Vol. 153, No. 3JOURNAL OF BACTERIOLOGY, Mar. 1983, p. 1493-15010021-9193/83/031493-09$02.00/0Copyright © 1983, American Society for Microbiology

Molecular Properties of Succinate Dehydrogenase Isolatedfrom Micrococcus luteus (lysodeikticus)

BRIAN A. CROWE AND PETER OWEN*

Department of Microbiology, Trinity College, Dublin 2, Ireland

Received 30 September 1982/Accepted 3 December 1982

Succinate dehydrogenase (EC 1.3.99.1) of Micrococcus luteus was selectivelyprecipitated from Triton X-100-solubilized membranes by using specific antiser-um. The precipitated enzyme contained equimolar amounts of four polypeptideswith apparent molecular weights of 72,000, 30,000, 17,000, and 15,000. The 72,000polypeptide possessed a covalently bound flavin prosthetic group and appeared tobe strongly antigenic as judged by immunoprinting experiments. Low-tempera-ture absorption spectroscopy revealed the presence of cytochrome b556 in theantigen complex. By analogy with succinate dehydrogenase purified from othersources, the 72,000 and 30,000 polypeptides were considered to representsubunits of the succinate dehydrogenase enzyme, whereas one (or both) of thelow-molecular-weight polypeptides was attributed to the apoprotein of the b-typecytochrome. A succinate dehydrogenase antigen cross-reacting with the M. luteusenzyme complex could be demonstrated in membranes of Micrococcus roseus,Micrococcusflavus, and Sarcina lutea, but not in the membranes isolated from awide variety of other gram-positive and gram-negative bacteria.

Succinate dehydrogenase (EC 1.3.99.1) is anintegral component of the respiratory electrontransfer chain of both eucaryotic and procaryot-ic cells. The enzyme catalyzes the oxidation ofsuccinate to fumarate and transfers reducingequivalents to the cytochrome chain (1, 14).Enzymes and enzyme complexes of variouscomplexities have been isolated from a numberof diverse sources, including mammalian andyeast mitochondria (7, 35), photosynthetic bac-teria (2, 8), and more recently from Bacillussubtilis (13) and Escherichia coli (21). The en-zyme has consistently been resolved as a me-talo-flavoprotein consisting of two subunits, thelarger of which has a molecular weight of 60,000to 70,000 and possesses a flavin prosthetic groupin covalent linkage. The smaller subunit has amolecular weight of 25,000 to 30,000. Both sub-units are thought to possess iron-sulfur centers(12, 14). Other low-molecular-weight compo-nents have often been observed in associationwith succinate dehydrogenase, and in severalinstances these have been shown to correspondto b-type cytochromes (11, 12, 35). In addition,the mammalian enzyme complex is known tocontain a ubiquinone-binding protein, which isthought to be essential for succinate-ubiquinonereductase activity (36, 37).For a number of years we have been interest-

ed in the succinate dehydrogenase found withinthe plasma membranes of the model gram-posi-tive aerobic bacterium Micrococcus luteus. Ear-

ly investigations on this topic dealt primarilywith the properties of the membrane-bound orpartially purified enzyme but failed to resolve itin molecular detail (17, 23, 29). More recently,crossed immunoelectrophoresis (CIE) of appro-priately labeled membranes has made it possibleto identify succinate dehydrogenase as a majorenzyme-active immunogen possessing covalent-ly bound flavin and iron (4). Moreover, theresults of progressive immunoadsorption experi-ments conducted with protoplasts and isolatedmembranes (27, 28) have indicated that themajor determinants of this enzyme antigen areexpressed on the cytoplasmic face of the mem-brane, as suggested by Mitchell (20).

In this communication -we extend our immu-nological approach to the analysis of M. luteussuccinate dehydrogenase. Specific immunoglob-ulins were used to precipitate the enzyme selec-tively from Triton X-100-solubilized mem-branes, thereby facilitating an examination ofthe molecular properties of this important mem-brane-bound immunogen.(A preliminary account of this work has been

presented at the 94th Ordinary Meeting, Societyfor General Microbiology, Cambridge, England,1982, p. 4.)

MATERIALS AND METHODS

Growth of cells. Bacteria were grown aerobically inan orbital incubator at 200 rpm, except where indicat-ed. Details of growth conditions are as follows: M.

