the journal of chemistry no. 5, issue of 15, pp. of ... · glycine decarboxylase multienzyme...

Val. 261, No. 5, Issue of February 15, pp. 2214-2221,1986 Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Glycine Decarboxylase Multienzyme Complex PURIFICATION AND PARTIAL CHARACTERIZATION FROM PEA LEAF MITOCHONDRIA*

(Received for publication, August 16, 1985)

Joan L. Walker and David J. Oliver From the Department of Bacteriology and Biochemistry, University of Idaho, Moscow, Idaho 83843

The P, H, and T proteins of the glycine cleavage system have been purified separately from pea leaf mitochondria and demonstrate molecular weights of 98,000, 15,500, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of P protein by gel filtration was 210,000, indicating that this enzyme has a native homodimer conformation. Reconstitution assays containing purified P, H, and T proteins and yeast lipoamide dehydrogenase catalyze the oxidation of glycine and demonstrate a strict dependence on pyridoxal phosphate, tetrahydrofolate, NAD+, and dithiothreitol. The released COz, methylamine-H protein intermediate, and methylenetetrahydrofolate are produced in stoichiometric amounts from glycine during the cleavage reaction. H protein acts as co-substrate with glycine during the decarboxylation reaction, demonstrating an apparent K , value of 2.2 p ~ . P and H protein alone jointly catalyze the glycine carb~xy l -~~CO~ exchange reaction in the presence of pyridoxal phosphate and dithiothreitol. L protein of the glycine cleavage system was immunopurified using monoclonal antibodies. An- tigenic and molecular weight similarities of the L protein with the lipoamide dehydrogenase component of the pyruvate dehydrogenase complex were shown suggesting the possibility of common isomers of lipoamide dehydrogenase for the two enzyme complexes.

The glycine cleavage system (aminomethyltransferase; EC 2.1.2.10) catalyzes the reversible oxidation of glycine, yielding carbon dioxide, ammonia, and 5,lO-methylene-H4folate’ (Fig. 1). The 5,lO-methylene-H4folate produced reacts with a second mole of glycine to form serine in a reaction catalyzed by serine hydroxymethyltransferase. The glycine decarboxylase multienzyme complex has been isolated and extensively characterized from animal mitochondria (2-8) and consists of 4 enzymes, the P, H, T, and L proteins (designated PI, Pz, PB, and P4, respectively, in bacterial systems (9-14)). In these animal and bacterial systems, the P protein binds pyridoxal phosphate and catalyzes the release of COz from glycine (5). Glycine forms a Schiff base with the carbonyl group of pyridoxal phosphate (Fig. l), and the carboxyl carbon is released as carbon dioxide. The remaining aminomethyl moiety is

* This work was financed by a grant from the University of Idaho Research Foundation and is Publication 8559 of the Idaho Agricul- tural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: H,folate, tetrahydrofolate; MOPS, 4- morpholinepropanesulfonic acid; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis.

transferred to the lipoic acid group of the small heat-stable H protein (4,5: 15). Although the P protein singly facilitates the release of CO, from glycine only at very low rates, it is considered the true glycine decarboxylase (5). The H protein binds to the P protein prior to the CO, release and regulates the decarboxylase activity. The H and P proteins jointly catalyze a rate of COz release from glycine several orders of magnitude greater than that catalyzed by P protein alone (5 , 6, 11).

T protein catalyzes the release of ammonia from the methylamine intermediate bound to H protein (8,16). Tetrahydro- folate can serve as acceptor for the remaining one-carbon unit of glycine and form 5,lO-methylene-H4folate (Fig. 1). In the absence of H,folate, T protein catalyzes the degradation of the methylamine intermediate at a much slower rate producing ammonia and formaldehyde (16). The electrons accepted by the H protein during glycine oxidation are transferred to NAD+ via the L protein. The activity of this flavin-requiring lipoyl dehydrogenase is analogous to the electron transfer reaction in the decarboxylation of a-keto acids.

Collectively, the P, H, T, and L proteins catalyze the following reversible reaction (3, 9, 17).

Glycine + HIfolate + NAD+ c-) methylene-HAfolate

+ NHg + COz + NADH + H+

P and H protein alone jointly facilitate the glycine-bicarbonate exchange reaction whereby exogenous COz can replace the carboxyl carbon of glycine.

HgN+-CHz-CO; + l4COZ -+ H3N+-CHz-14CO; + COz

In higher plants, a substantial portion of the newly fixed carbohydrates is oxidized back to COz by the photorespiratory pathway (photosynthetic carbon oxidation cycle) (19,ZO). An obligate reaction of this cycle and the source of photorespiratory COz release is the oxidation of glycine via the glycine decarboxylase complex in the mitochondria. Even though electrons from glycine are used to reduce NAD+ to NADH and are, therefore, available to the mitochondrial electron transport chain, the photorespiratory cycle results in net energy consumption. Characterization of the glycine decarboxylase system in plant tissues has been limited to studies with either whole mitochondria (18-20) or a crude protein extract (21). Cofactor requirements, inhibitor sensitivities, and catalytic properties of the enzyme complex in these preparations are comparable to those demonstrated for the animal and bacterial enzyme systems. Attempts to isolate the glycine cleavage activities intact resulted in low yields, suggesting that the plant enzyme is a labile multicomponent complex (21). As a result we have attempted to isolate the enzyme components separately and reconstitute and charac- terize the system.

In this study we have succeeded in isolating the P, H, and

2214

Glycine Decarboxylase Complex from Pea Mitochondria 2215

T equivalents in the pea leaf glycine decarboxylase enzyme complex. Various aspects of the partial reactions, including cofactor requirements and stoichiometric ratios of enzymes and intermediates, were investigated using purified preparations of the proteins. Also in this paper we discuss the appli- cation of monoclonal antibodies to the purification of proteins and their use in these investigations to isolate pea leaf L protein. Approaches are outlined for the production of antibodies against a crude enzyme preparation and the selection of enzyme-specific hybridoma clones.

