Vitamin K-dependent carboxylase: Effect of detergent concentrations, vitamin K status, and added protein precursors on activity

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 222, No. 1, April 1, pp. 216-221, 1983

Vitamin K-Dependent Carboxylase: Effect of Detergent Concentrations, Vitamin K Status, and Added Protein Precursors on Activity’ D. V. SHAH, Department

of Biochemistry,

College

of Wiscmsin-Madison,

University

Received

J. C. SWANSON,

October

AND

of Agricultural

Madison,

8, 1982, and in revised

form

J. W. SUTTIE and Life Sciences, Wkwnsin 53706 December

6, 1982

Activity of the rat liver microsomal vitamin K-dependent carboxylase has been studied at various concentrations of detergent. The activity which could be solubilized by 0.25% Triton X-100 was low but could be greatly increased if vitamin K-deficient rats were given vitamin K a few minutes before they were killed. At higher concentrations of Triton, more activity was solubilized and this effect was not seen. In vitro carboxylation of endogenous microsomal proteins was decreased by 80-90% if vitamin K was administered 1 min before rats were killed, but the amount of assayable prothrombin precursor was decreased by only 20%. Decarboxylated vitamin K-dependent rat plasma proteins were not substrates for the carboxylase and did not influence peptide carboxylase activity significantly. Purified microsomal prothrombin precursors did, however, stimulate carboxylation of peptide substrate and were used as a substrate for the carboxylase in a preparation from precursor depleted vitamin K-deficient rats.

Vitamin K functions in the postribosoma1 modification of liver microsomal protein precursors to form biologically active prothrombin and the other vitamin K-dependent clotting factors (l-2). This modification involves the carboxylation of specific glutamyl residues in these proteins to y-carboxyglutamyl residues in the finished proteins. The carboxylation of these precursors and of low-molecular-weight synthetic peptide substrates has been demonstrated in detergent-solubilized microsomal preparations, and reviews of the current progress in this field are available (3-5). The available data (6-8) regarding the ability of the rat liver microsomal vitamin K-dependent carboxylase to utilize exogenous added protein substrates is limited

and contradictory. This report describes studies on the effects of thermally decarboxylated plasma prothrombin and immunologically purified rat liver prothrombin precursors on vitamin K-dependent carboxylation of peptide substrates, and the ability of these proteins to act as substrates. The reported effects of vitamin K status on the carboxylation of endogenous and exogenous substrates and the effects of detergent concentration on enzyme activity have also been variable, and these parameters have been reassessed. MATERIALS

0003-9861/83

$3.00

0 1983 by Academic Press. Inc. of reproduction in any form reserved.

METHODS

Male Holtzman strain rats, 220-225 g, were fed Purina Laboratory Chow and given drinking water containing 50 pg menadione/lOO ml to ensure vitamin K sufficiency or were fed a vitamin K-deficient diet (9) for 7-8 days in coprophagy-preventing cages (10). When vitamin K1 was administered, 1 mg/rat was given intracardially as a detergent-dispersed suspension of phylloquinone (Konakion). Rats were fasted for 18-20 h and killed by decapitation before microsomal pellets were prepared as previously described

1 This research was supported by the College of Agricultural and Life Sciences of the University of Wisconsin-Madison, and in part by Grant AM-14881 of the National Institutes of Health, Bethesda, Maryland.

Copyright All rights

AND

216

ACTIVITY

OF

VITAMIN

K-DEPENDENT

CARBOXYLASE

217

24

16

L Minutes

After

Phylloquinone

Treatment

FIG. 1. Effect of time after vitamin K administration to vitamin K-deficient hypoprothrombinemic rats on vitamin K-dependent carboxylase activity. The rats were administered phylloquinone (1 mg/rat, intracardially) 1 to 120 min prior to decapitation, and solubilized microsomes from each group of three to four rats were incubated in duplicate. Values shown are means f SEM for five experiments. Control animals were vitamin K-sufficient normal rats. Vitamin K-dependent carboxylation of an exogenous peptide substrate is shown as an open bar and of endogenous microsomal protein as a crosshatched bar. (A) Solubilized in 0.25% Triton; (B) solubilized in 0.5% Triton; (C) solubilized in 1.0% Triton; (D) solubilized in 1.5% Triton.