1493

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

1494 CROWE AND OWEN

luteus (lysodeikticus) NCTC 2665, MicrococcusflavusNCTC 7743, and Sarcina lutea (laboratory strain) inpeptone-water-yeast extract medium at 30°C; Micro-coccus roseus NCTC 7523, Planococcus citreus CCM316, B. subtilis NCTC 3610, Staphylococcus aureusNCTC 8532, and laboratory strains of Serratia marces-cens, Proteus vulgaris, and Pseudomonas aeruginosain nutrient broth at either 30°C (M. roseus and P.citreus) or 37°C (others); Streptococcus pyogenesNCTC 8198 on Todd-Hewitt broth (Oxoid Ltd.) at37°C without shaking; Leuconostoc mesenteroides(laboratory strain) on a glucose-tryptone-yeast ex-tract-based medium at 20°C; Lactobacillus caseiNCTC 10302 on de Man-Rogosa-Sharpe broth (OxoidLtd.) at 30°C; and E. coli ML308-225 on succinateminimal medium at 37°C (6). Both Clostridium perfrin-gens (laboratory strain) and Peptococcus indolicusPcR14 were grown anaerobically at 37°C on PRASpeptone-yeast extract-glucose medium (32). Cellswere harvested in the late exponential phase of growthand were washed twice in 50 mM Tris-hydrochloridebuffer (pH 7.5). To obtain cells of M. luteus uniformlylabeled with 14C and 35S, bacteria were grown indefined media (26) devoid of either unlabeled aminoacids or unlabeled sulfate but supplemented with uni-formly 14C-labeled protein hydrolyzate (3.3 p.Ci/ml) orH235S04 (2.8 to 5.7 1LCi/ml) as appropriate. 59Fe-labeled and D-[2-14C]riboflavin-labeled cells of M.luteus were obtained as previously described (4).

Preparation of membranes and membrane extracts.Cells of M. luteus, P. citreus, B. subtilis, and Staphy-lococcus aureus were lysed osmotically with the aid ofeither lysozyme (100 ,ug/ml) or lysostaphin (15 [Lg/ml)as appropriate, and membranes were prepared bystandard procedures (24). The remaining gram-posi-tive bacteria were lysed by several passages through aFrench press (32,000 lb/in2) in the presence of lyso-zyme (100 pg/ml) and DNase (50 p.g/ml). The mem-branes of these organisms were subsequently isolatedby centrifugation (48,000 x g) after the removal ofwhole cells at 6,000 x g (24). Membrane vesicles of E.coli were prepared as previously described (15), andenvelopes of the other gram-negative bacteria wereobtained by French pressing (20,000 lb/in2) cell sus-pensions in the presence of lysozyme and EDTA asdescribed elsewhere (3).Methods for preparing Triton X-100 extracts of

gram-positive membranes and for obtaining Triton X-100-EDTA extracts of gram-negative envelopes andfor the mild dispersal of Triton X-100-insoluble mem-brane residues in sodium dodecyl sulfate (SDS) havebeen described (4, 22, 25, 27).

Preparation of antisera. Antiserum to plasma mem-branes of M. luteus was raised in rabbits as outlinedpreviously (4, 27). Serum specific for succinate dehy-drogenase was obtained by procedures involving pre-cipitate excision (5). Succinate dehydrogenase-activeimmunoprecipitates from a total of 60 wet CIE immu-noplates were used to immunize six rabbits by intra-dermal and subcutaneous routes. Two weeks later,each rabbit was boosted with immunoprecipitatesfrom four more immunoplates. This process was re-peated at monthly intervals. The rabbits were bledevery 10 to 14 days, and immunoglobulins were frac-tionated and concentrated to about 1/10 serum volumeas described previously (4, 27).

Immunoprecipitation. Before immunoprecipitation,

Triton X-100-solubilized membrane extracts were cen-trifuged at 48,000 x g for 20 min to remove anysedimentable material which might impair analysis.Extracts (normally 20 to 50 i±l) were then mixed withappropriate volumes (see below) of anti-succinate de-hydrogenase serum and were incubated at 30°C for 1 to2 h. Immune complexes were pelleted by centrifuga-tion at 25,000 x g for 10 min and were washed twicewith a large excess of 0.1 M NaCl containing 0.5%(vol/vol) Triton X-100. Washed immunoprecipitateswere finally suspended by gentle sonication in a smallvolume of Laemmli sample buffer (16). If large quanti-ties of immunoprecipitated material were required(e.g., for absorption spectroscopy), the reaction wasscaled up 50-fold, and the incubation period wasextended to 48 h at 4°C. In these instances, immuno-precipitates were finally suspended by sonication in 50mM Tris-hydrochloride buffer (pH 7.5) containing0.5% (vol/vol) Triton X-100. These modifications didnot alter the polypeptide profile as judged by SDS-polyacrylamide gel electrophoresis.