EXPERIMENTAL PROCEDURES’

RESULTS

Purification of the P, H, and T Proteins of the Glycine Cleavage System-Precipitation of the enzyme complex by cold acetone produced a crude extract exhibiting stable glycine-bicarbonate exchange and C02 released from glycine activities. The C 0 2 release (Fig. 2) and glycine-bicarbonate

@CHO + C!+--COOH e &H=N-CH~-CCY)H I t NH2

coz

+ NH3

aSH + NAD+ SH

+

+ NADH + Hi

FIG. 1. Reaction scheme for glycine cleavage reaction (1).

a

FIG. 2. The effect of crude acetone-extracted mitochondrial protein concentration on the specific activity of the glycine decarboxylase complex. The rates of 14C02 release from [1-”C] glycine were measured as described under “Experimental Proce- dures.” Acetone-extracted protein was supplied in concentrations indicated.

* Portions of this paper (including “Experimental Procedures”) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-2759, cite the authors, and include a check or money order for $2.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

exchange (21) activities were assayed as a function of crude enzyme concentration. Fig. 2 indicates that a linear relation- ship between CO, release rates and the amount of enzyme occurred only at relatively high concentrations of the crude extract. The nonlinearity observed with low extract concentrations suggests that the complex is at least partially disso- ciating under dilute conditions and that one or more component enzymes are present in limiting amounts. Subsequent to the purification of P, H, and T proteins, these enzymes were separately supplied to the acetone-extracted enzyme complex. Fig. 3 demonstrates that the CO2 release activitiy is stimulated by exogenous H protein, indicating that the acetone extract contained limiting amounts of this enzyme and that it was dissociated from the complex. The dissociation of glycine decarboxylase complex during extraction was further evident when attempts to fractionate the crude protein extract with ammonium sulfate showed that no single fraction could catalyze the C02 release reaction and that only a reconstituted system composed of several of these fractions catalyzed glycine decarboxylation (21). The ammonium sulfate fractions essential for glycine decarboxylation were further purified in order to isolate separately the component enzymes of the glycine cleavage complex.

The glycine-bicarbonate exchange reaction could be catalyzed by a combination of the 50-60% and 75-100% saturated ammonium sulfate fractions. Enzyme activity in the 75-100% saturated ammonium sulfate fraction was heat stable and, therefore, assumed to contain the H protein. The P protein activity was isolated from the 50-60% saturated ammonium sulfate fraction. The glycine-bicarbonate activity of the P protein was sensitive to the concentration of H protein, so reactions contained saturating amounts of the heat-stable fraction. The P protein was purified 23-fold overall by a combination of ammonium sulfate and polyethylene glycol precipitations, gel filtration, and ion exchange chromatography (Table I). The decrease in specific activity during DEAE- cellulose anion exchange chromatography suggests some in- stability in the purified form of this enzyme (27). Analysis of the purified P protein by SDS-polyacrylamide gel electrophoresis demonstrated a predominant band with a molecular mass of 98 kDa and greater than 90% purity (Fig. 4). A molecular

0 2 4 6 8 1 0 1 2 H protein ( pM)

FIG. 3. The effect of H protein concentration on the rate of 14C02 release from [1-’*C]glycine catalyzed by crude enzyme extract. The assays contained 200 pg of acetone-extracted mitochondrial protein, purified H protein in the concentrations indicated, and were performed as described under “Experimental Procedures.” In assays where no H protein was supplied, the concentration of H protein in the crude enzyme was determined to be 0.3 GM according to direct linear plotting techniques (26), and the final H protein concentration was corrected for this value.

2216 Glycine Decarboxylase Complex from Pea Mitochondria

TABLE I Purification of the P, H, and Tproteins from pea leaf mitochondria

The enzymes were purified from 250 mg of acetone-extracted mitochondrial protein. P protein activity was quantitated throughout the purification by assaying glycine-bicarbonate exchange activity as described under “Experimental Procedures,” in the presence of the heat-stable H protein fraction of the crude acetone extract. H protein activity was measured similarly in the presence of purified P protein. T protein was assayed via the I4CO2 release from [l-I4C]glycine reaction as detailed under “Experimental Procedures,’’ in the presence of purified P protein, H protein, and yeast lipoamide dehydrogenase.

Enzyme

P protein

H protein

T protein

Purification procedure

Acetone powder 50-60% ammonium

14-20% PEG” sulfate

Sephadex G-150 DE-52


Heat treatment (90 “C,

Sephadex G-50


Sephadex G-100

sulfate

5 min)

sulfate

DE-52

Total units

2100 1800

1500 1200 900

1920 1620

1280

693

2480

1760 1410

Protein Units/ Purifi- mg cation

mg

34 52.9 7

12.4 121.0 16 6.2 193.5 26 5.2 173.1 23

276 7.6 ’

276 7.0 39 42 6

9.2 139 20

3.5 198 28

276 9 40 62 6.9

11 160 18 7 201 22

PEG, polyethylene glycol.

kDa A B C

9 2 1 45

31 21 t FIG. 4. Electrophoretic analysis of purified P, H, and T

proteins. 5-10 pg of P, H, and T proteins were separated on 10-20% gradient SDS-polyacrylamide slab gels. A, P protein (98 kDa); B, H protein (15.5 kDa); C, T protein (45 kDa).

mass of 210 kDa for the nondenatured protein, as determined by gel filtration (data not shown), suggests the existence of P protein homodimer. Based on P protein yields, this enzyme appears to comprise approximately 4% of the acetone-extracted protein.

The crude heat-stable H protein, obtained from the 75- 100% saturated ammonium sulfate precipitate, was further

fractionated for H protein activity subsequent to the purification of P protein. The H protein was purified 28-fold from the crude enzyme extract (Table I) and shown to be essentially pure with a molecular mass of 15.5 kDa as determined by SDS-polyacrylamide gel electrophoresis (Fig. 4). The H protein comprises approximately 3.5% of the total crude acetone extract as calculated from enzyme yields.

Since T protein does not participate in the glycine exchange reaction, its activity throughout the purification can only be assayed by its ability to bestow 14C02 release capacity on the purified P and H proteins and yeast lipoamide dehydrogenase in the presence of the required cofactors. Because the acetone powder exhibits CO, release activity, the T protein activity cannot be assessed there, and the initial concentration of T protein was back calculated from the 60-75% saturated ammonium sulfate fraction. T protein was purified 22-fold from the ammonium sulfate fraction by gel filtration and by anion exchange chormatography due to its basic nature at pH 7.0 (8) (Table I). Fig. 4 confirms that this 45-kDa protein is about 90% pure. The T protein comprises approximately 4% of the acetone powder based on final enzyme yields. These results suggest that the P, H, and T protein of the glycine decarboxylase complex together comprise at least 12% of the crude protein extract and probably represent a similar proportion of the protein in the mitochondrial matrix. Because of the multiple forms of lipoamide dehydrogenase (28) and the dif- ficulty of assigning a specific isoform of this enzyme to glycine decarboxylation, the L protein was not purified from pea mitochondria but was studied immunochemically (see below).

