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1 Proc. Nat. Acad. Sci. USA Vol. 68, No. 7, pp , July 1971 Mechanism of Action of Vitamin K: Evidence for the Conversion of a Precursor Protein to Prothrombin in the Rat (phylloquinone/baso4 adsorption/polyacrylamide gel electrophoresis) D. V. SHAH AND J. W. SUTTIE Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wis Communicated by K. P. Link, May 13, 1971 ABSTRACT Previous studies have shown that the vitamin K-induced synthesis of prothrombin in a vitamin K-deficient rat is only slightly inhibited by cycloheximide treatment. Rat prothrombin has now been purified by disc electrophoresis after BaSO4 adsorption and citrate elution. When cycloheximide (5 mg/kg) was given to vitamin K-deficient rats 30 min before vitamin K, about 70% of the amount of prothrombin seen in rats not treated with cycloheximide was produced (two-stage assay), and the prothrombin band could be seen on the electrophoretic gels. However, if radioactive amino acids are administered to the rats after cycloheximide treatment, the newly formed prothrombin contains no radioactivity. The isolated prothrombin does contain radioactivity if the vitamin K-deficient rats are treated with vitamin K but no cycloheximide. When radioactive amino acids were given to deficient rats 1 hr before cycloheximide and vitamin K, radioactivity was found in prothrombin. These data suggest that, in the intact rat, the action of vitamin K is to convert a protein precursor with a short biological half life to prothrombin. The existence of a fat-soluble dietary antihemorrhagic factor was established by Dam in 1935 (1) and subsequently named vitamin K. The only generally accepted function of this vitamin in animals is to regulate the rate of synthesis of the plasma protein, prothrombin, and the other vitamin K- dependent clotting factors (VII, IX, and X). It has previously been shown that the vitamin does not control the release of biologically active, previously formed, prothrombin from the liver (2) and that it is the rate of biosynthesis of prothrombin, not its degradation rate, that is controlled by the vitamin (3). How this synthetic rate is regulated is undetermined. The original postulate (4) that the vitamin exerted its action by controlling the synthesis of the specific messenger RNA for prothrombin synthesis has not been confirmed (5-7). There is, however, conflicting evidence as to whether the vitamin is acting by initiating de novo synthesis of prothrombin, or if it may act by mediating the conversion of some presently undefined precursor protein to the physiologically active form of prothrombin. Li et al. (8), utilizing rat liver perfusion, have obtained data that were interpreted as evidence that the vitamin is initiating de novo synthesis of the protein, and limited data from another system (9) are consistent with this mechanism of action. There are, however, a number of reports (3, 6, 10, 11) suggesting that protein synthesis is not required for the vitamin to express its activity. About half of the normal circulating levels of prothrombin can be restored during the first hour after the administration of vitamin K to hypoprothrombinemic, deficient rats. Suttie (12) has recently shown that this burst of prothrombin production is rather insensitive to the treatment of the animals with an amount of cycloheximide sufficient to block other indications of protein synthesis. Although such data strongly suggest that a protein precursor is being converted to prothrombin under the influence of the vitamin, experiments using inhibitors of protein biosynthesis in intact animals are inherently difficult to interpret. It has also been suggested by Kipfer et al. (13) that cycloheximide and vitamin K are somehow competing for the same site on the ribosomes that are synthesizing prothrombin. This report describes experiments demonstrating that the prothrombin formed in the hypoprothrombinemic rat during cycloheximide and vitamin K treatment does not contain newly-synthesized protein, and must therefore have been converted from a previously existing precursor protein pool. MATERIALS AND