Immunoprinting. A modification of the sensitiveimmunoprint procedure (1) was employed to deter-mine which membrane polypeptides reacted with theanti-succinate dehydrogenase serum. 35S-labeledmembrane polypeptides were initially resolved bySDS-polyacrylamide gel electrophoresis. To removethe SDS, unfixed gels were washed twice (20 min) indistilled water and subsequently in barbital-hydrochlo-ride buffer (I = 0.02, pH 8.6) containing 1% (vol/vol)Triton X-100. The washed gels were then overlaid with1% (wt/vol) agarose gels (1.2 mm thick) containingeither specific serum or preimmune serum and buff-ered with barbital-hydrochloride (I = 0.02, pH 8.6)containing 1% (vol/vol) Triton X-100 and 15 mMNaN3. After incubation in a humidified chamber at30°C for 16 h, overlay gels were removed and werewashed, pressed, and dried (34) before autoradiogra-phy.

Electrophoretic techniques. Procedures for perform-ing CIE and related techniques; for washing, drying,and staining immunoplates with Coomassie brilliantblue; and for performing succinate dehydrogenasezymograms have been described in detail elsewhere(4, 28).

SDS-polyacrylamide gel electrophoresis was per-formed by the method of Laemmli (16) with 12.5% (wt/vol) acrylamide separating gels and 4% (wt/vol) acryl-amide stacking gels. Radioactive polypeptides werevisualized by autoradiography, and their molecularweights were calculated in the standard fashion withthe following molecular weight markers: phosphory-lase A (94,000), bovine serum albumin (66,000), cata-lase (61,000), glutamate dehydrogenase (55,400), aldol-ase (40,000), E. coli matrix protein (37,200), carbonicanhydrase (30,000), chymotrypsinogen A (25,500),trypsin inhibitor (20,100), myoglobin (17,200), andlysozyme (14,300).

Difference spectroscopy. Difference spectra at a tem-perature of 77°K were obtained between 500 and 620nm with an Aminco DW-2 spectrophotometer fittedwith a liquid nitrogen dewar. For membranes andTriton X-100 extracts, ferricyanide-oxidized (1.25mM) versus dithionite-reduced (1.25 mM) spectrawere recorded at a scan speed of 0.75 nm/s. In the caseof the succinate dehydrogenase immunoprecipitate, abase line was initially obtained with two buffer blanks.

J. BACTERIOL.

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

SUCCINATE DEHYDROGENASE OF M. LUTEUS 1495

Ferricyanide-oxidized (1.25 mM), succinate-reduced(12.5 mM), and dithionite-reduced (1.25 mM) spectrawere then recorded consecutively against a bufferblank. Reduced minus oxidized spectra were obtainedby computer subtraction.

Analytical methods and radiochemicals. Succinatedehydrogenase and NADH dehydrogenase (EC1.6.99.3) activities were determined spectrophotomet-rically as described by Owen and Freer (23) and byGel'man et al. (10), respectively. Protein was estimat-ed by the method of Lowry et al. (18), modified (9) toeliminate interference by Triton X-100. Bovine serumalbumin was used as the standard. 59FeC13 (9.6 mCi/mg of iron), D-[2-14C]riboflavin (60.6 mCi/mmol),H235S04 (4.33 mCi/mmol) and uniformly 14C-labeledprotein hydrolysate (59 mCi per milligram-atomicweight of carbon) were obtained from the Radiochemi-cal Centre, Amersham.

RESULTSPreparation and specificity of anti-succinate

dehydrogenase serum. To obtain specific serumof good quality by precipitate excision and im-munization (5), it is desirable to work from CIEprofiles in which the immunoprecipitate in ques-tion is well resolved and free of other intersect-ing immunoprecipitates. We have reported pre-viously that such a situation occurs for the M.luteus succinate dehydrogenase antigen after theCIE analysis of Triton X-100-insoluble mem-brane residues which have been dispersed insubdenaturing concentrations of SDS (22). Atypical CIE immunoplate illustrating this pointand indicating the regions of the succinate dehy-drogenase-active immunoprecipitate which werechosen for excision is shown in Fig. 1A. A poolof over 60 such excised arcs provided the anti-gen source for raising the anti-succinate dehy-drogenase serum (see above).The specificity of the resultant serum was

assessed by CIE, by immunodiffusion, and byenzyme inhibition studies. A single immunopre-cipitate which possessed succinate dehydroge-nase activity was resolved when M. luteus mem-branes were tested by either CIE orimmunodiffusion against the anti-succinate de-hydrogenase serum (Fig. 1B; see also Fig. 7).Moreover, no additional antigens reacting weak-ly with the serum could be discerned by thesensitive procedure of CIE with intermediate gel(Fig. 1C and D), even when this was coupled byautoradiography with the use of 35S-labeledmembranes (data not shown) or with the use ofradioisotopes such as 59Fe (Fig. 1D and F) or[14C]riboflavin (data not shown) designed toselectively label prosthetic groups of iron-con-taining and flavoprotein antigens. In contrast tothe single immunoprecipitate observed in testsagainst the specific serum, up to 32 immunopre-cipitates could be resolved for the homologous(membrane-anti-membrane) reaction (Fig. 1C;ref. 4).