Requirements for I4CO2 Release from [I-l4C]Glycine and Glycine-Bicarbonate Exchange Reactions-The studies reported here were performed to compare the reconstituted plant glycine decarboxylase complex with systems already characterized in animal mitochondria (Fig. 1). The assays for 14C0, release from [l-14C]glycine and the exchange of 14C02 into glycine were performed as described under “Experimental Procedures,’’ using purified P, H, and T proteins, and yeast lipoamide dehydrogenase. As predicted by the model, glycine decarboxylation activity was maximal in the presence of all four enzymes and exhibited a strong dependence on pyridoxal phosphate, NAD+, H4folate, and DTT (Table 11). The CO, release reaction demonstrated an absolute requirement for the purified P protein and H protein, consistent with the proposal that these two enzymes jointly comprise the func-

TABLE I1 Enzyme and cofactor requirements for ‘‘CO, release from [l-“C/

glycine and glycine-bicarbonate exchange reactions Except for the deletions indicated, reactions contained 30 pM

pyridoxal phosphate, 0.5 mM H4folate, 2 mM DTT, 1 mM NAD, 10 mM glycine, and 20 mM bicarbonate. Purified P, H, and T proteins were supplied at approximately 100 units/assay. Assays contained 50 pg of yeast lipoamide dehydrogenase. Reactions were performed and quantitated as described under “Experimental Procedures.’’

NaH“C02 Condition [l-“C]glycine “Cor from [1-“ClGlycine from

Complete - P protein - H protein - T protein - L protein - P L Y - H4folate - NAH - DTT - Glycine

nmol/rnin 81 1 2

14 10 13 0

16 8

% control 100

1 2

18 12 16 0

20 10

nmol/min 114

0 2

184 78 16

238 95 81 0

% control 100

0 2

162 68 14

210 84 71 0

PLP, pyridoxal phosphate.


tional glycine decarboxylase. Low levels of activity (lo-20%) could be demonstrated in the absence of T protein or L protein where some portion of the intermediate-bound H protein may be participating in the exchange reaction between unlabeled CO, in the media and [1-14C]glycine, thereby releasing small amounts of WOZ.

The reconstituted pea leaf enzyme complex utilized exogenous yeast lipoamide dehydrogenase. Although the COZ release reaction showed a strong dependence upon the cofactors pyridoxal phosphate, H*folate, and NAD’, low rates of CO2 release were detectable in the absence of pyridoxal phosphate and NAD’ (Table II). The reason for this is not clear, but in the case of pyridoxal phosphate may indicate some enzyme- cofactor binding and co-purification (5). Deletion of DTT from the reconstituted system dramatically reduced the decarboxylation activity (Table II). The function of this reduc- ing reagent in this system has not been characterized.

Consistent with the model, the glycine-bicarbonate exchange reaction demonstrated dependence upon the presence of both the P and H proteins (Table II). Omission of pyridoxal phosphate reduced glycine synthesis dramatically, but low residual rates indicated again that some pyridoxal phosphate bound to the P protein survives enzyme purification (Table II). Deletion of T protein or H4folate stimulated the exchange reaction as much as Z-fold (Table II). This stimulation suggests that the methylamine-bound H protein, which readily acts as substrate for the synthesis of methylene-H4folate catalyzed by T protein, also participates in the P protein- catalyzed synthesis of glycine. By preventing the T protein- catalyzed breakdown of the methylamine-bound H protein, the intermediate is directed to the glycine-bicarbonate exchange reaction. The elimination of lipoamide dehydrogenase or NAD+ results in a decrease in bicarbonate exchange rates by 15-30% (Table II). The lipoamide dehydrogenase is not thought to be directly involved in the exchange activity, but these effects may be explained by its capacity to convert the inactive reduced form of H protein to the active oxidized state. If the purified H protein preparation contains a signif- icant proportion of reduced lipoic acid residues, which are unable to bind the methylamine moiety, the presence of lipoamide dehydrogenase and NAD+ may increase the number of oxidized lipoic acid prosthetic groups available.

Stoichiometric Analysis of the Glycine Decarboxylase Com- plex-The glycine decarboxylase enzyme complex catalyzes the release of CO, from giycine in a I:1 molar ratio. The carboxyl carbon of glycine, released as COZ, can be assayed by the quantitation of 14C02 produced from [l-14C]glycine. The methylamine moiety of glycine remaining after decarboxylation is transferred to the lipoic acid chains of H protein. This H protein-bound intermediate was measured by con- ducting an identical COZ release reaction with [2-l*C]glycine. The NH,-‘4CHz-bound H protein was isolated from the reaction mixture by gel filtration and quantitated by liquid scintillation counting. Labeled H protein intermediate was produced in a ratio of 0.85 with respect to CO, released (Table III). A small amount of radioactivity equivalent to 2.4 nmol of the methylamine-H protein intermediate was eluted with the 2 nmol of P protein in the assay. This may suggest that approximatley 1 mol of H protein binds to each mol of P protein subunit. The reaction between T protein and H protein was investigated by incubating the labeled methylamine- bound H protein with purified T protein in the presence of H,folate. Radioactivity was released from the protein and recovered in a compound that formed an adduct with dimedone. It is well known that the methylene moiety of 5,10- methylene-H4folate reacts with dimedone (16). The 5,10-

TABLE III

The role of H and T proteims: synthesis of the methylamine intermediate and methylerwH4folate

W02 from [I-‘%]glycine was measured in assays Containing 30 pM pyridoxal phosphate, 20 mM MOPS.KOH (pH 7.1), 2 mM DTT, 2.0 nmol of P protein, 66 nmol of H protein, and 10 mM [l-W] glycine. H3N+-14CH2-H protein was produced in assays identical to that described above except that 10 mM [2-‘*C]glycine was supplied as substrate. Labeled H-protein intermediate was isolated on a Seuhadex G-50 column and counted by liquid scintillation. 5,10- “e~Hz-&folate was produced in assays containing 20 mM MOPS. KOH (pH 7.0), 1.5 mM J&folate, 0.5 mg of T protein, and the labeled H protein intermediate produced in assay described above. Labeled methylene-bfolate was precipitated and quantitated as a dimedone adduct.

