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Kinetics of carboxylation of endogenous and exogenous substrates by the vitamin K-dependent carboxylase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 230, No. 1, April, pp. 294-299, 1984
Kinetics of Carboxylation of Endogenous and Exogenous by the Vitamin K-Dependent Carboxylase WILLIAM
K. KAPPEL’
AND
ROBERT
Substrates
E. OLSON1*2
Edward A. Daisy Lkpartnzent of Bimh.emistry, St. Lvuis University School of Medicine, St Louis, ikfk~~ri 63104, and Biochemistry Department, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261 Received April
19, 1983, and in revised form September
20, 1983
The vitamin K-dependent carboxylation of the exogenous pentapeptide, Phe-LeuGlu-Glu-Ile, and endogenous liver microsomal protein was studied in solubilized rat liver microsomes. The MnC12 stimulation of the vitamin K-dependent pentapeptide carboxylation rate, which is conducted at subsaturating concentrations of pentapeptide, is due to the cation’s ability to lower the Km of the substrate. Although there are clear kinetic differences observed between the carboxylation rates for the pentapeptide and the endogenous protein substrates, several lines of evidence suggest that the same carboxylase system is responsible for both. These points of evidence are (i) the initial velocity of endogenous protein carboxylation is lowered in the presence of 3 mM pentapeptide; (ii) the presence of endogenous microsomal protein substrate causes an initial lag in pentapeptide carboxylation; and (iii) this initial lag phase is not observed when the total endogenous substrate pool is carboxylated by a preincubation reaction prior to the addition of pentapeptide.
Rat prothrombin maturation involves the post-translational carboxylation of specific glutamic acid (G~u)~ residues to ycarboxyglutamic acid (Gla) residues. The microsomal vitamin KHz-dependent carboxylase responsible for this modification can be solubilized with nonionic detergents such as Triton X-100 (l-3). The carboxylase reaction requires a Glu-peptide substrate, 02, reduced vitamin K, and C02. The trichloroacetic acid-soluble pentapeptides Phe-Leu-Glu-Glu-Val (4) or Phe-LeuGlu-Glu-Ile (5), representing residues 51 Current address: Biochemistry Department, School of Medicine, University of Pittsburgh. zTo whom reprint requests should be addressed. * Abbreviations used: Hepes, 4-(Z-hydroxyethyl)-lpiperazineethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride: DTE, dithioerythritol; TCA, trichloroacetic acid; Glu, glutamic acid, Gla, y-carboxyglutamic acid; STI, soybean trypsin inhibitor. 0003-9861/84 $3.00 Copyright 0 1984by AcademicPress.Inc. All righta of reproductionin any form reserved.
9 of the uncarboxylated bovine and rat prothrombin molecules, respectively, have been used as artificial substrates. The active form of “COZ” is CO2 and not bicarbonate (6, 7). The carboxylation does not require ATP or biotin (6) and the mechanism of reaction is obscure. When compared to the carboxylation rate of the endogenous microsomal protein substrates, the pentapeptide carboxylase activity is more sensitive to the action of various activators and inhibitors (8). In particular, 10 mM MnCl, was shown to stimulate the rat liver pentapeptide carboxylase activity about 2-fold, while having little effect on the endogenous microsomal protein carboxylation rate (9). Uotila and Suttie (10) demonstrated that the adult ox and dicoumarol-treated calf liver vitamin K-dependent carboxylase activities were stimulated 9- and 22-fold, respectively, by 10 mM MnC12 under optimal conditions. 294
KINETICS
OF VITAMIN
K-DEPENDENT
They claimed that Mn2+ only increased the maximum velocity of the adult ox liver carboxylase reaction without any influence on the K,,, of the pentapeptide; however, the data was not shown. The kinetic properties of the adult ox liver enzyme were found to be substantially different from those of the rat liver carboxylase. Some of these differences were the ionic strength optima, the Km for vitamin KH2, the extent of pyridoxal-5’-P activation, and the fold stimulation by Mn2+. Our findings on the mechanism of Mn”+ activation of the solubilized rat liver vitamin K-dependent carboxylase from warfarinized rats are different from those previously reported (9, 10). We also have observed that exogenous pentapeptide competes with endogenous protein substrate for carboxylation by the vitamin Kdependent carboxylase. A preliminary report of this work has been published (11).