The effect of anti-succinate dehydrogenaseserum on the succinate dehydrogenase activityexpressed in Triton X-100 extracts of M. luteusmembranes is illustrated in Fig. 2. About 75% ofthe total succinate dehydrogenase activity couldbe inhibited and precipitated by the specificserum. As anticipated, the immunoprecipitatedmaterial expressed very low levels of succinatedehydrogenase activity. For reasons that areunclear at the present time, the residual succi-nate dehydrogenase activity in the supernatantfraction (25% of the total) was not inhibited atserum excess. This could reflect, among otherthings, the presence of (i) a related form of theenzyme which, by virtue of altered antigenicityor formation of soluble immune complexes, isnot inhibited or precipitated by antibody to theparent molecule, or (ii) a second unrelated succi-nate dehydrogenase-active enzyme which hasescaped detection by CIE so far (4, 27, 28).The specificity of the succinate dehydroge-

nase-anti-succinate dehydrogenase reaction wasfurther confirmed by the facts that (i) preim-mune serum had very little neutralizing activity,and (ii) NADH dehydrogenase activity of themembrane extract was virtually unaffected byconcentrations of anti-succinate dehydrogenaseserum that produced maximal inhibition of ho-mologous enzyme activity (Fig. 2). On the basisof these and other similar experiments, the spe-cific anti-succinate dehydrogenase serum wasjudged acceptable for use in further studies ofthe biochemical and biophysical properties ofthe enzyme complex.The concentration of serum for optimum im-

munoprecipitation was determined by experi-ments similar to that displayed in Fig. 2, inwhich the following parameters were monitoredas a function of antibody concentration: (i) inhi-bition of succinate dehydrogenase activity, (ii)the amount of 59Fe precipitated from Triton X-100 extracts of 59Fe-labeled membranes, and (iii)the amount of residual succinate dehydrogenaseantigen left after immune complexes were re-moved by centrifugation. This final parameterwas measured by CIE of supernatant fractionsand involved estimates of peak area. It wasobserved that the complete removal of succinatedehydrogenase antigen occurred at a point (Fig.2, arrow) which coincided with the maximumprecipitation of radiolabeled iron and maximalinhibition of enzyme activity (data not shown).

Analysis of the succinate dehydrogenase-anti-body complex by SDS-polyacrylamide gel electro-phoresis. The polypeptide composition of theimmunoprecipitated succinate dehydrogenasecomplex was ascertained by SDS-polyacryl-amide gel electrophoresis. To facilitate the reso-lution of polypeptides of bacterial origin (asopposed to those derived from the antiserum)

VOL. 153, 1983

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

1496 CROWE AND OWEN

A B

Jjks ,~~~~~~~.P-

I __

DC

.

FE

FIG. 1. Preparation and specificity of anti-succinate dehydrogenase serum. Succinate dehydrogenase-activeimmunoprecipitates were excised (A, dotted region) from CIE immunoplates in which a 0.15% (wt/vol) SDSextract (46 ,ug of protein) of Triton X-100-insoluble membrane residues had been analyzed against anti-membraneserum. After immunization, the specificity of the resultant serum was tested by CIE (B) and by CIE withintermediate gel (C through F) by using Triton X-100 extracts of unlabeled (B, 74 ,ug of protein) and 59Fe-labeled(C through F, 42 pLg of protein) membranes. The main antibody gels of A and C through F contained anti-membrane serum (4.5 mg of protein per ml), and the main gel of B together with intermediate gels ofD through Fcontained anti-succinate dehydrogenase immunoglobulins (3.6 mg of protein per ml). Immunoprecipitates werevisualized by protein staining (A through D), enzyme staining for succinate dehydrogenase activity (E), andautoradiography (F). Note that only a single antigen (no. 8 of the CIE reference immunoprecipitate profile; ref.27) appears to react with the specific serum (B, D, and F) and that this component possesses succinatedehydrogenase activity (E) and iron (F). Anode is to the left and top of all gels.