Partial reactions Total nmol Ratios (substrate + product) of product (substrate/product)

Glycine + CO2 33 1.0 Glycine + methylamine-H protein 28 0.85 Methylamine-H protein + meth- 26 0.93

ylene-Hrfolate

methylene-Hlfolate was produced in a ratio of 0.93 with respect to the labeled H protein intermediate and 0.79 with respect to the ‘*CO2 initially released (Table III). These results demonstrate that CO*, methylamine intermediate, and 5,10- methylene-H4folate are produced during the oxidation of gly- tine in equimolar amounts as predicted by the model. The pea leaf H protein acts as co-substrate with glycine during the decarboxylation catalyzed by P protein, accepts the methylamine moiety, and donates the intermediate to the T protein.

Hybridoma Selection-Hybridoma technology allows for the production of antibodies specific for an unpurified protein and their use in isolating the protein from crude preparations. Monoclonal antibodies were necessarily produced to a crude preparation of mitochondrial proteins because components of the pea leaf glycine decarboxylase system had not been identified or purified. Detection of positive clones was based on the ability of their secreted immunoglobulins to inhibit the decarboxylation of glycine. Several clones were found to affect enzyme activity, but the investigations described here em- ployed only the clone-producing antibodies of highest affinity and consistent inhibition characteristics.

Antigen identification and Purification-The antigen was isolated by immunoaffinity chromatography. Monoclonal antibodies were immobilized on Staphyloccocus Protein A conjugated to Sepharose. The crude enzyme was applied to the column and eluted in a purified antibody-antigen complex. Electrophoretic analysis of the immune complex (Fig. 5) indicated that the monoclonal antibody specifically recognized a 59-kDa protein. The immynoadsorption technique also isolated smaller more variable amounts of the 15.5-, 98-, and 45- kDa proteins (data not shown). The decreased amounts of these enzymes compared to the immunopurified 59-kDa protein suggests extensive dissociation of the glycine decarboxylase complex under dilute conditions.

The antigen was identified as L protein based on antibody- dependent inhibition of partial reactions catalyzed by the glycine decarboxylase complex (Fig. 6A). The glycine-bicarbonate exchange activity, which requires only P and H proteins, was not affected by the presence of monoclonal antibody. Lipoamide dehydrogenase activity associated with the acetone powder was inhibited more than 90% with antibody concentrations identical to those required for comparable inhibition of glycine decarboxylation. This tentative identification of the 59-kDa protein as L protein was confirmed when yeast lipoamide dehydrogenase added in excess reversed the

2218 Glycine Decarboxylase Complex from Pea Mitochondria

1 L protein (59 kDu)

? 'IgG H

* -1gG L

FIG. 5. Electrophoretic analysis of immunoprecipitated L protein. The lipoamide dehydrogenase was isolated on a Sepharose CL-4B column as described under "Experimental Procedures." The enzyme was co-eluted with the monoclonal antibody and separated on a 15% SDS-polyacrylamide slab gel. The electrophoretic pattern indicated the presence of heavy (IgG H) and light (IgG L) chains of immunoglobulins, and the 59-kDa L protein.

antibody-dependent inhibition of C 0 2 release from glycine (Fig. 6 B ) . Neither excess H, P, or T protein affected the inhibitory characteristics of the immunoglobulin.

The lipoamide dehydrogenase of the pyruvate dehydrogenase complex exhibits a molecular weight of 56 kDa by electrophoretic analysis in SDS (29). Molecular weight similarities between the lipoamide dehydrogenase of this enzyme system and the glycine decarboxylase complex prompted studies of antigenic homology. Incubation of the two complexes with equal concentrations of antibody produced indistinguishable inhibition profiles for both lipoamide dehydrogenase-containing enzyme complexes (Fig. 7). These results indicate that the immunoglobulin binds to lipoamide dehydrogenase at or near an antigenic site common to both enzyme complexes, possibly at the active site.

DISCUSSION

We have reported studies on the purification and partial characterization of the reversible glycine cleavage complex in pea leaf mitochondria. Our results indicate that the plant enzyme complex is composed of four enzymes catalytically equivalent to the animal components: the pyridoxal phosphate-dependent P protein, the lipoic acid-bound H protein, the H4folate-dependent T protein, and the lipoamide dehydrogenase L protein. Together these enzymes catalyze the oxidative decarboxylation of glycine. In addition, the isolated enzymes demonstrate several partial reactions: the P and H proteins jointly catalyze the synthesis of glycine from bicarbonate and a methylamine reaction intermediate, and T protein facilitates the degradation of the H protein-bound methylamine intermediate to ammonia, simultaneously catalyzing the formation of 5,lO-methylene-H4folate. Motokawa and Kikuchi (1) have proposed a model for reaction mechanisms based on the animal enzyme system (Fig. 1) that is also consistent with some bacterial studies (2, 10). Our investigations of the partial reactions, cofactor requirements, and stoichiometric ratios of enzymes and intermediates indicate that this model adequately describes the mechanism of glycine cleavage in pea leaf mitochondria.

LlPOAMlDE DEHYDROGENASE -rn v)

80- t >

N z W

A

Z

c m = 20 z

0

o-o.-7"o, , , , , , I

2 4 6 8 10 12 MONOCLONAL ANTIBODY (1-19)

PROTEIN (pg)

FIG. 6. Identification of the antigenic component of the glycine decarboxylase enzyme complex. A, inhibition of the partial reactions of glycine decarboxylase by monoclonal antibody. Assays containing 150 pg of crude enzyme extract were incubated with indicated amounts of purified antibody for 1 h on ice. Control rates for the COz release from glycine and glycine-bicarbonate exchange reactions were 20 nmol/min.mg and 10 nmol/min.mg, respectively. Lipoamide dehydrogenase activity was measured as described (3) , and the control rate of NADH oxidation in the crude enzyme extract was 12 nmol/min.mg. B, effects of purified P, H, and T protein and lipoamide dehydrogenase on antibody inhibition of COZ release from glycine activity. Assays containing 150 pg of crude enzyme extract, 3 pg of purified monoclonal antibody, and P, H, and T protein, and lipoamide dehydrogenase in the amounts indicated were incubated for 1 h on ice. I4CO2 released from [l-"C]glycine was measured as described under "Experimental Procedures." W, P protein; A-A, H protein; M, T protein; U, lipoamide dehydrogenase.