METHODS Purification of rat prothrombin Polyacrylamide gel electrophoresis was used to provide microgram quantities of prothrombin for analyses. Oxalated pooled rat plasma was treated with 35 mg/ml of citratewashed BaSO4 while being slowly stirred (14, 15) to adsorb the clotting proteins. The BaSO4 sediments obtained by centrifugation at 1500 X g for 20 min were thoroughly washed by suspending the pellet in one-half the plasma volume of cold 0.1 M K oxalate-0.15 M NaCl. The suspension was centrifuged and the sediment was washed a second time in 0.15 M NaCl. Adsorbed proteins were eluted from the BaSO4 by gently stirring the suspension with 0.17 M, ph 8.0 sodium citrate (0.067 ml per ml of oxalated plasma) for 30 min and then centrifuging. The elution was repeated, and both eluates were pooled and centrifuged again at 2000 x g for 10 min to remove traces of BaSO4. All steps were carried out in a cold room at 0-40C. The pooled citrate eluate from BaSO4 will be referred to as "the eluate". Protein concentrations were measured by a modification (16) of the method of Lowry et al., and prothrombin concentrations were measured by the twostage method of Ware and Seegers as modified by Shapiro and Waugh (17). Disc gel electrophoresis of the eluate was performed with Canalco equipment (Canal Industrial Corp., Bethesda, Md.) in Tris-glycine (18) or in ph 7 Veronal-Tris (19); the gels were stained and fixed in 1.0% Amido black in 7% (v/v) acetic acid for 1 hr at room temperature and electrophoretically destained. Densitometric tracings of the gels were made on a Joyce-Loeble Chromoscan densitometer, and the tracings were quantitated (20). 1653

2 1654 Biochemistry: Shah and Suttie FIG. 1. Disc electrophoretic patterns of 0 ud of BaSO4- citrate eluate (30-35 Aog of protein) obtained from the plasma of normal rats (left) and of purified rat prothrombin. The protein obtained by elution from gel sections of the b bands of normal eluates was again electrophoresed (100,d on gel) after storage at -20'C (middle) and at 4VC (right) for 24 hr. The protein was resolved in a 6 X 65 mm separating gel of 7% acrylamide; Trisglycine buffer, ph ; and a constant current of 3 ma per disc, running time 75 min. The gels were stained and fixed, and densitometry was performed. Treatment of animals Male 200-g rats of the Holtzman strain were housed in coprophagy-preventing cages (21) and fed a diet low in vitamin K (22) for 6-8 days to reduce their plasma prothrombin concentrations to 20 units/ml plasma (normal values are units). To measure the response to vitamin K, we gave phylloquinone (vitamin K1) and drew blood after 1 hr. When the rats were given cycloheximide, 5 mg/kg was administered intraperitoneally 30 min prior to vitamin K, and TABLE 1. Prothrombin Proithrombi Type of in prothrombin Expt. rat Plasma Eluate (dpm) Total radioactivity I Normal Deficient II Normal Deficient There were three to four rats in each group and the amino-acid dose was given in two equal doses 30 min apart. The amino-acid mixture used in Expt. I was "4C-labeled, 50.uCi per rat, given by stomach tube, and in Expt. II, 3H-labeled, 150 uci per rat, given by intraperitoneal injection. Blood was obtained by cardiac puncture 70 min after the first amino-acid dose. A total of 150 JAI of pooled eluate was placed on three gels, which was the equivalent of 1.12 ml of plasma. I 1 i Proc. Nat. Acad. Sci. USA 68 (1971) 'H-labeled or "C-labeled reconstituted amino-acid mixture (Schwarz BioResearch Inc.) was administered intraperitoneally or by stomach tube at times indicated in the tables. Determination of radioactivity Stained acrylamide gels were sliced transversely into 5-mm segments with a razor blade. The gel slices were placed in a counting vial and dried overnight over PAO,. Then 0.3 ml of 30% hydrogen peroxide was added to each vial (23), which was tightly capped and incubated for 6 hr at 50 C. After solubilization, 1.5 ml of an organic base (NCS) was added to each vial, which was agitated for 1 hr at room temperature before the addition of 15 ml of scintillation fluid (0.5% PPO and 0.012% dimethyl POPOP dissolved in toluene). Efficiency of 14C