CARBOXYLATION
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at 27,000~ for 20 min. The pelleted microsomes were solubilized in 50 mM Hepes, 0.5 M KCl, 250 rnM sucrose, 1 mM PMSF, 50 pg/ml STI, 2% Triton X-100 (pH 7.4) at a ratio of 1.0 ml buffer per initial gram of liver by the aid of 10 strokes in a Teflon homogenizer. All steps were carried out as quickly as possible at O4°C. The solubilized microsomes were stored at -70°C in 1.5-ml plastic-capped centrifuge tubes. Under these conditions the enzyme was stable for at least 6 months. Carblase assays. The standard endogenous protein carboxylation assay was conducted in 13 X lOOmm tubes at 20°C in a total volume of 0.3 ml containing solubilized microsomes (0.1 ml), 24 mM vitamin KHZ (0.01 ml), NaI-I”COs (0.02 ml), 0.3 M DTE (0.005 ml), and any test substances. The solution was made to 0.3 ml with 0.1 M Hepes (pH 7.4). The reaction was stopped with 8 ml of 10% TCA (0°C) and kept at 0°C for 15 min. Protein was collected by centrifugation and washed once with 5 ml 10% TCA. The tubes were stored overnight under vacuum at room temperature to remove residual 14COZ.Protosol(l.0 ml) was added and the tubes were sealed with four layers of parafllm. The tubes were incubated with constant agitation at 50°C until the protein was completely dissolved (2 h). The solubilized protein was quantitatively transferred to 20-ml plastic scintillation vials with two EXPERIMENTAL PROCEDURES washes of 4 ml of Omnifluor. The samples were made Materials Vitamin K1, 3-(a-acetonylbenzyl)-4-hyto 12 ml with Omnifluor and then counted after 2 h. droxy coumarin (warfarin), Triton X-100, fibrinogen, The standard TCA-soluble peptide carboxylation Echis cm-in&us, and Hepes buffer were bought from was conducted at 18°C in 12 X 75-mm tubes in a total Sigma. The sodium [“Clbicarbonate (55-60 mCi/mol; volume of 0.3 ml containing solubilized microsomes 2 mCi/ml) and ACS were purchased from Amersham. (0.1 ml), 24 mM vitamin KH, (0.01 ml), 20 mM penOmnifluor (gal-pak) and protosol were obtained from tapeptide (0.015 ml), 0.3 M DTE! (0.005 ml), 0.3 M MnCl, New England Nuclear. Scintillation-grade toluene was (0.01 ml), and any test substances. The samples were from Baker. Palladium on activated carbon was purmade to 0.3 ml with 0.1 M Hepes (pH 7.4). The reaction chased from Aldrich. The Sprague-Dawley rats were was stopped after 15 min by the addition of 0.7 ml obtained from Sasco, Inc. All other reagents were of of 10% TCA (O’C) and kept at 0°C for 15 minutes. the highest possible commercial grade. The protein was removed by centrifugation. At this Carboxylase preparation The rat liver microsomes point, either 0.5 ml of the supernatant was transferred were prepared by a modified procedure of Schenkman to a 6-ml scintillation vial and left in an exhaust hood and Cinti (12). Sprague-Dawley rats (150-300 g) were overnight at room temperature to remove residual injected intraperitoneally with sodium warfarin (10 14C02 or the entire supernatant was poured off into mg/kg) and fasted for 18 h in raised-bottom cages 12 X 75-mm tubes. In the later case, COz gas was to reduce coprophagy. A stock warfarin solution (30 bubbled through the supernatant for 5 min and 0.5 mg/ml) was made in 0.15 M NaOH. This was then ml of the gassed sample was then placed in the vials. diluted to 10 mg/ml with 0.1 M sodium phosphate (pH In either case, the samples were counted in 4 ml 7.0) and used for injections. The livers were removed of ACS. from exsanguinated rats and placed in 30 ml of 10 All reactions were initiated by the addition of vimM Hepes, 250 mM sucrose, 1 mM phenylmethylsultamin KHZ, followed by gentle mixing. A control assay was always run in the absence of added vitamin KH, fonyl fluoride (PMSF), 50 pg/ml soybean trypsin inhibitor (STI) (pH 7.4) and weighed, and then lightly and was subtracted from the values obtained in the minced with scissors. The liver was homogenized with presence of added vitamin KH,. The radioactivity insix strokes at medium speed with a Teflon homogecorporated into protein or pentapeptide Gla was stable nizer. Pooled homogenates were centrifuged at 12,OOOg under the above conditions for at least 48 h. Any for 15 min. The supernatant was diluted to 80 ml per deviation from these standard assay conditions will liver with homogenization buffer, and 0.9 ml of 0.8 M be described in the text. CaCl, was added to each 80 ml of protein solution vitamin K preparation The stock 24 mM- vitamin and thoroughly mixed. This solution was centrifuged K solutions were prepared by making a 10.87 mg/ml
296
KAPPEL
AND
OLSON
tivation of the pentapeptide carboxylation is nonadditive and is probably general cation action. These same cations exhibited a reproducible, but much smaller activation (11-W%) of the endogenous protein carboxylation rate. The sodium salts of F-, II, Br-, or Pi were found to have no effect or to be slightly inhibitory in either of the two carboxylation assays and were not studied further. The mechanism responsible for the cation stimulation of the pentapeptide carboxylation was investigated in more detail, and the effect of MnC12 on the K, of the pentapeptide substrate is shown in Fig. 1. The data clearly demonstrate that MnC12 does not effect the V,,, of the reaction, but that it effectively lowers the apparent K, of the pentapeptide threefold. All of the previous pentapeptide carboxylation studies were run at only 1 mM substrate conRESULTS centration due to its expense. Therefore, The carboxylation rate of the pentapep- the observed activating effect of MnC12 on tide substrate by the solubilized vitamin the solubilized rat liver carboxylase apK-dependent carboxylase was found to be pears to be due to an effect on the Km of stimulated from 250 to 300% by 10 mM the enzyme. MgC12 was also found to exMn2+, Mgaf, Ca2+,and S?. The cation ac- hibit identical effects on Km and V,,,.
vitamin K solution in the absolute ethanol. This solution was stored at -15°C. The yellow quinone was reduced to the colorless hydroquinone using palladium on activated carbon (10 mg/ml) by gassing with hydrogen during constant agitation for 2 min. The vitamin KHZ was stored under hydrogen in the dark until needed. The sample was clarified by centrifugation immediately before use. The concentration of vitamin K was determined from its absorbance at 248 nm in absolute ethanol using Ei”z = 419 (13). The molar extinction coefficient was calculated to be 18,885. were deterOther methods. Protein concentrations mined by the method of Bradford (19) using bovine serum albumin as a standard. The pentapeptide, PheLeu-Glu-Glu-Ile (Af, 649.8). was synthesized by the solid-phase procedure of Merrifield (29). A stock 20 mM peptide solution was made by dissolving the peptide in 0.15 M NaOH, followed by adjustment of the pH to 7.4 (pH paper) with HCI. This solution was adjusted to volume and stored at 4°C.