In reconstitution assays, the purified pea leaf enzymes of the glycine cleavage system catalyze the same reactions dem-


o.............,..I 2 4 6 8 x) 12 14 16 18 20

MONOCLONAL ANTIBODY (pg)

FIG. 7. Monoclonal antibody inhibition profiles for the glycine decarboxylase and pyruvate dehydrogenase enzyme complexes. Increasing concentrations of purified monoclonal antibody were incubated with 0.2 mg of acetone-extracted mitochondrial protein for 1 h on ice. Rates of 14C02 release from [l-’4C]glycine were measured as described under “Experimental Procedures.” Pyruvate dehydrogenase was assayed by measuring pyruvate-dependent NAD+ reduction at 340 nm as described (30). Control rates for the glycine decarboxylase and pyruvate dehydrogenase complexes were 24 and 9.5 nmol/min . mg, respectively.

onstrated in earlier plant tissue work using whole mitochondria and crude enzyme extracts (21, 23). Together, the P, H, T, and L (yeast lipoamide dehydrogenase) proteins in the presence of pyridoxal phosphate, NAD+, H4folate, and DTT catalyze maximal rates of COz release from glycine. The reaction exhibits an absolute requirement for P and H protein consistent with the model in Fig. 1 (Table 11). As predicted, the decarboxylation activity of P protein is enhanced dramatically by the presence of H protein, suggesting a role for the heat-stable protein in leaf mitochondria comparable to that observed in animal systems. Studies with plant glycine decarboxylase components confirm the binding of P protein and H protein as demonstrated by coelution during gel filtration. The H protein appears to dissociate from the complex under dilute conditions as evidenced by the fact that exogenously supplied H protein will stimulate the COz release and bicarbonate exchange activity in the acetone extract (Fig. 3). Addition of purified P and T proteins and yeast lipoamide dehydrogenase did not stimulate these activities in the crude enzyme preparation (data not shown).

Kinetic studies of avian glycine decarboxylase activity indicated that both glycine and H protein function as co- substrates for P protein activity (15). The P protein displayed Michaelis-Menten kinetics with respect to both substrates. Fujiwara and Motokawa (15) proposed a random Bi Bi mechanism for the decarboxylase reaction catalyzed by P protein where glycine and H protein bind independently a t separate sites on the P protein prior to the release of any products. Fig. 3 indicates that the H protein concentration is limiting in the crude acetone-extracted leaf enzyme. Rates of CO, release from glycine exhibit typical saturation kinetics with increasing concentrations of exogenously supplied H protein, demonstrating an apparent K, of approximately 2.2 p ~ . This indicates that this enzyme may be acting as co-substrate to the P, T, and L proteins: as co-substrate with glycine for P protein activity, as methylamine-bound intermediate with Hafolate for T protein, or in reduced form with NAD+ for L protein activity.

Both the small heat-stable H protein, with a molecular mass of 15.5 kDa, and the more labile P protein, with a subunit molecular mass of 98 kDa, appear to be very similar to the corresponding animal enzymes (4, 5). The H protein

binds to the P protein subunits with a molar ratio of one. The P protein demonstrated a molecular mass of 210 kDa by gel filtration, possibly indicating a native homodimer conformation, similar to that described for the animal enzyme (5). It follows from the stoichiometric analysis that two H proteins bind to each P protein dimer. These results are consistent with avian liver studies (5) but differ from some bacterial investigations (27) where the P protein (heat-labile component) is an && tetramer of M, = 230,000. This bacterial enzyme is thought to be primarily operative in the direction of glycine synthesis, and these authors (27) suggest that the biochemical difference between the animal and bacterial glycine cleavage systems may reflect different physiological roles of the enzyme.

Characterization of the animal (4) and bacterial (10, 13) glycine cleavage complexes established that the H protein binds the aminomethyl intermediate via covalently bound lipoic acid chains. In reconstitution assays, lipoic acid could at least partially substitute for H protein activity in these systems’ (10, 13). The presence of lipoic acid was earlier indicated in studies with crude pea leaf enzyme preparations where H protein activity was inhibited by arsenite, presum- ably by reacting with lipoamide (21).

Based on the molecular weight determinations, the pea leaf T protein is composed of a single polypeptide with a molecular mass of 45 kDa (Fig. 4). The value is dissimilar to that of 33 kDa determined for rat and 38 kDa for avian T protein (3, 16). While this implies physical differences as yet uncharac- terized, T proteins from both plant and animal sources appear to be catalytically equivalent. H protein has been shown to readily associate with T protein to form a fairly stable complex in animals (16). In our studies with purified H and T proteins, one-carbon units were produced in stoichiometric amounts equal to the methylamine intermediate and COz released (Table 111). As in animal systems (16), the T protein-mediated degradation of methylamine is strictly dependent on H4folate (Table 11). Previous studies with crude enzyme preparations have confirmed the reversibility of this reaction (21).

Our studies show that monoclonal antibodies are applicable to the isolation and purification of a specific component from a crude tissue extract. Screening of fusion products for clones producing antibodies specific for the desired protein requires a fast conclusive activity inhibition assay. This approach will necessarily select only those clones whose secreted immunoglobulins bind to the protein so as to affect its activity. Monoclonal antibodies were produced against glycine decarboxylase, selected on the basis of their capacity to inhibit the C 0 2 release reaction, and later established to be specific for the L protein. We were unsuccessful in isolating the entire pea leaf glycine cleavage system by immunoaffinity techniques due to the dissociation of the enzyme complex under dilute conditions. The monoclonal antibody was used to isolate the lipoamide dehydrogenase, but high antibody-antigen affinity prevented the dissociation of the immune complex and investigation of L protein catalytic properties. L protein exhibited a molecular mass of 59 kDa, similar to the lipoamide dehydrogenase of pyruvate dehydrogenase complex, and the antibody inhibited both enzymes equally. Current knowledge of the lipoamide dehydrogenase in animals indicates the existence of several major forms (28). Depending on isolation and assay conditions, 2-13 isomers have been detected. The chemical basis of the differences among the molecular forms is uncertain, but they often demonstrate similar electrophoretic patterns and catalytic and immunochemical properties. Whether the pea leaf glycine decarboxylase complex utilizes one or more of the lipoamide dehydrogenase isomers in com-

Glycine Decarboxylase Complex from Pea Mitochondria

mon with pyruvate dehydrogenase cannot be determined from our studies.