counting was 88-89%; for 'H it was 28-30%. Counting efficiencies of gels containing both labels were 55-58% for 14C and 24-25% for 'H % of the radioactivity in the 50 Iul of eluate loaded in a single gel was recovered by this method. RESULTS Separation of rat prothrombin When ml of pooled oxalated rat plasma ( Iowa units of prothrombin per mg of protein) was treated as described above, about 60% of the prothrombin was recovered in an eluate having a specific activity of Iowa units/mg protein and mg protein per ml. This eluate was stable for at least 1 month if stored in small aliquots at -20 C. Freezing and thawing or storage at 0-4 C results in loss of activity. Fig. 1 shows the result of acrylamide gel electrophoresis of 50 ul of eluate from normal rats. A major band b and a faint following band a and leading band c were seen. No additional protein bands were observed when up to 100,ug of protein was loaded on 6-mm acrylamide gels. It was estimated by the degree of staining that less than 10% of the protein failed to enter the gel. Six unfixed gels were chilled rapidly to -20 C and the a, b, and c bands were cut out, a stained gel being used as a guide. Six blank gel slices of the same size were also obtained. The gel slices were pulverized, extracted for 1 hr in 2-3 vol of 0.17 M, ph 8.0 sodium citrate and filtered from the gel extract through nylon netting. No prothrombin activity was observed in eluates of blank gels or in eluates from the a and c bands. The eluate from the b band contained prothrombin activity; we electrophoresed it again on polyacrylamide gel to study its mobility and resolution. As seen in Fig. 1, this extract gave only a single band corresponding to the b band of the normal eluate, and only one peak was observed on a densitometer trace. The specific activity of the prothrombin eluted from the b band was Iowa units/mg protein. About 55-60% of the prothrombin activity in the BaSO4-citrate eluate that was placed on the gels could be recovered by elution of this band. When the eluates were electrophoresed at ph 7.0, the separation was similar to that of the first system, except that the proteins migrated more slowly in the second system. Effect of vitamin K and cycloheximide on prothrombin production Plasma was obtained from control rats, vitamin K-deficient rats, and deficient rats given vitamin K with or without cycloheximide. BaSO4 eluates were prepared from similar amounts of plasma and electrophoresed as described. The

3 Proc. Nat. Acad. Sci. USA 68 (1971) Mechanism of Action of Vitamin K _ e..t A.<.; A. w. :2ffi,: s_.. \; B FIG. 2. Disc electrophoretic patterns of 50,l of BaSO4c citrate eluate obtained from plasma of A, normal rats; B, vitamin K-deficient rats; C, deficient rats given vitamin K1; and D, deficient rats given cycloheximide and vitamin Ki. The details of the conditions are given in Expt. I, Table 2. stained gels are shown in Fig. 2 and densitometer tracings of these gels in Fig. 3. The prothrombin band b is very faint in K-deficient rats compared to normal rats; it is strongly intensified 1 hr after the administration of vitamin K, to deficient rats. When deficient rats were treated with cycloheximide 30 min before vitamin K,, there was again an increase in the prothrombin band. The b band from the last two gels was eluted and found to contain prothrombin activity. From the densitometer tracings, the amount of prothrombin (b band) on the gels relative to total protein on the gels was determined. These values, for the normal, K-deficient, and phylloquinonetreated deficient rats with or without cycloheximide, were: 75, 35, and 60% of the total protein on the gel present as prothrombin. It was also observed (Figs. 2 and 3) that deficient rat plasma contained a fourth, minor, electrophoretic band, running ahead of the c band, that was not C D FIG. 3. Densitometer traces of the acrylamide gels (A-D) in Fig. 2. The top of the gel is on the right. found in normal rat plasma. The amount of this band was variable from one experiment to another, and did not completely disappear upon vitamin K administration. These data indicate that the increase in