J. BACTERIOL.

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

SUCCINATE DEHYDROGENASE OF M. LUTEUS 1497

and to allow a determination of the stoichiom-etry of the individual subunits, uniformly 14C-labeled membrane extracts were employed. Fig.3 shows the protein-stained profiles (tracks 1and 2) and corresponding autoradiograms(tracks 3 and 4) of a Triton X-100 extract of 14C-labeled membranes (tracks 1 and 3) and 14C-labeled immunoprecipitated material (tracks 2and 4). It is clear from these results that theaddition of monospecific serum to Triton X-100-solubilized extracts of M. luteus membranesresults in the selective precipitation offour poly-peptides with molecular weights of 72,000,30,000, 17,000, and 15,000. The subunit of high-est molecular weight could often be resolved asa doublet. Radioactive measurements made onexcised polypeptide bands indicate a stoichiom-etry of 1:1:1:1 for the four polypeptides (Fig. 3).The apparent stoichiometry was unaffected byeither incorporation into the Triton X-100 ex-traction buffers of, or pretreatment of antiserumfor 1 h with, a cocktail of protease inhibitorspresent in the following final concentrations:phenylmethylsulfonyl fluoride (1 mM); N-ot-p-tosyl-1-lysine chloromethyl ketone (1 mM),EDTA (5 mM), trypsin inhibitor (0.5 mg/ml), andAprotinin (0.6 trypsin inhibitor units (TIU)/ml).We have previously shown that the M. luteus

succinate dehydrogenase antigen possesses co-valently bound flavin (4), a feature shared bysimilar enzymes from other sources (12, 14). Itwas therefore of interest to establish to which ofthe resolved subunits this prosthetic group wasbound. Accordingly, D-[2-14C]riboflavin-labeled

100

80

c

°60 -

-c 40_

0 30 60 90 120

pl SerumFIG. 2. Inhibition of enzyme activity by anti-succi-

nate dehydrogenase serum. Triton X-100 extracts (120,ug of protein) of M. luteus membranes were incubatedwith 0 to 120 ,ul of anti-succinate dehydrogenase serum(120 mg of protein per ml, ) or equivalent amountspreimmune serum (-----) in a total volume of 140 pul.After 1 h at room temperature, samples (17 to 34 ,ug ofmembrane protein) were assayed for succinate dehy-drogenase (0) or NADH dehydrogenase (U) activities.Arrow indicates point at which complete precipitationof succinate dehydrogenase antigen and maximal pre-cipitation of bound iron were observed (see text forfurther details).

12 3 4

^_ ~~~~~~*72

-~~~~~~~~-o3

A= ._

4-0

s-17

.15

FIG. 3. Polypeptide composition of the M. luteussuccinate dehydrogenase antigen complex. A TritonX-100 extract (23 p,g of protein) of "4C-labeled mem-branes (track 1) and succinate dehydrogenase complexprecipitated from twice the amount of Triton X-100extract used in track 1 (track 2) were analyzed by SDS-polyacrylamide gel electrophoresis, and the resolvedpolypeptides were stained for protein. Direct autora-diograms of tracks 1 and 2 are displayed in tracks 3 and4. The positions and apparent molecular masses (inkilodaltons) of the four polypeptides resolved for theenzyme complex are indicated. For a typical prepara-tion, excision of individual polypeptides from pairs oftracks similar to track 2 followed by peroxide treat-ment and scintillation counting yielded (over a back-ground of 35 cpm) the following counts for the foursubunits; 599 cpm (72,000 molecular weight subunit),251 cpm (30,000 molecular weight subunit), 143 cpm(17,000 molecular weight subunit), and 129 cpm(15,000 molecular weight subunit).

membranes and material precipitated therefromby anti-succinate dehydrogenase serum wereanalyzed by SDS-polyacrylamide gel electro-phoresis in conjunction with autoradiography(Fig. 4). A single radiolabeled polypeptide with amolecular weight of 72,000 was observed forboth preparations (Fig. 4, tracks 3 and 4), estab-lishing the highest molecular weight subunit ofthe succinate dehydrogenase complex as themajor polypeptide possessing covalently boundflavin. In instances in which this polypeptide

VOL. 153, 1983

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

1498 CROWE AND OWEN

difference spectrum of the immunoprecipitatedsuccinate dehydrogenase complex (Fig. 5C).However, succinate was ineffective in reducingcytochrome b556 in the precipitated complex.This contrasts with the situation in isolatedmembranes and Triton X-100 extracts in whichsuccinate and other respiratory substrates suchas NADH2 and malate (10) can be shown byspectrophotometry to effect the reduction ofcytochrome b556 and other cytochromes presentin the preparations (data not shown).