Our studies indicate that the glycine decarboxylase multienzyme complex exists in solution predominantly as four separate enzymes following acetone extraction. This is sup- ported by the lack of linearity observed for CO, release rates as a function of crude enzyme concentration (Fig. 2), and the ability of exogenously supplied H protein to stimulate the rates of glycine decarboxylation (Fig. 3). In addition, the P, H, and T proteins can be cleanly separated by ammonium sulfate fractionation and an antibody specific for the lipoamide dehydrogenase precipitates negligible quantities of these three enzymes (Fig. 5).

As in the animal system (31,32) the glycine cleavage system is confined to the mitochondria, either as peripheral proteins bound to the inner face of the inner mitochondrial membrane or free in the matrix. Whether the enzymes of the glycine decarboxylase system exist as a complex cannot be deduced from our studies under dilute conditions. The minimal dis- tances required for H protein movement between P, T, and L proteins and, therefore, rapid catalytic rates may be achieved due to the small inner mitochondrial volume and resulting high local concentrations of the enzymes.

1.

2. 3.

4.

5.

6.

7.

8.

9.

REFERENCES Motokawa, Y., and Kikuchi, G. (1974) Arch. Biochem. Biophys.

Kikuchi, G. (1973) Mol. Cell. Biochem. 1 , 169-187 Motokawa, Y., and Kikuchi, G. (1974) Arch. Biochem. Biophys.

Fujiwara, K., Okamura, K., and Motokawa, Y. (1979) Arch.

Hiraga, K., and Klkuchi, G. (1980) J. Biol. Chem. 2 5 5 , 11664-

Hiraga, K., and Kikuchi, G. (1980) J. Biol. Chem. 255, 11671-

Kikuchi, G., and Hiraga, K. (1982) Mol. Cell. Bioehem. 4 5 , 137-

Okamura-Ikeda, K., Fujiwara, K., and Motokawa, Y. (1982) J.

Kochi, H., and Kikuchi, G. (1969) Arch. Biochem. Biophys. 132,

164,634-640

164,624-633

Biochem. Biophys. 197,454-462

11670

11676

149

Bid. Chem. 2 5 7 , 135-139

359-369

10. Kochi, H., and Kikuchi, G. (1974) J. Biochem. (Tokyo) 75,1113-

11. Klein, L. M., and Sagers, R. D. (1966) J. Biol. Chem. 2 4 1 , 197-

12. Klein, S. M., and Sagers, R. D. (1966) J. Biol. Chem. 241 , 206-

13. Klein, S. M., and Sagers, R. D. (1967) J. Biol. Chem. 2 4 2 , 301-

14. Robinson, J. R., Klein, S. M., and Sagers, R. D. (1973) J. Biol.

15. Fujiwara, K., and Motokawa, Y. (1983) J. Biol. Chem. 258,8156-

16. Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1984) J.

17. Kawasaki, H., Sato, T., and Kikuchi, G. (1966) Biochem. Biophys.

18. Walker, G. H., Oliver, D. J., and Sarojini, G. (1982) Plant Physiol.

19. Woo, K. C., and Osmond, C. B. (1976) A u t . J. Plant Physiol. 3 ,

20. Keys, A. J. (1980) in The Biochemistry of Plants. A Comprehensive Treatise (Strumpf, P. K., and Conn, E. E., eds) Vol. 5, pp. 359- 374, Academic Press, New York

21. Sarojini, G., and Oliver, D. J. (1983) Plant Physiol. 7 2 , 194-199 22. Laemmli, U. K. (1970) Nature 227,680-685 23. Walker, G. H., Sarojini, G., and Oliver, D. J. (1982) Biochem.

Biophys. Res. Commun. 107,856-861 24. Miskell, B. B., and Shiigi, S. M. (eds) (1980) Selected Methods in

Cellular Immunologypp. 351-372, W. H. Freeman and Co., San Francisco

25. Davis, J. M., Lennington, J. E., Kubler, A. M., and Conscience, J. F. (1982) J. Immunol. Methods 50, 161-171

26. Eisenthal, R., and Cornish-Bowden, A. (1974) Biochern. J. 139 ,

27. Garboldi, R. T., and Drake, H. L. (1984) J. Biol. Chem. 259 ,

28. McManus, I. R., and Cohen, M. L. (1975) in isozymes I-Mokc- ular Structure (Markert, C. L., ed) pp . 621-636, Academic Press, New York

29. Furuta, S., Shindo, Y., and Hashimoto, T. (1977) J. Biochem.

30. Randall, D. D. (1982) Methods Enzymol. 89,408-414 31. Hiraga, K., Kochi, H., Motokawa, Y., and Kikuchi, G. (1972) J.

32. Motokawa, Y., and Kikuchi, G. (1971) Arch. Bwchem. Biophys.

1127

205

209

305

Chem. 248,5319-5323

8162

Biol. Chem. 259,10664-10668

Res. Commun. 23,227-233

70,1465-1469

771-785

715-720

6085-6089

(Tokyo) 81,1839-1847

Biochem. (Tokyo) 72,1285-1289

146,461-466

Glycine Decarboxylase Complex from Pea Mitochondria 2221 Supplementary Material to

Glycine Decarboxylase Multienzyme complex: Purification and Partial Characterization from pea Leaf Mitochondria