two-stage prothrombin units that was previously seen in deficient rats treated with vitamin K and cycloheximide (12) is associated with the actual production of prothrombin, and was not some artifact associated with prothrombin assay in this system. We have previously shown that the vitamin K-dependent synthesis of prothrombin in the hypoprothrombinemic rat is inhibited only 25-30% by an amount of cycloheximide that will decrease the amino-acid incorporation into plasma proteins by 85-95%. To establish that this dose of cycloheximide would also block the biosynthesis of prothrombin, we measured the incorporation of radioactive amino acids into the prothrombin band on the polyacrylamide gels. Rats were given the doses of radioactive amino acids indicated in Tables 1 and TABLE 2. Prothrombin (units/mi) Total radioactivity in prothrombin (Dpm prothrombin)/ Expt. Type of rat Plasma Eluate (dpm) (units of prothrombin) I Deficient + vitamin K, Deficient + cycloheximide + vitamin K II Deficient + vitamin K, Deficient + cycloheximide + vitamin K Vitamin K-deficient rats (3 or 4 per group) were administered a divided dose of an amino-acid mixture at zero time and at 30 min. They were given phylloquinone 10 min after the first amino-acid dose. When cycloheximide was administered, 5 mg/kg was given 20 min prior to the first dose of amino-acid mixture. Blood was drawn by cardiac puncture 60 min after vitamin K was given. In Expt. I, the amino-acid mixture was "C-labeled (50 uci per rat by stomach tube), and 100 pg/kg of vitamin K was given intravenously. In Expt. II, the amino-acid mixture was 'H-labeled (150 pci per rat given intraperitoneally), and 5 mg/kg vitamin K1 was given intramuscularly. The amount of eluate on the gels was the same as in Table 1.

4 1656 Biochemistry: Shah and Suttie Proc. Nat. Acad. Sci. USA 68 (1971) 70 Lo u I iii Ul 60 cycloheximide 60- rn 50-0 X free amino acids protein free amino acids protein Intraperitoneal Stomach Tube FIG. 4. Effect of cycloheximide treatment on the incorporation of 4C-labeled amino acids into rat liver proteins. 4,uCi was administered to control rats and to rats given 5 mg/kg cycloheximide 20 min before the amino acids. The animals were killed 60 min after the administration of the amino acids. The livers were homogenized in saline and the homogenate was precipitated with an equal volume of 10% trichloroacetic acid. The radioactivity found in the precipitate is expressed as protein and that in the supernatant as free amino acids. There were three rats per group; results are means + SE. 2, and BaSO4 eluates were prepared. From 50 to 65% of the total radioactivity loaded on the gels was distributed between the three or four readily observable bands. Table 1 indicates that the incorporation of 4C- or 3Hlabeled amino acids into prothrombin was significantly influenced by the vitamin K status of the animals. The amounts of radioactivity found in the prothrombin band of the polyacrylamide gel prepared from deficient rat plasma was about 10% of that seen in control animals. There was no difference in the amount of radioactive amino acids incorporated into the total plasma proteins in the two groups of rats. The incorporation of radioactivity into prothrombin formed in the presence or absence of cycloheximide during the first hour after the administration of vitamin K to hypoprothrombinemic rats was then determined. We also determined the radioactivity in both the trichloroacetic acidsoluble and the trichloroacetic acid-precipitable portion of a liver homogenate (Fig. 4) to see if the dose of cycloheximide used had any effect on amino-acid absorption or on hepatic pools of free amino acid. These data showed a high incorporation of labeled amino acids into liver proteins in control rats as compared to cycloheximide-treated rats and an increase in the free amino-acid pool after cycloheximide administration. These results provided additional confirmation that the dose of cycloheximide used was sufficient to block general protein synthesis, and established that the treatment did not interfere with the uptake of radioactive amino acids by the liver. The results