Vitamin K-dependent carboxylase: Partial purification of the enzyme by antibody affinity techniques

THROMBOSIS RESEARCH 56; 317-323, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.

CARBOXYLASE: PARTIAL PURIFICATION OF THE ENZYME BY ANTIBODY AFFINITY TECHNIQUES

VITAMIN K-DEPENDENT

Mark C. Harbeck, Alex Y. Cheung, and J. W. Suttie Department of Biochemistry College of Agricultural and Life Sciences University of Wisconsin-Madison Madison, Wisconsin 53706, USA

(Received 19.1.1989; accepted in original form 30.1.1989 by Editor C.T. Esmon) (Received in final form by Executive Editorial Office 7.9.1989) ABSTRACT The vitamin K-dependent carboxylase activity of bovine liver microsomes has been purified 500-fold by adsorption to an antiprothrombin column and elution with a dodeca peptide which competes with a prothrombin precursor enzyme recognition site. The purified enzyme is devoid of bound precursors, and has the same ratio of vitamin K epoxidase activity to carboxylase activity as the crude microsomal preparation.

INTRODUCTION The liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of specific Glu residues in the intracellular precursors of a limited number of proteins to r-carboxyglutamyl (Gla) residues in mature proinclude plasma clotting factors II, VII, IX, and X as well as teins. These K plasma proteins C and S. The enzyme requires the reduced form of vitamin (vitamin KHz), O,, and CO, as co-substrates and forms the 2,3-epoxide of the Although plausible vitamin (vitamin K epoxide) as a second product (192). of the enzyme have been proposed, the lack of a mechanisms for the action purified enzyme has precluded an understanding of the detailed action of this carboxylase at the molecular level. of the enzyme has been reported by a number of inPartial purification vestigators (3-6), but no highly reproducible preparation is available. It has been clearly demonstrated that microsomal precursors to vitamin K-dependent proteins increase when vitamin K action is blocked (7) and that antibodies to these proteins will immobilize the active enzyme on an inert support (8-10). Efforts to purify the enzyme by dissociation of the presumed

Key Words: antibody.

Vitamin

K;

carboxylase;

I-carboxyglutamic

317

acid;

prothrombin

318

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antibody/precursor/enzyme complex have been unsuccessful. Precursors of vitamin K-dependent proteins have now been shown (11) to contain a propeptide region between the signal peptide and the amino terminus of the mature proteins which appear to serve as a recognition site for the carboxylase (12-16) and to also modulate the activity of the carboxylase A peptide con(17). the propeptide taining region has now been used to effect a substantial and reproducible purification of the enzyme.