Reaction of individual subunits with anti-succi-nate dehydrogenase serum. The reactivity of theindividual subunits of the succinate dehydroge-nase complex with monospecific serum wasassessed by an immunoprint technique (seeabove) in which SDS-polyacrylamide slab gelscontaining resolved 5S-labeled polypeptideswere overlaid with antibody-containing agarosegels. Overlay experiments conducted with mem-branes and membrane extracts (Fig. 6) and also

FIG. 4. Analysis of [14C]riboflavin-labeled mem-brane polypeptides from M. luteus by SDS-polyacryl-amide gel electrophoresis. The figure shows the poly-peptides resolved by protein staining (tracks 1 and 2)and by autoradiography (tracks 3 and 4) for [14C]ribo-flavin-labeled membranes (58 ,ug of protein, tracks 1and 3) and for the succinate dehydrogenase antigencomplex (track 2 and 4) immunoprecipitated from aTriton X-100 extract (110 1lg of protein) of [14C]ribofla-vin-labeled membranes. The identity of the singleradiolabeled band seen in tracks 3 and 4 is indicated byan arrow in the full spectrum of membrane polypep-tides (track 1).

was resolved as two closely migrating bands,both components of the doublet possessed fla-vin.

Presence of cytochrome b556. Preliminary re-sults based upon CIE analysis of membranesprepared from cells grown in the presence of theheme precursor [14C]aminolevulinic acid haveindicated that the succinate dehydrogenase anti-gen of M. luteus may be associated with cyto-chrome (4). To confirm this and to ascertain thenature of any cytochrome species, an analysis ofM. luteus membranes, Triton X-100 membraneextracts, and immunoprecipitated succinate de-hydrogenase was carried out by low-tempera-ture difference spectroscopy (Fig. 5). Of the fourcytochromes known (10, 19) to be present in M.luteus membranes (cytochromes c550, b556, b560,and a601; Fig. SA), only cytochrome b556 parti-tioned to a significant extent into the Triton X-100-soluble fraction (Fig. SB). It is notable thatcytochrome b556 could also be detected in thedithionite-reduced versus ferricyanide-oxidized

FIG. 5. Low-temperature cytochrome differencespectra (dithionite reduced minus ferricyanide oxi-dized). (A) M. luteus membranes (11.6 mg of proteinper ml); (B) Triton X-100 extract of M. luteus mem-branes (6.2 mg of protein per ml); (C) succinatedehydrogenase antigen complex immunoprecipitatedfrom a Triton X-100 membrane extract similar to thatused in B. The identities of the cytochromes giving riseto the individual oa-Soret absorption bands are indicat-ed.

1 2 3 4

-0 _o db, ..:.?...

do l.

__immfLw

J. BACTERIOL.

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

SUCCINATE DEHYDROGENASE OF M. LUTEUS 1499

with immunoprecipitated succinate dehydroge-nase complex (data not shown) revealed thatonly the subunit with a molecular weight of72,000 was capable of interacting with the mo-mospecific serum to form an immunoprecipitate.It seems likely, therefore, that the 72,000-molec-ular-weight flavopeptide is the major antigenicspecies of the succinate dehydrogenase com-plex.

Cross-reactivity of M. luteus succinate dehy-drogenase with similar enzymes from othermicroorganisms. To assess the degree of antigen-ic similarity between M. luteus succinate dehy-drogenase and similar enzymes from othermicroorganisms, membranes from a wide rangeof bacteria were tested by immunodiffusionagainst anti-M. luteus succinate dehydrogenaseserum. Of the membranes tested (those from M.

1 2 3 4

721=

FIG. 6. Reaction of membrane polypeptides withanti-succinate dehydrogenase serum. 35S-labeledmembranes (58 ,ug of protein, track 1) and a Triton X-100 extract (52 ,ug of protein) of "S-labeled mem-branes (track 2) were analyzed by SDS-polyacryl-amide gel electrophoresis. Gels were either fixed(tracks 1 and 2) or overlaid with an agarose gelcontaining anti-succinate dehydrogenase serum (5.4mg of protein per ml of gel) as detailed in the text.Autoradiograms of the fixed gel (tracks 1 and 2) and ofthe corresponding immunoprint (tracks 3 and 4) arepresented. The identity and molecular mass (in kilo-daltons) of the single polypeptide reacting with themonospecific serum is indicated at the side of thefigure.