75
IO
5
[PEPTIDE].
mM
FIG. 1. The effect of MnClz on the kinetics of pentapeptide carboxylation by soiubiiized microsomes containing the vitamin K-dependent carboxylase. The carboxylase assays were done in duplicate at 18°C for 15 min in the presence (0) and absence (0) of LO rnM MnClz, using the indicated pentapeptide concentrations. A separate blank for each of the different pentapeptide concentrations was run in the absence of added vitamin KHz, and was used to determine the net vitamin Kdependent “COz incorporation rate. The insert represents a replot of the original data in the form of l/Vvs l/S. The apparent K,‘s for the pentapeptide were determined to be 1.7 and 6.2 mu in the presence and absence of 10 mru MnClz, respectively.
KINETICS
OF VITAMIN
K-DEPENDENT
The rate of 14C02 incorporation into TCA-precipitable material was found to be very rapid at. 20-30°C and was totally dependent on vitamin KHz. The reaction exhibited a linear rate for only about 45 s and was essentially complete within 5 min. The rapidity of the reaction necessitated employment of short, accurate assay times to remain in the linear portion of the curve. The initial reaction velocity of endogenous protein carboxylation increased linearly with protein concentration up to 0.2 ml microsomes. The cessation of the carboxylation rate after’5 min was not due to the oxidation of vitamin KHz, since the addition of more vitamin KHz at 5-min intervals over a 15-min period did not enhance the total radioactivity found in the precipitated protein. The carboxylase itself is stable over this time period, as evidenced by linear peptide carboxylation under identical conditions. Doubling the microsomal protein concentration also increases the total precipitable radioactivity twofold. It is therefore assumed that the endogenous microsomal protein substrate is completely utilized within 5 to 10 min. The effect of the pentapeptide substrate on the initial velocity of endogenous protein carboxylation was investigated using 3 mM pentapeptide in the presence of 5 mM DTE and 10 mM MnCl, at 2O’C. Two important results were obtained under these conditions. Figure 2 shows that the initial rate of endogenous protein carboxylation was lowered in the presence of 3 mM pentapeptide by about 1476,and that the final total radioactive incorporation into the protein was equivalent after 3 min and then remains equal. Secondly, it is also apparent that there is an initial lag of about 45 s in pentapeptide carboxylation under these conditions. The decrease in the endogenous protein carboxylation rate and the observed lag in peptide Gla formation is reproducible and significant (P < 0.025). The observed lag in pentapeptide carboxylation could be a result of competition for the carboxylase enzyme by the presence of uncarboxylated endogenous protein substrate. This possibility of substrate competition was investigated by running the penta-
297
CARBOXYLATION I
I
I
I
I
I
15
MINUTEi
FIG. 2. The effects of pentapeptide on the initial rate of vitamin K-dependent endogenous protein carboxylation. The assays were done in triplicate at 20°C in the presence of 10 mM MnClp as described. The assays were initiated with vitamin KH, and terminated at the indicated times with 0.7 ml of 10% TCA. A O.&ml aliquot of the supernatant was removed and counted to quantitate the TCA-soluble pentapeptide carboxylation in the presence (0 0) and absence (0- - -0) of 3 ml pentapeptide. The total radioactivity incorporated into the TCA-precipitable protein in the presence (a- - -0) and absence (0 0) of 3 mM pentapeptide was quantitated after it was thoroughly washed four times with 10% TCA to completely remove any contaminating pentapeptide. The decrease in the initial velocity of endogenous protein carboxylation due to the presence of pentapeptide was reproducible and significant (P < 0.025).
peptide carboxylating system in the presence and absence of the uncarboxylated protein precursor. The endogenous, uncarboxylated protein substrate was first carboxylated for ‘7min in the presence of 14C02 and excess vitamin KHz. After this preincubation, the pentapeptide was added to initiate pentapeptide carboxylation and samples were removed at the indicated times. Figure 3 presents the data obtained. The zero-minute preincubation control, which still contains all of the uncarboxylated endogenous protein precursor, exhibited the same lag in pentapeptide carboxylation and the same slightly depressed rate of endogenous protein carboxylation as seen in Fig. 2. No lag in pentapeptide
KAPPEL
AND OLSON
activated as much as 400% by divalent cations, while the carboxylation rate of the endogenous protein substrate is stimulated only marginally. Since the pentapeptide carboxylation assays are usually conducted at subsaturating substrate concentrations (1 mM), the observed increase in pentapeptide carboxylation rate can be explained by the threefold lowering of the apparent I& for the pentapeptide to l-2 mM (Fig. 1). We have recently observed a similar effect on the K, of the enzyme by the activator pyridoxal-5’-phosphate (21). These kinetic results are in contrast to those observed for the MnClz stimulation of the ox liver carbohydrates (10) and further demI 2 3 onstrate the differences between the enMINUTES zymes from different species. FIG. 3. The effect of carboxylated and uncarboxylThe minimal activation of the endogeated endogenous protein precursor on the initial rate nous protein carboxylation rate by cations of vitamin K-dependent pentapeptide carboxylation. could be a result of a much lower Km value The assays were done in triplicate at 20°C in the presence of 10 mM MnClz, 3 mM