(6, 11). The microsomal pellets were solubilized in SIK-DTTz buffer (0.25 M sucrose/O.025 M imidazole/ 0.5 M KCl, pH 7.2,l mM dithiothreitol) containing0.25 to 1.50% (w/v) Triton X-100 and centrifuged at

‘Abbreviations used: SIK-DTT buffer, 0.25 M sucrose/0.025 M imidazole/0.5 M KCl, pH 7.2, and 1 mM dithiothreitol; ECV, Echis carinatus venom.

105,OOOg for 60 min to remove the small amount of insoluble material. When not specified, 1.50% Triton X-100 was used to solubilize the microsomal pellet. Assays for vitamin K-dependent carboxylase contained 0.4 ml of solubilized microsomal supernatant, 20 ~1 of NaHi4C03 (10 &i/incubation), 0.1 ml of SIKDTT buffer or SIK-DTT buffer containing 0.5 mM PheLeu-Glu-Glu-Leu (Vega Fox Biochemicals, Tucson, Ariz.), and 25 ~1 of 0.025 M imidazole buffer or this

218

SHAH,

Minutes

SWANSON,

After

AND

Phylloquinone

SUTTIE

Treatment

FIG. 2. Effect of time after vitamin K administration to hypoprothrombinemic rats on liver microsomal venom-generated thrombin activity. Soluble microsomal fractions were prepared in 0.25% Triton following vitamin K administration to vitamin K-deficient rats as described in Fig. 1. Venom-generated thrombin activity before BaS04 adsorption is shown as open bar and after BaSO, adsorption as horizontally hatched bar. Values shown are means + SEM for five experiments. buffer containing 4-16 NIH units of either immunologically purified liver prothrombin precursor or thermally decarboxylated plasma prothrombin. The reaction was initiated by the addition of 10 pl of vitamin K1 hydroquinone in ethanol. Control reactions received only ethanol. All tubes were incubated at 17°C for 30 min. Incorporation of radioactivity into peptide substrates (peptide carboxylase activity) or endogenous microsomal proteins (protein carboxylase activity) were measured as described earlier (11). Double antibody precipitations of prothrombin were performed with the Ca*+-independent antibodies described by Swanson and Suttie (12) and goat antirabbit IgG. Precipitates of radioactive prothrombin were washed four times with Tris buffer, dissolved in NaHCOs, reprecipitated, and radioactivity determined as for endogenous microsomal protein radioactivity. Generation of thrombin from prothrombin by E&is carinatus venom (ECV) is not dependent on the presence of Gla residue and can be used as a measure of prothrombin precursor concentration (13). Microsomal prothrombin precursor activity was determined after suspending the washed microsomal pellet in 0.25 M sucrose, 0.025 M imidazole, pH 7.2, containing 0.25% Triton X-100. Insoluble particulate matter was removed by centrifugation and the soluble microsomal fraction was assayed for total venom generated thrombin activity (before BaS04 adsorption) and precursor activity (after BaSOl adsorption) using ECV as described earlier (13). Vitamin K-dependent rat plasma proteins were purified as described earlier (14). The citrate eluate of the BaS04

adsorbate was dialyzed overnight against 0.09% NaCl and then freeze-dried. Partial protein decarboxylation was carried out by heating the protein in wczcuo at 110°C for 5 h as described by Poser and Price (15). This resulted in 50-70% decarboxylation as estimated by amino acid analysis following alkaline hydrolysis. Rat liver prothrombin precursor from solubilized microsomal fraction of vitamin K-deficient rat was purified using antiprothrombin agarose affinity chromatography (12).