of the amino-acid incorporation study are shown in Table 2. Although the deficient rats given vitamin K and cycloheximide produced 65-75% as much prothrombin in 1 hr as the rats given the vitamin alone, there was very little incorporation of radioactive amino acid into the prothrombin. These data offer conclusive evidence that the amount of cycloheximide used in these experiments will block the incorporation of amino acids into prothrombin as well as into total liver or plasma proteins. The formation of prothrombin under these conditions therefore strongly suggested that a previously formed protein molecule was converted to prothrombin in the presence of vitamin K. To substantiate this finding, we performed a double-labeled experiment, using ['H]- and [14C]aminoacids. Vitamin K-deficient rats were administered ['H]aminoacids before they were given vitamin K and cycloheximide, and ["4C]aminoacids after this treatment. There was (Table 3) a significant incorporation of ['H]aminoacids into prothrombin in both the control and cycloheximide-treated groups. The decreased incorporation of [PHJaminoacids into the prothrombin from the cycloheximide-treated rats is presumably due to the shorter period of incorporation that resulted from cycloheximide administration 1 hr after the tritiated amino acids. As was seen in Table 2, the administration of cycloheximide again prevented the incorporation of amino acids into the prothrombin produced after the administration of the antibiotic. There was a substantial incorporation of ['Hiaminoacids before cycloheximide and vitamin K treatment, and an insignificant TABLE 3. Incorporation of [3H]- and [14C]aminoacids into prothrombin Total radioactivity in (Dpm prothrombin)/ Prothrombin prothrombin (units of (units/ml) (dpm) prothrombin) 3H: 14C Treatment of deficient rats Plasma Eluate 3H 14C 3H 14C ratio Vitamin K, Cycloheximide + vitamin K, Vitamin K-deficient rats were given 150 jsci of [3H]aminoacids by intraperitoneal injection at zero time, and 50 1ACi of ['4C]aminoacids in two equal doses at 80 min and 110 min. Vitamin K (5 mg/kg) was injected intramuscularly 10 min after, and 5 mg/kg cycloheximide was injected intraperitoneally 20 min prior to the first dose of [14C]aminoacids. Blood was drawn 60 min after the administration of vitamin K1. Both groups had prothrombin concentrations of 10 units/ml before vitamin K administration. The amount of eluate on the gels was the same as in Table 1.

5 Proc. Nat. Acad. Sci. USA 68 (1971) incorporation of subsequently administered ["4C]aminoacids into the prothrombin produced in response to vitamin K. This clearly suggests that a precursor protein is built up before the administration of cycloheximide and that this precursor is converted to active prothrombin by the action of vitamin K. Cycloheximide blocked the incorporation of ['4C]aminoacids, and hence the ratio of 3H: 14C is quite high in cycloheximide-treated rats compared to the control group. Similar experiments have been carried out in rats in which the hypoprothrombinemia was produced by warfarin treatment rather than vitamin K depletion, with essentially the same results. DISCUSSION AND CONCLUSIONS These data suggest that a protein precursor to prothrombin is present in the liver of vitamin K-deficient rats, and that this precursor is converted to prothrombin after administration of the vitamin. Because of the known mechanism of action of cycloheximide (24), the evidence presented here indicates that the conversion does not require ribosomal protein synthesis, but it does not indicate what the nature of the conversion is. These data are not consistent with the report of Li et al. (8) that the vitamin stimulates de novo synthesis of prothrombin. If that were the case, the production of prothrombin in the presence of cycloheximide could be explained only by postulating that for some reason the antibiotic is ineffective in blocking the synthesis of this particular protein. The data on the amino-acid incorporation into prothrombin presented here definitely indicate that this is not the case. Some experiments on intact animals (3, 6, 12) have indicated