pentapeptide, and 1.2 for the natural, endogenous substrate, as Soute et ~2. (22) have shown that the apmhl vitamin KHz. The zero-minute preincubation control was run exactly as described in Fig. 2. The parent Km for added decarboxylated ‘I-min preincubation test reaction was performed by plasma prothrombin and the decarboxylincubating the microsomes in the absence of pentaated NHz-terminal residues 13-29 are lopeptide but in the presence of “CO, and excess vitamin and lOOO-fold lower, respectively, than the KHz for 7 min to carboxylate fully all available en- Km for the pentapeptide. An average predogenous substrate. Pentapeptide carboxylation was prothrombin concentration of about 0.2 ~.JM then initiated by the addition of peptide. The samples can be calculated in the microsomes from were then analyzed at the indicated times for ‘“COz the &his assays. The carboxylase system incorporation into pentapeptide and endogenous probeing studied is a very crude system and tein substrate as described in Fig. 2. The radioactivity other explanations for the lack of effect of incorporated into TCA-precipitable protein for the cations on the endogenous protein carboxzero-minute (o- - -0) and %min (O 0) preinylation reaction could be valid, including cubations, and into the TCA-soluble pentapeptide for zero-minute (O-O) and ‘I-min (0- - -0) preinthat the endogenous substrates are already cubations, are shown. tightly bound to the enzyme. The data showing competition between the synthetic pentapeptide substrate and carboxylation was observed after the ‘I-min the endogenous microsomal protein subpreincubation period designed to eliminate strate for the solubilized rat liver vitamin the uncarboxylated endogenous micro- K-dependent carboxylase is an expansion somal precursor. Virtually all of the en- of the initial report (11) of the phenomedogenous, uncarboxylated protein sub- non. The data presented in Figs. 2 and 3 strate was carboxylated during the 7-min clearly indicate that the endogenous propreincubation period. This fact was dem- tein carboxylation rate is inhibited by the onstrated by the large amount of radio- presence of 3 mM pentapeptide, and that activity present in the TCA-precipitable the presence of uncarboxylated endogenous protein at zero time. protein substrate causes an initial lag in the pentapeptide carboxylation rate. AlDISCUSSION though kinetic differences between penThe solubilized rat liver vitamin K-de- tapeptide and endogenous protein carboxpendent pentapeptide carboxylase can be ylation have been observed, it is believed
KINETICS
OF VITAMIN
K-DEPENDENT
that only a single microsomal vitamin KHz-dependent carboxylase is responsible for the carboxylation of both substrates.4 The K, values for CO2 and vitamin KHz are essentially the same for the two reactions, and thermal denaturation studies show identical rates of denaturation of carboxylase when either the pentapeptide or endogenous protein substrates are used (data not shown). The initial average rate of protein carboxylation was calculated to be 43.4 pmol min-’ mg protein-’ and is 5.6fold higher than that observed for the pentapeptide carboxylation rate in the presence of MnC12. ACKNOWLEDGMENTS The authors wish to thank Mr. Steve Smith for excellent technical assistance during this project. This work was supported by NIH Grants HL-25196 and AM-09992. REFERENCES 1. ESMON, C. T., AND SUTTIE, J. W. (1976) J. Bid Chem. 251,6238. 2. MACK, D. O., SUEN, E. T., GIRARD~T, J. M., MILLER, J. A., DELANEY, R., AND JOHNSON, B. G. (1976) J. BioL Ch.em 251,3269. 3. OLSON, R. E., JONES, J. P., GARDNER, E. S., HOUSER, R. M., KOB~LKA, P., AND LEE, L. C. (1976) in Proceedings, Xth Int. Congr. Biochem., Hamburg, p. 153. 4. SUSIE, J. W., HAGEMAN, J. M., LEHRMAN, S. R., AND RIC:H, P. H. (1976) J. Bid Chem 251, 5867.
’ After this manuscript was submitted, Shah and Suttie (23) published a report in which they arrived at the same conclusion by a somewhat different approach.
CARBOXYLATION
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5. HOUSER, R. M., CAREY, D. J., Duo, K. M., MARSHALL, G. R., AND OLSON, R. E. (1977) FEBS. La&t. 75. 226. 6. JONES, J. P., GARDNER, E. J., C~UPER, T. G., AND OLSON, R. E. (1977) J. BioL Chem 252.7738. 7. OLSON, R. E., HOUSER, R. M., SEARCEY, M. L., GARDNER, E. J., SCHEINBUK, S. J., SUBBA RAO, G. N., JONES, J. P., AND HALL, A. L. (1978) Fed
Proc 37,261O. 8. SUTTIE, J. W., LEHRYAN, S. R., GEWEJEM, L. O., HAGEMAN, J. M., AND RICH, D. H. (1979)
Biochem Biophys. Res. Ccvnmun 36,500~507. 9. LARSON, A. E., AND SUITIE, J. W. (1980) FEBS Lett 118, 95-98. 10. UOTILA, L., AND SUITIE, J. W. (1982) Biochxm. J. 201,249-258. 11. KAPPEL, W. K., ANDOLSON, R. E. (1982) Fed Proc 41,500. 12. SCHENKMAN, J. B., AND CINTI, D. L. (1978) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 53, pp. 83-89, Academic Press, New York. 13. SOMMER, P. AND KOFLER, M. (1976) Vitomin Hwrmes 24, 349-399. 14. GRAVES, C. B., GRABAU, G. G., OLSON, R. E., AND MUNNS, T. W. (1980) Biochemistry 19,266-272. 15. SHAH. D. V., AND SUITIE, J. W. (1971) Proc. N&L
Acad Sci. USA 68.1653-1657. 16. CARLISLE, T. L., SHAH, D. V., SCHLEGEL, R., AND SUSIE, J. W. (1975) Proc Sot Exp. Bid Med 148,140-144. 17. LAEMBILI, U. K. (1970) Nature (London) 277, 680. 18. MATSUMMURA, T., AND NODA, H. (1973) AnaL Biochem 56, 571-575. 19. BRADFORD, M. M. (1976) Anal Biochem 72,248251. 20. MERRIFIELD, R. B. (1965) Science 150, 178-185. 21. KAPPEL, W. K., ANDOLSON, R. E. (1983) Fed Proc 42,1929. 22. SOUTE, B. A. M., VERMEER, C., MEIZ, M., HEMKER, H. C., AND LIJNEN, H. R. (1981) B&him B&I+ phys. Acta 676,101-107. 23. SHAH, D. V., AND SUTTIE, J. W. (1983) Proc Sot. Exp. BioL Med 173,148-152.