RESULTS

Vitamin K-dependent activities were studied using detergent-solubilized (0.25 to 1.50%) microsomal preparations obtained from control, vitamin K-deficient nutritionally hypoprothrombinemic rats, and prothrombin precursor-depleted vitamin K-deficient rats. Microsomal precursors were depleted by an intracardial injection of vitamin K. Both peptide and protein carboxylase activities were increased in vitamin K deficiency, but the extent of increase was dependent on the Triton concentration used to solubilize the pellets (Fig. 1). Maximum activity of the carboxylase was seen only at 1.0 and 1.5% Triton, and the enzyme was apparently not completely solubilized by the lower con-

ACTIVITY

Control

Vit.K

def

OF

def+Vit.K 15 min.

VITAMIN

K-DEPENDENT

def+Vit.K 120min

FIG. 3. Effect of the addition of thermally decarboxylated rat plasma prothrombin on vitamin K-dependent carboxylase activity. An amount of thermally decarboxylated rat plasma prothrombin equivalent to the prothrombin precursor level of a vitamin K-deficient microsomal preparation was added to the reaction mixture solubilized in 1.5% Triton. Carboxylase activity of 7-day vitamin K-deficient rats was set at 100%. Vitamin K-dependent carboxylase activity within the addition of decarboxy prothrombin is shown as an open bar (peptide), diagonally hatched bar (protein), while activity observed following the addition is shown as a lightly screened bar (peptide), and heavily screened bar (protein). Other details were as outlined in Fig. 1. Values shown are means f SEM for three experiments.

centrations. When only 0.25% Triton was utilized, the amount of peptide carboxylase activity solubilized was greatly increased following vitamin K injection. The amount of endogenous precursor proteins in the preparation which could act as a substrate for the carboxylase was apparently greatly decreased if the rats were killed as soon as 1 min after a vitamin K injection. Greater than 80% of the protein carboxylase activity was lost in these animals, independent of the Triton concentration of the incubation. Prothrombin precursors represent about 25% of the total microsomal protein precursor pool (12), and changes in these proteins were followed in an attempt to explain the rapid loss of protein carboxylation observed in Fig. 1. Solubilized microsomal fractions were assayed for

CARBOXYLASE

219

ECV-generated thrombin activity before and after BaS04 adsorption (Fig. 2). More than 80% of the total thrombin activity measured in the microsomes of vitamin Kdeficient rats by ECV assay was still present 15 min after vitamin K treatment. The data in Fig. 2 indicate that over half of this pool was sufficiently carboxylated in 15 min to bind to BaS04, but at early time periods much of it was apparently not yet carboxylated. However, the data in Fig. 1 indicate that these proteins do not serve as substrates for carboxylation, suggesting that the liver precursors may be rapidly modified in an unknown fashion when vitamin K is administered. This microsomal pool of carboxylated and uncarboxylated prothrombin precursor decreased rapidly 15 min after vitamin K treatment, and its level fell to 13% of uninjected rats by 2 h. Although the ability to carboxylate endogenous protein was lost from microsomal preparations 120 min after vitamin K treatment, about 60% of the peptide carboxylase activity remained. An amount of thermally decarboxylated (des y-carboxy) vitamin K-dependent plasma prothrombins equivalent to the prothrombin precursor present in the solubilized microsomal system prepared from vitamin K-deficient rats was added to the solubilized microsomal preparations of control, vitamin K-deficient, and vitamin Ktreated, vitamin K-deficient rats (Fig. 3). This decarboxylated rat plasma vitamin K-dependent protein preparation was not a substrate for the carboxylase, even in precursor-depleted microsomal preparations, and did not significantly influence peptide carboxylase activity. Both peptide and protein carboxylation could, however, be stimulated by immunologically purified prothrombin precursors. When increasing amounts of purified precursors were added to the solubilized liver microsomal preparation, protein carboxylation was stimulated in both control and precursor-depleted rat liver microsomes (Fig. 4). Carboxylase preparations from precursor-depleted vitamin K-deficient rat livers utilized exogenous precursors as substrates much better than