FIG. 7. The reactions of membrane antigens fromM. luteus, M. roseus, M.flavus, and S. lutea with anti-M. luteus succinate dehydrogenase serum as deter-mined by immunodiffusion. Triton X-100 extracts ofmembranes from M. luteus (wells 1, 3, and 5; 60 ,ug ofprotein), M. roseus (well 2; 54 ,ug of protein), M.flavus (well 4; 65 jig of protein), and S. lutea (well 6; 65±g of protein) were tested against anti-M. luteussuccinate dehydrogenase immunoglobulins (centralwell, 1.2 mg of protein).

roseus, M. flavus, S. lutea, Staphylococcus au-reus, P. citreus, B. subtilis, Lactobacillus casei,Streptococcus pyogenes, Leucondstoc mesen-teroides, C. perfringens, Peptococcus indolicus,E. coli, Serratia marcescens, Proteus vulgaris,Pseudomonas aeruginosa, and Rhodopseudo-monas sphaeroides), only three (i.e., those ofM. roseus, M. flavus, and S. Iutea) gave positivereactions. The reacting antigens all gave lines ofpartial identity with the M. luteus enzyme (Fig.7). Moreover, tests of M. roseus, M. flavus, andS. lutea membranes by CIE revealed that ineach case, succinate dehydrogenase was one ofa maximum of only four antigens capable ofreacting with anti-M. luteus membrane serum(compared with 32 antigens for M. luteus mem-branes; Fig. 1C, ref. 4).

DISCUSSIONFrom the results presented above and else-

where (4), it is apparent that the Triton X-100-solubilized succinate dehydrogenase antigen ofM. luteus is remarkably similar in its molecularproperties to corresponding enzyme complexesisolated from other sources (12, 14). These simi-larities include the facts that (i) it is a metalo-flavoantigen possessing nonheme iron and cova-lently bound flavin (4); (ii) its two largestsubunits are present in equimolar amounts andare in the molecular weight ranges of 70,000 and30,000; (iii) the largest (72,000-molecular-

VOL. 153, 1983

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

1500 CROWE AND OWEN

weight) subunit contains the covalently boundflavin prosthetic group; and (iv) the complexcontains additional low-molecular-weight sub-units, including a b-type cytochrome, cyto-chrome b556. It is notable that this cytochromehas previously been shown to copurify with twoother enzymes of the M. luteus respiratorychain, namely NADH dehydrogenase and ma-late dehydrogenase (EC 1.1.1.37; ref. 19, 33).

In most, but not all, instances, b-type cyto-chromes have been shown to be composed ofsingle polypeptide chains, and in several dehy-drogenase-cytochrome b complexes they havebeen implicated not only in electron transfer butalso in attachment of the dehydrogenase to themembrane (11, 14). However, at the presenttime it is unclear which (or indeed whether both)of the low-molecular-weight subunits consistent-ly observed for the M. luteus antigen complexrepresents cytochrome b556. Certainly, the ob-served 1:1:1:1 stoichiometry of the four sub-units, together with results of experiments con-ducted with protease inhibitors, would seem torule out the possibility that they are artifactscaused by proteolytic degradation.An additional candidate for one of the low-

molecular-weight subunits might be a quinone-binding protein similar to that observed for thesuccinate-ubiquinone oxidoreductase (complexII) of beef heart mitochondria (36, 37). In themitochondrial complex, the ubiquinone-bindingprotein is of low molecular weight and is neces-sary for the expression by succinate dehydroge-nase of succinate-ubiquinone oxidoreductase ac-tivity (36, 37). It is not inconceivable thatanalogous menaquinone-binding proteins arepresent in M. luteus membranes and in theprecipitated succinate dehydrogenase enzymecomplex. Certainly we have been able to demon-strate for both the membrane-bound and deter-gent-solubilized forms of the enzyme complexnot only succinate-cytochrome b556 reductaseactivity but also succinate-ubiquinone (Q-1) re-ductase activity. Unfortunately, we have beenunable as yet to detect either of these activitiesfor the immunoprecipitated enzyme complex.The following observations suggest that this isprobably a result of antibody-induced inhibitionof catalytic activity: (i) monospecific serum al-most totally inhibits phenazine methosulphate-mediated succinate-dichlorophenolindophenolreductase activity; (ii) the 72,000-dalton flavo-protein which probably houses the catalytic siteof-the enzyme (12) appears to be strongly anti-genic; and (iii) electron spin resonance studieshave indicated that iron-sulfur centers S-1 and S-3 are only partially reduced by succinate in theimmunoprecipitated form of the enzyme com-plex but are fully reduced by substrate in themembrane-bound and Triton X-100-solubilized

forms of the enzyme (B. A. Crowe et al., manu-script in preparation).The similarities that are evident in the molecu-