Kinetics of Carboxylation of Endogenous and Exogenous by the Vitamin K-Dependent Carboxylase WILLIAM
K. KAPPEL’
AND
ROBERT
Substrates
E. OLSON1*2
Edward A. Daisy Lkpartnzent of Bimh.emistry, St. Lvuis University School of Medicine, St Louis, ikfk~~ri 63104, and Biochemistry Department, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261 Received April
19, 1983, and in revised form September
20, 1983
The vitamin K-dependent carboxylation of the exogenous pentapeptide, Phe-LeuGlu-Glu-Ile, and endogenous liver microsomal protein was studied in solubilized rat liver microsomes. The MnC12 stimulation of the vitamin K-dependent pentapeptide carboxylation rate, which is conducted at subsaturating concentrations of pentapeptide, is due to the cation’s ability to lower the Km of the substrate. Although there are clear kinetic differences observed between the carboxylation rates for the pentapeptide and the endogenous protein substrates, several lines of evidence suggest that the same carboxylase system is responsible for both. These points of evidence are (i) the initial velocity of endogenous protein carboxylation is lowered in the presence of 3 mM pentapeptide; (ii) the presence of endogenous microsomal protein substrate causes an initial lag in pentapeptide carboxylation; and (iii) this initial lag phase is not observed when the total endogenous substrate pool is carboxylated by a preincubation reaction prior to the addition of pentapeptide.
Rat prothrombin maturation involves the post-translational carboxylation of specific glutamic acid (G~u)~ residues to ycarboxyglutamic acid (Gla) residues. The microsomal vitamin KHz-dependent carboxylase responsible for this modification can be solubilized with nonionic detergents such as Triton X-100 (l-3). The carboxylase reaction requires a Glu-peptide substrate, 02, reduced vitamin K, and C02. The trichloroacetic acid-soluble pentapeptides Phe-Leu-Glu-Glu-Val (4) or Phe-LeuGlu-Glu-Ile (5), representing residues 51 Current address: Biochemistry Department, School of Medicine, University of Pittsburgh. zTo whom reprint requests should be addressed. * Abbreviations used: Hepes, 4-(Z-hydroxyethyl)-lpiperazineethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride: DTE, dithioerythritol; TCA, trichloroacetic acid; Glu, glutamic acid, Gla, y-carboxyglutamic acid; STI, soybean trypsin inhibitor. 0003-9861/84 $3.00 Copyright 0 1984by AcademicPress.Inc. All righta of reproductionin any form reserved.
9 of the uncarboxylated bovine and rat prothrombin molecules, respectively, have been used as artificial substrates. The active form of “COZ” is CO2 and not bicarbonate (6, 7). The carboxylation does not require ATP or biotin (6) and the mechanism of reaction is obscure. When compared to the carboxylation rate of the endogenous microsomal protein substrates, the pentapeptide carboxylase activity is more sensitive to the action of various activators and inhibitors (8). In particular, 10 mM MnCl, was shown to stimulate the rat liver pentapeptide carboxylase activity about 2-fold, while having little effect on the endogenous microsomal protein carboxylation rate (9). Uotila and Suttie (10) demonstrated that the adult ox and dicoumarol-treated calf liver vitamin K-dependent carboxylase activities were stimulated 9- and 22-fold, respectively, by 10 mM MnC12 under optimal conditions. 294
KINETICS
OF VITAMIN
K-DEPENDENT
They claimed that Mn2+ only increased the maximum velocity of the adult ox liver carboxylase reaction without any influence on the K,,, of the pentapeptide; however, the data was not shown. The kinetic properties of the adult ox liver enzyme were found to be substantially different from those of the rat liver carboxylase. Some of these differences were the ionic strength optima, the Km for vitamin KH2, the extent of pyridoxal-5’-P activation, and the fold stimulation by Mn2+. Our findings on the mechanism of Mn”+ activation of the solubilized rat liver vitamin K-dependent carboxylase from warfarinized rats are different from those previously reported (9, 10). We also have observed that exogenous pentapeptide competes with endogenous protein substrate for carboxylation by the vitamin Kdependent carboxylase. A preliminary report of this work has been published (11).