that there can be some production of prothrombin without protein synthesis, while studies of isolated perfused livers have yielded conflicting results (5, 13, 25, 26). It is significant that the rapid initial burst of prothrombin seen in deficient animals after vitamin K administration (3, 6, 12) is not seen in the liver-perfusion studies, and that cycloheximide does block prothrombin production in vitamin K-deficient rats during the second hour after vitamin administration (12). These observations indicate that the precursor pool may be very short-lived, and that this might not be detected by the experimental design of the studies that have utilized perfused livers. A number of groups (26-30) have previously reported the presence of a new BaSO4-adsorbable protein in the plasma of warfarin-treated or vitamin K-deficient animals. There was a suggestion of this response in the present study. The electrophoretic gels prepared from the eluate of deficient rat plasma consistently contained a fourth, minor, protein band. What relationship this protein has to the possible excretion of a prothrombin precursor or altered prothrombin by the liver is not clear, and at the present time there is little agreement as to the general action of the vitamin (31-33). Mechanism of Action of Vitamin K 1657 We acknowledge the technical assistance of Mrs. Kathleen Nelson in these experiments. This research was supported in part by Grant AM from the National Institutes of Health, U.S. Public Health Service. 1. Dam, H., Vitamins Hormones, 24, 295 (1965). 2. Anderson, G. F., and M. I. Barnhart, Amer. J. Physiol., 206, 929 (1964). 3. Bell, R. G., and J. T. Matschiner, Arch. Biochem. Biophys., 135, 153 (1969). 4. Olson, R. E., Science, 145, 926 (1964). 5. Suttie, J. W., Arch. Biochem. Biophys., 118, 166 (1967). 6. Hill, R. B., S. Gaetani, A. M. Paolucci, P. B. RamaRao, R. Alden, G. S. Ranhotra, D. V. Shah, V. K. Shah, and B. C. Johnson, J. Biol. Chem., 243, 3930 (1968). 7. Lowenthal, J., and E. L. Simmons, Experientia, 23, 421 (1967). 8. Li, L. F., R. K. Kipfer, and R. E. Olson, Arch. Biochem. Biophys., 137, 494 (1970). 9. Suttie, J. W., Fed. Proc., 28, 1696 (1969). 10. Babior, B. M., Biochim. Biophys. Acta, 123, 606 (1966). 11. Babior, B. M., and R. S. Kipnes, Biochemistry, 9, 2564 (1970). 12. Suttie, J. W., Arch. Biochem. Biophys., 141, 571 (1970). 13. Kipfer, R. K., and R. E. Olson, Biochim. Biophys. Acta, 38, 1041 (1970). 14. Goldstein, R.., A. LeBolloc'h, B. Alexander, and E. Zonderman, J. Biol. Chem., 234, 2857 (1959). 15. Li, L. F., and R. E. Olson, J. Biol. Chem., 242, 5611 (1967). 16. Omaya, V. I., and H. Eagle, Proc. Soc. Exp. Biol. Med., 91, 305 (1956). 17. Shapiro, S. S., and D. F. Waugh, Thromb. Diath. Haemorrh., 16, 469 (1966). 18. Davis, B. J., Ann. N.Y. Acad. Sci., 121, 404 (1964). 19. Williams, D. E., and R. A. Reisfeld, Ann. N.Y. Acad. Sci., 121, 373 (1964). 20. Gorovsky, M. A., K. Carlson, and J. L. Rosenbaum, Anal. Biochem., 35, 359 (1970). 21. Metta, V. C., L. Nash, and B. C. Johnson, J. Nutr., 74, 473 (1961). 22. Mameesh, M. S., and B. C. Johnson, Proc. Soc. Exp. Biol. Med., 101, 467 (1959). 23. Tishler, P. V., and C. J. Epstein, Anal. Biochem., 22, 89 (1968). 24. Wettstein, E. 0., H. Noll, and S. Penman, Biochim. Biophys. Acta, 87, 525 (1964). 25. Olson, J. P., L. L. Miller, and S. B. Troup, J. Clin. Invest., 45, 690 (1966). 26. Dodds, W. J., Fed. Proc., 29, 381 (1970). 27. Losito, R., Acta Chem. Scand., 19, 2229 (1965). 28. Hemker, H. C., and A. D. Muller, Thromb. Diath. Haemorrh., 20, 78 (1968). 29. Dulock, M. A., and S. N. Kolman, Thromb. Diath. Haemorrh., 20, 136 (1968). 30. Ranhotra, G. S., and B. C. Johnson, Life Sci., 9, 79 (1970). 31. Suttie, J. W., in The Fat-Soluble Vitamins, ed. H. F. DeLuca and J. W. Suttie (The University of Wisconsin Press, Madison, 1970), p Olson, R. E., in The Fat-Soluble Vitamins, ed. H. D. DeLuca and J. W. Suttie (The University of Wisconsin Press, Madison, 1970), p Johnson, B. C., in The Fat-Soluble Vitamins, ed. H. F. DeLuca and J. W. Suttie (The University of Wisconsin Press, Madison, 1970), p. 491.

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