MATERIALS

AND METHODS

Preparation of bovine microsomes. Liver obtained from a cow administered 1.85 mg/kg of dicoumarol on days 1, 2, and 5, and killed on day 7 was transported on ice to a cold room and the outer membranes and connective tissue removed before homogenization. The liver was blended with an equal volume of 0.25 M sucrose, 0.025 M imidazole, pH 7.3 (SI buffer) using a Waring blender and filtered through cheesecloth. The filtrate was homogenized in a PotterElvehjem homogenizer and centrifuged at 10,000 x g for 15 min. Aliquots (8 ml) of the supernatant were centrifuged at 105,000 x g for 60 min and pellets washed twice by resuspension with a Dounce homogenizer in SI buffer containing 0.1 M KC1 and centrifuged as before. As a final wash, the pellets were resuspended in SI buffer containing 1 M KCl, centrifuged at 105,000 x g for 60 min, frozen in liquid nitrogen, and stored at -70" C. White male New Zealand rabbits were Preoaration of antiorothrombin Seoharose. immunized with bovine prothrombin purified from a barium citrate precipitate of bovine plasma (18) and maintained at high antibody titers by boosting with antigen as required. The antiprothrombin antibodies were purified from ammonium sulfate fractionated immune sera by immunoaffinity chromatography over bovine prothrombin Sepharose and coupled to Sepharose-4B by cyanogen bromide activation (19). Vitamin K-dependent carboxylations of the peptide substrate were Assays. carried out by adding 100 ~1 of enzyme preparations to 100 ~1 of other reactants such that the final 200 ~1 reaction volume contained 0.25 M sucrose, 0.5 M KCl, 0.025 M imidazole, pH 7.3, 8 mM DTT, 0.5 mM Boc-Glu-Glu-Leu-OMe CA), 0.62 M (NH,)zSO,, and 10 &i H"C0,. Reactions were (Bachem, Torrance, initiated by the addition of 10 ~1 of 5-7 mg/ml vitamin KH, in ethanol, incubated at 25" C for 60 min, quenched by the addition of 1 ml ice cold 10% TCA, and centrifuged to remove acid insoluble material. The supernatant was bubbled with CO, (from dry ice) to remove excess labeled Hl'CO, and 200 ~1 of the quenched, degassed sample was mixed with 3.8 ml Bio-Safe-II (RPI, Elk Grove, IL), and radioactivity determined in a liquid scintillation spectrophotometer. Samples containing antibody gel were added to the reaction mixture as a suspension of 50 ~1 gel in 50 ~1 of buffer. The carboxylation of endogenous substrates (precursor proteins) was assayed as the incorporation of '4c0, into trichloracetic acid precipitable material (20) using assay conditions identical to those described above. Protein concentration of fractions was assayed by the Peterson modification of the Lowry method (21). Vitamin K epoxidase activity was measured under the same incubation conditions as those used to measure carboxylase activity, and epoxide formed measured as previously described (22). Prooeotide. The dodeca peptide KSLFIRREQANNILARVTRA which contains the 18 residue propeptide region of human factor X was synthesized as previously described (17) and is referred to as propeptide.

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319

RESULTS Microsomes from a dicoumarol-treated cow were solubilized in detergent, equilibrated with an anti-prothrombin sepharose preparation as described in Methods, and subjected to the purification procedure described in the legend to Table I. The antibody gel was collected by low-speed centrifugation and both the gel and supernatant assayed for carboxylase activity. Total activity was increased, and about 35% of the activity present was associated with the antibody gel. Washing the gel with buffer was ineffective in removing carboxylase activity, but elution of the gel with 1 mM propeptide displaced the enzyme from the antibody gel in a sharp front that tailed off to achieve a 470-fold increase in specific activity in a single step. The procedure is reproducible with 400-600-fold purifications achieved in a series of prepaover a three-month rations conducted period. Because of the high concentration of propeptide in the elution buffer, accurate protein assays are difficult, and the extent of purification should be considered a minimum. The preparation procedure used should have dissociated the carboxylase from precursors which would remain bound to the antibody column. The data in Table II indicate that the purified preparation showed no significant protein carboxylase activity (vitamin K-dependent incorporation of "COz into protein) and provide evidence that the bound precursors have been effectively removed. The general properties of the crude microsomal carboxylase have been extensively studied (2), and current evidence suggests that the enzyme is a linked carboxylase/epoxidase. The data in Table III demonstrate that the purified preparation is capable of converting vitamin KHz to vitamin K epoxide

Purification

Table I of the Vitamin K-dependent Carboxylase Antiprothrombin-Sephadex Column

Fraction

Volume (ml) 10.0

A

Solubilized

Bl

Prothrombin antibody gel Antibody gel supernatant

1.0 10.0

c2

Buffer eluate of gel Propeptide eluate of gel

15.0 3.6

D

Post elution antibody

B, C,

microsomes

gel

1.0

Protein ( e) 34:

320 1.5 0.36

on an

Carboxylase (units) 439 199 379 45 224

Sp Act U/me Prot 1.29

1.18 30 608

86

fixed in the standard Carboxylase units are expressed as 10' dpm of "CO, All fractions other than C, were assayed after the addition of 100 PM assay. Microsomal pellets were solubilized in 0.25 M sucrose, 0.5 M KCl, propeptide. and 1 mM phenylmethyl0.025 M imidazole, pH 7.3, 0.4% CHAPS, 0.01% NaN,, sulfonyl fluoride (buffer A) to a protein concentration of 30 mg/ml. After 30 min at 4" C, the solubilized microsomes were centrifuged at 105,000 x g for 60 min to remove insoluble material and 10 ml of the solubilized microsomes incubated with one ml of the antiprothrombin sepharose overnight at 4" C were The antibody gel was collected by on a Clay Adams (Parsippany, NJ) nutator. centrifugation, loaded onto a 0.7 x 4.0 cm column, and washed with low speed phosphatidylcholine 15 ml buffer A plus 5 mM dithiothreitol and 5 mg/ml (buffer B) at 4" C. Carboxylase activity was eluted from the antibody column of buffer B at room temperature at a rate of 0.2 ml/min using eight volumes containing 1 mM propeptide and held at 4" C until assay.

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and that the ratio of carboxylase to epoxidase activity of the crude and purified preparations is similar. Chloro-K (2-Cl-3-phytyl-1,4-naphthoquinone) and tetrachloropy,ridinolare well characterized inhibitors of the carboxylase (2). These were more potent inhibitors of the purified preparation.

Table II Protein Precursors in Crude and Purified Carboxylase Preparations

Incubation

Peptide Carboxylase Activity_3 (DPM x 10 ) Crude Purified

- Vit KHz

0.6

0.6

+ Vit KHz

50.2

16.0

Protein Carboxylase Activity_3 (DPM x 10 ) Crude Purified

0.40

0.40

6.4

0.45

The crude preparation (solubilized microsomes) had 100 PM propeptide added to the incubations. The purified preparation was propeptide eluate (fraction C, of Table I). Incubations were for 1 h at 25" C, and the values are means of duplicate incubations differing by less than 10%.

Table III Epoxidase and Carboxylase Activity of Crude and Purified Preparations

Conditions

KO (nmole) Crude Purified

14C0, Fixed (DPM x 10m3) Crude Purified

No substrate

0.06

<0.02

0.1

0.1

6 mM tripeptide

0.64

0.15

18.8

3.7

Carboxylation of an exogenous peptide substrate (peptide carboxylase activity) and vitamin K epoxide (KO) formation was assessed in a purified carboxylase preparation (fraction C, of Table I). Incubations were for 1 h at 25" C, and values are the means of duplicate incubations differing by less than 10%.

DISCUSSION Previous attempts to purify the liver microsomal vitamin K-dependent carboxylase have resulted in some enrichment of activity (2-6) but have tended to be variable. These preparations have not been extensively used in studies of the mechanism of this unique enzyme. The preparation described here is rapidly reproducible, and results in a stable enzyme preparation that can be used for more detailed studies. The total recovery of enzyme units in the various fractions assayed was nearly double that of starting microsomes. Previous data have suggested the presence of an inhibitor of the enzyme in crude microsomes (3), and an increase in total activity is therefore not

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unexpected. There are indications (8,23) that prothrombin precursors are not the major precursor species in bovine microsomes, and it is likely that an affinity column comprised of antibodies to all vitamin K-dependent plasma proteins (a barium salt eluate) would substantially increase the yield in the first step of the reaction. These data have provided a direct confirmation of the general hypothesis that the propeptide region of the vitamin K-dependent proteins provides a recognition site for the carboxylase. The assumption that the antibody bound carboxylase preparation consists of antibody-precursor-enzyme is strongly supported by observations (Table II) that no precursors are bound to the purified preparation and supports the working hypothesis that the propeptide has effectively competed for a binding site independent of the catalytic site on the enzyme and has dissociated the precursor-enzyme complex. The data in Table III provide further confirmation of the dual role of the enzyme as a carboxylase/epoxidase. Previous studies of these two activities (22) have been carried out in crude microsomes and it is possible that a second enzyme was responsible for the activity. Maintenance of a constant ratio of carboxylase to epoxidase activity through a 500-fold purification is consistent with the location of these two activities on the same enzyme. The availability of this preparation should provide material for a more detailed investigation of the properties of the enzyme and serve as a starting point for further efforts at fractionation.

ACKNOWLEDGEMENTS This research was supported by the College of Agricultural and Life Sciences of the University of Wisconsin-Madison, and in part by grants DK14881 and HL-29586 from the National Institutes of Health, Bethesda, Maryland.

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