CARBOXYLATION
295
at 27,000~ for 20 min. The pelleted microsomes were solubilized in 50 mM Hepes, 0.5 M KCl, 250 rnM sucrose, 1 mM PMSF, 50 pg/ml STI, 2% Triton X-100 (pH 7.4) at a ratio of 1.0 ml buffer per initial gram of liver by the aid of 10 strokes in a Teflon homogenizer. All steps were carried out as quickly as possible at O4°C. The solubilized microsomes were stored at -70°C in 1.5-ml plastic-capped centrifuge tubes. Under these conditions the enzyme was stable for at least 6 months. Carblase assays. The standard endogenous protein carboxylation assay was conducted in 13 X lOOmm tubes at 20°C in a total volume of 0.3 ml containing solubilized microsomes (0.1 ml), 24 mM vitamin KHZ (0.01 ml), NaI-I”COs (0.02 ml), 0.3 M DTE (0.005 ml), and any test substances. The solution was made to 0.3 ml with 0.1 M Hepes (pH 7.4). The reaction was stopped with 8 ml of 10% TCA (0°C) and kept at 0°C for 15 min. Protein was collected by centrifugation and washed once with 5 ml 10% TCA. The tubes were stored overnight under vacuum at room temperature to remove residual 14COZ.Protosol(l.0 ml) was added and the tubes were sealed with four layers of parafllm. The tubes were incubated with constant agitation at 50°C until the protein was completely dissolved (2 h). The solubilized protein was quantitatively transferred to 20-ml plastic scintillation vials with two EXPERIMENTAL PROCEDURES washes of 4 ml of Omnifluor. The samples were made Materials Vitamin K1, 3-(a-acetonylbenzyl)-4-hyto 12 ml with Omnifluor and then counted after 2 h. droxy coumarin (warfarin), Triton X-100, fibrinogen, The standard TCA-soluble peptide carboxylation Echis cm-in&us, and Hepes buffer were bought from was conducted at 18°C in 12 X 75-mm tubes in a total Sigma. The sodium [“Clbicarbonate (55-60 mCi/mol; volume of 0.3 ml containing solubilized microsomes 2 mCi/ml) and ACS were purchased from Amersham. (0.1 ml), 24 mM vitamin KH, (0.01 ml), 20 mM penOmnifluor (gal-pak) and protosol were obtained from tapeptide (0.015 ml), 0.3 M DTE! (0.005 ml), 0.3 M MnCl, New England Nuclear. Scintillation-grade toluene was (0.01 ml), and any test substances. The samples were from Baker. Palladium on activated carbon was purmade to 0.3 ml with 0.1 M Hepes (pH 7.4). The reaction chased from Aldrich. The Sprague-Dawley rats were was stopped after 15 min by the addition of 0.7 ml obtained from Sasco, Inc. All other reagents were of of 10% TCA (O’C) and kept at 0°C for 15 minutes. the highest possible commercial grade. The protein was removed by centrifugation. At this Carboxylase preparation The rat liver microsomes point, either 0.5 ml of the supernatant was transferred were prepared by a modified procedure of Schenkman to a 6-ml scintillation vial and left in an exhaust hood and Cinti (12). Sprague-Dawley rats (150-300 g) were overnight at room temperature to remove residual injected intraperitoneally with sodium warfarin (10 14C02 or the entire supernatant was poured off into mg/kg) and fasted for 18 h in raised-bottom cages 12 X 75-mm tubes. In the later case, COz gas was to reduce coprophagy. A stock warfarin solution (30 bubbled through the supernatant for 5 min and 0.5 mg/ml) was made in 0.15 M NaOH. This was then ml of the gassed sample was then placed in the vials. diluted to 10 mg/ml with 0.1 M sodium phosphate (pH In either case, the samples were counted in 4 ml 7.0) and used for injections. The livers were removed of ACS. from exsanguinated rats and placed in 30 ml of 10 All reactions were initiated by the addition of vimM Hepes, 250 mM sucrose, 1 mM phenylmethylsultamin KHZ, followed by gentle mixing. A control assay was always run in the absence of added vitamin KH, fonyl fluoride (PMSF), 50 pg/ml soybean trypsin inhibitor (STI) (pH 7.4) and weighed, and then lightly and was subtracted from the values obtained in the minced with scissors. The liver was homogenized with presence of added vitamin KH,. The radioactivity insix strokes at medium speed with a Teflon homogecorporated into protein or pentapeptide Gla was stable nizer. Pooled homogenates were centrifuged at 12,OOOg under the above conditions for at least 48 h. Any for 15 min. The supernatant was diluted to 80 ml per deviation from these standard assay conditions will liver with homogenization buffer, and 0.9 ml of 0.8 M be described in the text. CaCl, was added to each 80 ml of protein solution vitamin K preparation The stock 24 mM- vitamin and thoroughly mixed. This solution was centrifuged K solutions were prepared by making a 10.87 mg/ml
296
KAPPEL
AND
OLSON
tivation of the pentapeptide carboxylation is nonadditive and is probably general cation action. These same cations exhibited a reproducible, but much smaller activation (11-W%) of the endogenous protein carboxylation rate. The sodium salts of F-, II, Br-, or Pi were found to have no effect or to be slightly inhibitory in either of the two carboxylation assays and were not studied further. The mechanism responsible for the cation stimulation of the pentapeptide carboxylation was investigated in more detail, and the effect of MnC12 on the K, of the pentapeptide substrate is shown in Fig. 1. The data clearly demonstrate that MnC12 does not effect the V,,, of the reaction, but that it effectively lowers the apparent K, of the pentapeptide threefold. All of the previous pentapeptide carboxylation studies were run at only 1 mM substrate conRESULTS centration due to its expense. Therefore, The carboxylation rate of the pentapep- the observed activating effect of MnC12 on tide substrate by the solubilized vitamin the solubilized rat liver carboxylase apK-dependent carboxylase was found to be pears to be due to an effect on the Km of stimulated from 250 to 300% by 10 mM the enzyme. MgC12 was also found to exMn2+, Mgaf, Ca2+,and S?. The cation ac- hibit identical effects on Km and V,,,.
vitamin K solution in the absolute ethanol. This solution was stored at -15°C. The yellow quinone was reduced to the colorless hydroquinone using palladium on activated carbon (10 mg/ml) by gassing with hydrogen during constant agitation for 2 min. The vitamin KHZ was stored under hydrogen in the dark until needed. The sample was clarified by centrifugation immediately before use. The concentration of vitamin K was determined from its absorbance at 248 nm in absolute ethanol using Ei”z = 419 (13). The molar extinction coefficient was calculated to be 18,885. were deterOther methods. Protein concentrations mined by the method of Bradford (19) using bovine serum albumin as a standard. The pentapeptide, PheLeu-Glu-Glu-Ile (Af, 649.8). was synthesized by the solid-phase procedure of Merrifield (29). A stock 20 mM peptide solution was made by dissolving the peptide in 0.15 M NaOH, followed by adjustment of the pH to 7.4 (pH paper) with HCI. This solution was adjusted to volume and stored at 4°C.