Joan L. Walker and David J. Oliver

EXPERIMENTAL PROCEDURES

. . ~ ~ ~ . . . ~ ~~ ~

3-pk-old plants. The tissue was rinsed in distilled Water and chilled to 4 C Subsequent homogenization and centrifugation Steps were performed at 0-4 'C according to (18). The leaf tissue was ground in a Waring blender in a medium with 0.5 H Sorbitol 30 mM MOPS-KOH (pH 7.51, 2 mM EDTA, and 0.2 % bovine S e m m albumin, then flltered successively through one layer of nylon mesh and two layers of Miracloth. The filtrate was centrifuged at 2,000 x g for 5 min to remove chloroplastic membranes. Mitochondria were collected from the supernatant fluid at 10,000 x g for 15 min. The differential centrifllmation stem3 were reveated to remove additional Chloroplast contamination. The crude iitochondrial pellet was resuspended to approximately 1 mg chlorophylllml. The glycine decarboxylase complex was extracted from the mitochondria with acetone. The mitochondrial susgensian w a s dripped slowly into a ten-fold excess Of 95% acetone at -20 C with Constant stirring. The precipitating protein was allowed to settle and collected on Whatman NO. 42 filter paper under vacuum. The greenish

.~~~~~~~ ~~ ~

precipitate was rinsed several times- Gith fresh acetone to remove additional lipids, then thoroughly dried under vacuum at 4 'C. Filter papers were either stored at -20 c or resuspended into a minimal amount of 20 mM MOPS-KOH (pH 7.01, 1 mM DTT (MOPS-DTT buffer) on ice. Filter paper fragments and undissolved proteins were removed by centrifugation. and the straw-colored suDernatant fluid containinq the enzyme complex was stored at

&;paration was used as i ,starting point for purifying the component enzymes Of the glycine decarboxylase complex.

extraction yielder approximately 250 mg of crude enzyme which. was Purification of the Glycine Decarboxylase Component Enzymes. Acetone

'C unless otherwise specified. Ammonium sulfate fractionations were esuspended to 5 mglml. All purification procedures were performed at 0-4

carried Out by the gradual addition of solid Salt to the acetone powder

precipitates were collected by centrifugation at 10,000 x g for 15 min, supernatant fluids followed by stirring for 30 min on ice. Protein

resuspended in a minimal volume of MOPS-DTT buffer and dialyzed against the same. (We gonsidered a 100% saturated ammonium sulfate solution to be 4.1 M at 0-4 C.1 Unless Otherwise stated, all gel filtration and anion exchange columns were equilibrated and developed with the MOPS-DTT buffer. Protein separation by SDS polyacrylamide gel electrophoresis was on 10-20% gradient slab gels essentially according to 122).

Purification of P protein: The crude enzyme extract was fractionated with 50 to 60% saturated ammonium sulfate. The protein precipitate was resuspended, dialyzed, and further fractionated with 14 to 20% PEG 6000

buffer and Separated on a Sephadex G-150 column (1.5 x 80 cml. The active (w/v). The precipitate was resuspended into a minimal amount Of MOPS-DTT

developed with MOPS-DTT buffer containing a linear gradient of 0-0.4 M KC1. fractions were applied to a DE-52 column (1.0 x 20 cml. The column was

P protein activity was determined throughout the purification by assaying under the conditions described for the slvcine-bicarbonate exchange reaction and in the presence of the heat stabl; H protein fraction of the crude enzyme. The purity of P protein was confirmed by SDS polyacrylamide gel electrophoresis. The molecular weight was determined by SDS-PAGE and gel filtration on Sephadex G-150.

extract with 75 to 100% saturated ammonium sulfate. The protein Purification Of H protein: H protein was fractionated from the crude

precipitate was resuspended and incubated at 90 C for 5 mi". The preparation was subjected to one or more freeze-thaw cycles to promote protein precipitation. The precipitate was removed by centrifugation and the clear Supernatant fluid which contained H protein was applied to a Sephadex G-50 column 11.5 x 80 cm) and developed with MOPS-DTT buffer. Active fractions were pooled, and purity and molecular weight were estimated by SDS-PAGE. H procein activity was estimated throughout the purification procedure by assaying for glycine-bicarbonate exchange activity in the presence of purified P protein.

Purification of T protein: The crude acetone extract was fractionated with 60 to 75% saturated ammonium sulfate. The dialyzed protein precipitate was applied to a Sephadex 6-100 column (2.5 x 80 c m l and the active fractions were collectively applied to a DE-52 column (1 x 20 cml equilibrated with MOPS-DTT buffer. The activity was contained in the

concentrated by precipitation in 75% saturated ammonium Sulfate and the initial pass and wash which were collected and pooled. T protein was

resuspended pellet dialyzed. The molecular weight of T protein was determined by SDS-PAGE. T protein activity was estimated throughout the

'%o -release from (1- Clglycine in the presence of purified P protein, H rification (with the4 exception of the acetone extract) by assaying the

protain and yeast lipoamide dehydrogenase.

Glycine Decarboxylase Activitr Assays. Glycine-Bicarbonate Exchange Reaction: The 200 p l reaction contained 20 mM MOPS-KOH (pH 7.01. 0.1 mM pyridoxal phosphate, 20 mM glycine, and 2 mM DTT. Assays contained either the crude enzyme extract or preparations of P protein and H protein. Reaptions were initiated by the addition of 4 pmoles of freshly prepared NaH CO at pH 7 2 (56 mCilmmol). Exchange reactions were terminated after

driven off and the acid-stable (1- Clglycine estimated by Itquid 30 min3at 25 %'by adding 25 p l of glacifa acetic acid. Dissolved CO was

Scintillation counting a5 described 1231.

