERT,M. L. SCOTTand A. L. TAPPEL,Peroxidation and lysosomes in nutritional muscular dystrophy of chicks, Proc. Soc. Exptl. Biol. and Med. 115, 462-466 (1964). 4. G. WOLFand B. C. JOHNSON,Vitamin A and mucopolysaccharide biosynthesis, Vitamins and Hormones 18, 439--442 (1960). 5. C. M~,,tTIUSand D. NITz-LITzOW,Oxydativephosphorylierung und vitamin K rnangel, Biochim. Biophys. Acta 13, 152-153 (1954). 6. R. E. BEYSRand E. D. K~I,~,p.SON,Relationship between prothrombin time and oxidative phosphorylation in chick liver mitochondria, Arch. Biochem. Biophys. 84, 63-70 (1959). 7. A.M. PAOLUCCI,P. B. R. RAOand B. C. JOHNSON,Vitamin K deficiencyand oxidative phosphorylation, J. Nutr. 81, 17-22 (1963). 8. M.L. Sgn,a~g, Effect of ethionine on the response of vitamin K-deficient chicks to vitamin K, Thesis M. Sc. (Hyg.), University of Pittsburgh (1964). 9. F. JACOBand J. MONOD,Genetic regulatory mechanism in the synthesis of proteins, J. Mol. Biol. 3, 318-356 (1961). 10. R. E. OLSON,Vitamin K induced prothrombin formation: antagonism by actinomycin D, Science 145, 926-928 (1964).

196 ll. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

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