75
IO
5
[PEPTIDE].
mM
FIG. 1. The effect of MnClz on the kinetics of pentapeptide carboxylation by soiubiiized microsomes containing the vitamin K-dependent carboxylase. The carboxylase assays were done in duplicate at 18°C for 15 min in the presence (0) and absence (0) of LO rnM MnClz, using the indicated pentapeptide concentrations. A separate blank for each of the different pentapeptide concentrations was run in the absence of added vitamin KHz, and was used to determine the net vitamin Kdependent “COz incorporation rate. The insert represents a replot of the original data in the form of l/Vvs l/S. The apparent K,‘s for the pentapeptide were determined to be 1.7 and 6.2 mu in the presence and absence of 10 mru MnClz, respectively.
KINETICS
OF VITAMIN
K-DEPENDENT
The rate of 14C02 incorporation into TCA-precipitable material was found to be very rapid at. 20-30°C and was totally dependent on vitamin KHz. The reaction exhibited a linear rate for only about 45 s and was essentially complete within 5 min. The rapidity of the reaction necessitated employment of short, accurate assay times to remain in the linear portion of the curve. The initial reaction velocity of endogenous protein carboxylation increased linearly with protein concentration up to 0.2 ml microsomes. The cessation of the carboxylation rate after’5 min was not due to the oxidation of vitamin KHz, since the addition of more vitamin KHz at 5-min intervals over a 15-min period did not enhance the total radioactivity found in the precipitated protein. The carboxylase itself is stable over this time period, as evidenced by linear peptide carboxylation under identical conditions. Doubling the microsomal protein concentration also increases the total precipitable radioactivity twofold. It is therefore assumed that the endogenous microsomal protein substrate is completely utilized within 5 to 10 min. The effect of the pentapeptide substrate on the initial velocity of endogenous protein carboxylation was investigated using 3 mM pentapeptide in the presence of 5 mM DTE and 10 mM MnCl, at 2O’C. Two important results were obtained under these conditions. Figure 2 shows that the initial rate of endogenous protein carboxylation was lowered in the presence of 3 mM pentapeptide by about 1476,and that the final total radioactive incorporation into the protein was equivalent after 3 min and then remains equal. Secondly, it is also apparent that there is an initial lag of about 45 s in pentapeptide carboxylation under these conditions. The decrease in the endogenous protein carboxylation rate and the observed lag in peptide Gla formation is reproducible and significant (P < 0.025). The observed lag in pentapeptide carboxylation could be a result of competition for the carboxylase enzyme by the presence of uncarboxylated endogenous protein substrate. This possibility of substrate competition was investigated by running the penta-
297
CARBOXYLATION I
I
I
I
I
I
15
MINUTEi
FIG. 2. The effects of pentapeptide on the initial rate of vitamin K-dependent endogenous protein carboxylation. The assays were done in triplicate at 20°C in the presence of 10 mM MnClp as described. The assays were initiated with vitamin KH, and terminated at the indicated times with 0.7 ml of 10% TCA. A O.&ml aliquot of the supernatant was removed and counted to quantitate the TCA-soluble pentapeptide carboxylation in the presence (0 0) and absence (0- - -0) of 3 ml pentapeptide. The total radioactivity incorporated into the TCA-precipitable protein in the presence (a- - -0) and absence (0 0) of 3 mM pentapeptide was quantitated after it was thoroughly washed four times with 10% TCA to completely remove any contaminating pentapeptide. The decrease in the initial velocity of endogenous protein carboxylation due to the presence of pentapeptide was reproducible and significant (P < 0.025).
peptide carboxylating system in the presence and absence of the uncarboxylated protein precursor. The endogenous, uncarboxylated protein substrate was first carboxylated for ‘7min in the presence of 14C02 and excess vitamin KHz. After this preincubation, the pentapeptide was added to initiate pentapeptide carboxylation and samples were removed at the indicated times. Figure 3 presents the data obtained. The zero-minute preincubation control, which still contains all of the uncarboxylated endogenous protein precursor, exhibited the same lag in pentapeptide carboxylation and the same slightly depressed rate of endogenous protein carboxylation as seen in Fig. 2. No lag in pentapeptide
KAPPEL
AND OLSON
activated as much as 400% by divalent cations, while the carboxylation rate of the endogenous protein substrate is stimulated only marginally. Since the pentapeptide carboxylation assays are usually conducted at subsaturating substrate concentrations (1 mM), the observed increase in pentapeptide carboxylation rate can be explained by the threefold lowering of the apparent I& for the pentapeptide to l-2 mM (Fig. 1). We have recently observed a similar effect on the K, of the enzyme by the activator pyridoxal-5’-phosphate (21). These kinetic results are in contrast to those observed for the MnClz stimulation of the ox liver carbohydrates (10) and further demI 2 3 onstrate the differences between the enMINUTES zymes from different species. FIG. 3. The effect of carboxylated and uncarboxylThe minimal activation of the endogeated endogenous protein precursor on the initial rate nous protein carboxylation rate by cations of vitamin K-dependent pentapeptide carboxylation. could be a result of a much lower Km value The assays were done in triplicate at 20°C in the presence of 10 mM MnClz, 3 mM