14C0.-Release from ll-14CIGlvcine Reaction: The 1.0 rnl reaction contained '20 mM MOPS-KOH (pH 7.01, 30 pM pyridoxal phosphate, 0.5 mM H folate, 1 mM NAD , and 2.0 mM DTT. The assay contained either crude &vme extracts or reconstituted svstems cornnosed Of P. H. and T Drotein preparations. In reconstituted reictions, y'east lipoamide dehydrbgenase (50 ~glassay) was supplied in the absence of isolated L prypin. The reactmns were initiated by the addition of lOpoles of (1- Clglycine

H so The co was trapppg and estimated as described 1231. A unit of (49.5 mCi/mmol14and terminated after 10 mi" at 25 C by adding 100 pl of 2N

a&i$ity is definad as "mol Co released per min, and reaction rates. are expressed in unitslmg protein.

~~~~ 1~ ~ ~~~~~ ~~~

Stoichiometric Anal sis Of GI cine Decarbox lase Rssay. Formation of co and H protein Bount; IntZ%tedyate from Glyc%e: The CO -release a6say wag performed as described above except that 200 pg of P p3otein (2 "moll and 1.0 ma 166 nmoll Of ourified H-orotein were added. NO T Drotein, lipoamide deh$drogenasb, H folate, or -NAD+ were included in tie assay media. The reaction was4 initiated by the addition of 10 pmoles Of

second CO2-release assay was simultaneously performed with conditions 11- Clglycine and terminated after 60 rnin by acidification as above. A

umoles of 12- c141vcine. The reaction was terminated on ice after 60 mi". identical tollthe first except that the reaction was initiated with 10

$he entire second- reaction mixture was applied to a Sephadex G-50 column

radioactivity by liquid Scintillation counting. 11.5 x 80 cml and the H protein fractions pooled and assayed for associated

The column-purified H pro& labelled from (2- CIglyain% was incubated in Formation of 5,10-14CH -Tetrahydrofolate frPt I4CH NH -bound H Protein:

a 1.0 m 1 reaction mixture containing 20 mM MOPS-KOH (pH 7.01, 1.5 mM H folate and 0.5 mg T protein. The reaction was terminated after 60 mi" b$ addiiion of 0.1 ml of 50% trichloroacetic acid, and the DreciDitated p;ote*$ was removed by centrifugation. 5.10- CH -tetrahydrofolate was recovered from the supernatant fluid as a

The- rkleased

dimedone gdduct 1161 and estimated by liquid scintillation counting.

G1 clne Decarbox lase Com lex. Production, Selection and Purification of Monoclonal Antibodies A ainst

1m;uiization Proczdure: A n t i b o d h r e produced to whole mitochondria Antigen P r e p a r a t i h

isolated from ea leaves. A crude mitochondrial suswnsion was obtained by the differential centrifugation procedure described above. Remaining chloroplast contamination was removed from the mitochondria by fractionation on discontinuous Percoll gradients (181. The purified mitochondria were resuspended in 10 mM sodium phosphate (pH 7.61, 0.15 M NaCl (PBS) to a final protein concentration of 5 mglml.

Four-to-Six-Week-Old BALBIC mice were injected with about 200 pg of Percoll-purified pea leaf mitochondria emulsified with an equal volume of complete Freund's adiuvant. Each leq received an injection of 0.05 ml delivered intramuscu1a;ly. The mice were-re-immunized 15-and 20 days later with 200 pg of mitochondrial protein emulsified in Freund's incomplete adjuvant. Serum samplps taken by heart punfture were assayed for their ability to inhibit CO -release from (1- Clglycine by crude acetone extracts under incubation 'and reaction conditions identical to those described below for hybridoma screening. Cell fusions were performed with spleen cells from animals exhibiting the Strongest serum-dependent inhibition of glycine decarboxylase activity. The fusion of spleen cells with the P3X63Ag8.653 myeloma cells and hybridoma cloning procedures in semi-solid medium were performed as described (24.251.

decarboxylase were selected on the basis of their ability to inhibit the Hybridoma Screening: Clones producing antibody against glycine

release of 14c0 from ~l-~~c~glycine I" a 1.0 ml reaction mixture 100-ZOO pg of acatone extracted mitochoAdria1 protein was incubated on i d with 0.1 ml of the untreated hybridoma culture supernatant fluids for 1 h. At the end of the incubation period, reaction mixtures were adjusted to 30 r M , pyridoxal phospppte, 0.5 mM H folate and 1 mM NAD. The reactfpns Were

measured by liquid scintillation counting? Inhibition studies were mltlated with (1- Clglycine as4far the CO -release assay, and C02 was

repeated with affinity-purified immunoglobulins from clones found to be positive.

Purification of Monoclonal Antibodies: Positive clones were Cultured in 500 ml tissue culture flasks or injected intraperitoneally in BALBlc mice

concentrated from the hybridoma medium or ascitic fluid by precipitating in for ascites tumor production in BALBIC mice. Immunoglobulins were

50% saturated ammonium sulfate, collecting the protein at 20,000 x g for 15 min and dialyzing against PBS IpH 7.61. The antibodies were purified from

column equilibrated with PBS IpH 7.61. After a 30 mi" incubation at room the dialysate on a Sepharose CL-4B Staphylococcal protein A-conjugated

were eluted with 0.1 M Sodium citrate (pH 5.5). The protein-containing temperature, the column was washed wlth PBS and the monoclonal antibodies

fractions were pooled and the pH adjusted to pH 7.6 with TriS buffer. The column-purified antibodies were concentratgd by precipitation with 50% Saturated ammonium sulfate and stored at "20 C.

with an immunoaffinity column. Concentrated immunoglobulins were bound to Antigen Purification: Glycine decarboxylase components were purified

washing with PES (pH 7.61, acetone-extracted mitochondrial protein was a Sepharose CL-48 protein A-conjugated column as described above. After

applied to the column and allowed to incubate for 1 h at room temperature. Extraneous proteins were eluted with PBS and the antibodylantigen complexes removed in 0.1 H sodium citrate (pH 5.5!. Eluted Samples were precipitated in 10% trichloroacetic acid on ~ c e . Pellets were collected by centrifugation and Washed in cold acetone. The protein precipitates were dissolved with boiling in a Sample buffer containing 1.5% each of SDS and

Materials. Radiolabeled (1-l4C1 and (2-14Clglycine and NaH14C03 were purchased from Research Products, Inc. All polyacrylamide gel electrophoresis reagents were of highest grades available from Bio-Rad Laboratories. Lipoamide dehydrogenase from yeast was purchased from Sigma. Complete and incomplete Freund's adjuvant were obtained from Difca Laboratories. Percoll, Sephadex, and Staphylococcal Protein A-conjugated

chemicals were of the purest grade available from commercial suppliers. Sepharose CL-48 were obtained from Pharmacia Fine Chemicals. A11 Other

t-mercaptoethanol and analyzed on 15% polyacrylamide slab gels.

the journal of chemistry no. 5, issue of 15, pp. of ... · glycine decarboxylase multienzyme...

Documents