220

SHAH,

Prothrombm

SWANSON,

AND

Precursor

Added

SUTTIE

( NIH

Units

I

FIG. 4. Effect of the addition of isolated prothrombin precursors on vitamin K-dependent carboxylase activity. The prothrombin precursor preparation used was purified 1300-fold from solubilized microsomes and had a specific activity of 1350 NIH thrombin units/mg protein. (A) carboxylation of protein; (B) carboxylation of peptide substrates. 0, Microsomes from vitamin Ksufficient rats; 0, microsomes from vitamin K-deficient rats administered vitamin K to deplete endogenous precursor pool.

preparations from normal rat livers. Exogenous precursors also resulted in a stimulation of peptide carboxylation in precursor-depleted vitamin K-deficient rat microsomes but not in normal vitamin Ksufficient microsomes. In a subsequent experiment (Table I), the effect of addition of immunologically purified prothrombin precursors equivalent to those present in incubations from vitamin K-deficient rats to solubilized microsomal preparations of vitamin K-treated, K-deficient rats was

again assessed. A similar increase in protein and peptide carboxylase activities was seen, and it was demonstrated that about 30% of the radioactivity observed in the protein precipitates was present as immunochemically adsorbable prothrombin. DISCUSSION

These data demonstrate that reported activities of the vitamin K-dependent carboxylase are readily altered by changes in

TABLE EFFECT

OF RAT LIVER

PROTHROMBIN

PRECURSOR Vitamin

Precursor added (NIH units)

I ON THE VITAMIN

CARBOXYLATION

K-dependent carboxylation (dpm X 10m3/g liver)

Peptide Activity

K-DEPENDENT

Prothrombin

Protein % Inc

Activity

7% Inc

Activity

5% Inc

None

34.6

-

1.44

-

0.12

-

8.0 16.0

41.6 48.3

20 40

4.93 8.73

242 506

1.30 2.77

983 2208

Note. Vitamin K-deficient rats were administered 1 mg of phylloquinone The precursor preparation that was described in Fig. 4 and incorporation after double antibody precipitation.

ic 120 min prior into prothrombin

to decapitation. was measured

ACTIVITY

OF

VITAMIN

K-DEPENDENT

detergent concentration, and that the extent to which the enzymes are solubilized by low concentrations of Triton X-100 is influenced by prior treatment of the rats with vitamin K. Even though protein carboxylase activity is abolished in microsomes prepared from rats injected with vitamin K 1 min before they were killed, the total amount of potential thrombin activity in these preparations was unaltered. The protein pool was not BaS04 adsorbable, and did not therefore contain an appreciable content of free y-carboxyglutamy1 groups. Either vitamin K administration caused some change in the precursor pool to render it unavailable as a substrate, or carboxylation has been followed by a second event which prevents those proteins from BaS04 adsorption. The large stimulation of peptide carboxylase activity by decarboxylated rat prothrombin reported by Dubin et al. (7) could not be repeated. Attempts to reproduce the response using the conditions described by Dubin et al. (7) and Mack et al. (16) were also unsuccessful. Repeated attempts resulted in only small increases in either peptide or protein carboxylase activity. Similar results have been observed by Soute et al. (8). These data again support our previous finding (6) that the presence of protein substrates stabilizes the enzyme rather than activates it. In contrast to the results with decarboxylated rat prothrombin, a preparation of rat liver prothrombin precursor did act as a substrate and also stimulated carboxylase activity. The response was concentration dependent and was more pronounced when microsomes from precursor-depleted vitamin K-deficient rats were used rather than microsomes from vitamin K-sufficient rats. This observation would again suggest that there is more total carboxylase in the vitamin K-deficient rat.

221

CARBOXYLASE ACKNOWLEDGMENTS

We are grateful to Dr. T. L. Carlisle (Washington University School of Medicine, St. Louis, MO.) for providing amino acid analyses on alkaline hydrolyzed thermally decarboxylated plasma prothrombin.

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9. 10. 11.

12.

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METZ,

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PRICE,

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D. O., WOLFENSBERGER, M., GIRARDOT, MILLER, J. A., AND JOHNSON, B. C. (1979)

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