pentapeptide, and 1.2 for the natural, endogenous substrate, as Soute et ~2. (22) have shown that the apmhl vitamin KHz. The zero-minute preincubation control was run exactly as described in Fig. 2. The parent Km for added decarboxylated ‘I-min preincubation test reaction was performed by plasma prothrombin and the decarboxylincubating the microsomes in the absence of pentaated NHz-terminal residues 13-29 are lopeptide but in the presence of “CO, and excess vitamin and lOOO-fold lower, respectively, than the KHz for 7 min to carboxylate fully all available en- Km for the pentapeptide. An average predogenous substrate. Pentapeptide carboxylation was prothrombin concentration of about 0.2 ~.JM then initiated by the addition of peptide. The samples can be calculated in the microsomes from were then analyzed at the indicated times for ‘“COz the &his assays. The carboxylase system incorporation into pentapeptide and endogenous probeing studied is a very crude system and tein substrate as described in Fig. 2. The radioactivity other explanations for the lack of effect of incorporated into TCA-precipitable protein for the cations on the endogenous protein carboxzero-minute (o- - -0) and %min (O 0) preinylation reaction could be valid, including cubations, and into the TCA-soluble pentapeptide for zero-minute (O-O) and ‘I-min (0- - -0) preinthat the endogenous substrates are already cubations, are shown. tightly bound to the enzyme. The data showing competition between the synthetic pentapeptide substrate and carboxylation was observed after the ‘I-min the endogenous microsomal protein subpreincubation period designed to eliminate strate for the solubilized rat liver vitamin the uncarboxylated endogenous micro- K-dependent carboxylase is an expansion somal precursor. Virtually all of the en- of the initial report (11) of the phenomedogenous, uncarboxylated protein sub- non. The data presented in Figs. 2 and 3 strate was carboxylated during the 7-min clearly indicate that the endogenous propreincubation period. This fact was dem- tein carboxylation rate is inhibited by the onstrated by the large amount of radio- presence of 3 mM pentapeptide, and that activity present in the TCA-precipitable the presence of uncarboxylated endogenous protein at zero time. protein substrate causes an initial lag in the pentapeptide carboxylation rate. AlDISCUSSION though kinetic differences between penThe solubilized rat liver vitamin K-de- tapeptide and endogenous protein carboxpendent pentapeptide carboxylase can be ylation have been observed, it is believed
KINETICS
OF VITAMIN
K-DEPENDENT
that only a single microsomal vitamin KHz-dependent carboxylase is responsible for the carboxylation of both substrates.4 The K, values for CO2 and vitamin KHz are essentially the same for the two reactions, and thermal denaturation studies show identical rates of denaturation of carboxylase when either the pentapeptide or endogenous protein substrates are used (data not shown). The initial average rate of protein carboxylation was calculated to be 43.4 pmol min-’ mg protein-’ and is 5.6fold higher than that observed for the pentapeptide carboxylation rate in the presence of MnC12. ACKNOWLEDGMENTS The authors wish to thank Mr. Steve Smith for excellent technical assistance during this project. This work was supported by NIH Grants HL-25196 and AM-09992. REFERENCES 1. ESMON, C. T., AND SUTTIE, J. W. (1976) J. Bid Chem. 251,6238. 2. MACK, D. O., SUEN, E. T., GIRARD~T, J. M., MILLER, J. A., DELANEY, R., AND JOHNSON, B. G. (1976) J. BioL Ch.em 251,3269. 3. OLSON, R. E., JONES, J. P., GARDNER, E. S., HOUSER, R. M., KOB~LKA, P., AND LEE, L. C. (1976) in Proceedings, Xth Int. Congr. Biochem., Hamburg, p. 153. 4. SUSIE, J. W., HAGEMAN, J. M., LEHRMAN, S. R., AND RIC:H, P. H. (1976) J. Bid Chem 251, 5867.
’ After this manuscript was submitted, Shah and Suttie (23) published a report in which they arrived at the same conclusion by a somewhat different approach.
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5. HOUSER, R. M., CAREY, D. J., Duo, K. M., MARSHALL, G. R., AND OLSON, R. E. (1977) FEBS. La&t. 75. 226. 6. JONES, J. P., GARDNER, E. J., C~UPER, T. G., AND OLSON, R. E. (1977) J. BioL Chem 252.7738. 7. OLSON, R. E., HOUSER, R. M., SEARCEY, M. L., GARDNER, E. J., SCHEINBUK, S. J., SUBBA RAO, G. N., JONES, J. P., AND HALL, A. L. (1978) Fed
Proc 37,261O. 8. SUTTIE, J. W., LEHRYAN, S. R., GEWEJEM, L. O., HAGEMAN, J. M., AND RICH, D. H. (1979)
Biochem Biophys. Res. Ccvnmun 36,500~507. 9. LARSON, A. E., AND SUITIE, J. W. (1980) FEBS Lett 118, 95-98. 10. UOTILA, L., AND SUITIE, J. W. (1982) Biochxm. J. 201,249-258. 11. KAPPEL, W. K., ANDOLSON, R. E. (1982) Fed Proc 41,500. 12. SCHENKMAN, J. B., AND CINTI, D. L. (1978) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 53, pp. 83-89, Academic Press, New York. 13. SOMMER, P. AND KOFLER, M. (1976) Vitomin Hwrmes 24, 349-399. 14. GRAVES, C. B., GRABAU, G. G., OLSON, R. E., AND MUNNS, T. W. (1980) Biochemistry 19,266-272. 15. SHAH. D. V., AND SUITIE, J. W. (1971) Proc. N&L
Acad Sci. USA 68.1653-1657. 16. CARLISLE, T. L., SHAH, D. V., SCHLEGEL, R., AND SUSIE, J. W. (1975) Proc Sot Exp. Bid Med 148,140-144. 17. LAEMBILI, U. K. (1970) Nature (London) 277, 680. 18. MATSUMMURA, T., AND NODA, H. (1973) AnaL Biochem 56, 571-575. 19. BRADFORD, M. M. (1976) Anal Biochem 72,248251. 20. MERRIFIELD, R. B. (1965) Science 150, 178-185. 21. KAPPEL, W. K., ANDOLSON, R. E. (1983) Fed Proc 42,1929. 22. SOUTE, B. A. M., VERMEER, C., MEIZ, M., HEMKER, H. C., AND LIJNEN, H. R. (1981) B&him B&I+ phys. Acta 676,101-107. 23. SHAH, D. V., AND SUTTIE, J. W. (1983) Proc Sot. Exp. BioL Med 173,148-152.