lar properties of succinate dehydrogenases iso-lated from M. luteus and other sources do notappear to be reflected in the cross-relatedness ofthe enzymes as determined by methods of agargel precipitation. In the current survey, antigensreacting with anti-M. luteus succinate dehydrog-enase serum were detected in members of thegenus Micrococcus only. Even other membersof the family Micrococcaceae (Staphylococcusaureus and P. citreus) failed to show cross-reactive material. A similar survey conductedrecently with anti-E. coli succinate dehydroge-nase has shown slightly broader specificity, withcross-reactive antigens observed in all membersof the Enterobacteriaceae tested (3). The E. coliantiserum, however, failed to react with mem-branes from other bacteria, including M. luteus(3), an observation which confirms the results ofthe reciprocal test performed in the presentstudy. It should be remembered, however, thatreactions of partial identity or nonidentity inimmunodiffusion tests often reflect relativelysmall changes in amino acid sequence. Reac-tions of partial identity, for example, can occurfor molecules which cross-react by 80% asjudged from quantitative immunoprecipitintests-a situation which can reflect less than10% difference in sequence homology. Further-more, molecules which differ from each other insequence by 30 to 40% or more have been shownto exhibit no immunological cross-reactivity (30,31).The present investigation extends to three the

number of bacterial succinate dehydrogenaseswhose molecular properties have been probedsuccessfully with the aid of specific immuno-globulins and without resort to classical proce-dures of protein purification (3, 14, 21). Therelevance of this approach to the analysis ofother membrane immunogens which are refrac-tory to purification should be obvious.

ACKNOWLEDGMENTSWe express our gratitude to Marieke G. L. Elferink of the

University of Grbningen for the generous gift of Triton X-100-solubilized membranes from R. sphaeroides and to RichardCammack of the University of London for assistance withcomputer subtraction.

LITERATURE CITED1. Arnaud, P., G. B. Wilson, J. Koistinen, and H. H. Funden-

berg. 1977. Immunofixation after electrofocusing: im-proved method for specific detection of serum proteinswith determination of isoelectric points. 1. Immunofixa-tion print technique for detection of alpha-l-proteaseinhibitor. J. Immunol. Methods 16:221-231.

2. Carithers, R. P., D. C. Yoch, and D. I. Arnon. 1977.Isolation and characterization of bound iron-sulfur pro-teins from bacterial photosynthetic membranes. II. Succi-

J. BACTERIOL.

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

SUCCINATE DEHYDROGENASE OF M. LUTEUS 1501

nate dehydrogenase from Rhodospirillum ruibrum chro-matophores. J. Biol. Chem. 252:7453-7460.

3. Condon, C., and P. Owen. 1982. Succinate dehydroge-nase: a major cross-reacting antigen in the Enterobacte-riaceae. FEMS Microbiol. Lett. 15:109-113.

4. Crowe, B. A., and P. Owen. 1983. Immunochemicalanalysis of respiratory-chain components of Micrococcusluteus (lysodeikticus). J. Bacteriol. 153:498-505.

5. Crowle, A. J., G. J. Revis, and K. Jarret. 1972. Prepara-tory electroimmunodiffusion for making precipitins toselected native antigens. Immunol. Commun. 1:325-336.

6. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Esche-richia coli requiring methionine or vitamin B12. J. Bacte-riol. 60:17-28.

7. Davis, K. A., and Y. Hatefi. 1971. Succinate dehydroge-nase. 1. Purification, molecular properties and substruc-ture. Biochemistry 10:2509-2516.

8. Davis, K. A., Y. Hatefi, I. P. Crawford, and H. Balts-cheffsky. 1977. Purification, molecular properties and ami-no acid composition of the subunits of Rhodospirillumrubrum succinate dehydrogenase. Arch. Biochem.Biophys. 180:459-464.

9. Dulley, J. R., and P. A. Grieve. 1975. A simple techniquefor eliminating interference by detergents in the Lowrymethod of protein determination. Anal. Biochem. 64:136-141.

10. Gel'man, N. S., G. V. Tikhonova, I. M. Simakova, M. A.Lukoyanova, S. D. Taptykova, and H. M. Mikelsaar. 1970.Fragmentation by detergents of the respiratory chain ofMic rococcus lysodeikticus membranes. Biochim.Biophys. Acta 223:321-331.

11. Hatefi, Y., and Y. M. Galante. 1980. Isolation of cyto-chrome b560 from complex II (succinate-ubiquinone oxi-doreductase) and its reconstitution with succinate dehy-drogenase. J. Biol. Chem. 255:5530-5537.

12. Hatefi, Y., and D. L. Stiggall. 1976. Metal-containingflavoprotein, p. 175-297. In P. D. Boyer (ed.), Theenzymes, vol. 13. Academic Press, Inc., New York.

13. Hederstedt, L., E. Holmgren, and L. Rutberg. 1979.Characterization of a succinate dehydrogenase complexsolubilized from the cytoplasmic membrane of Bacillussubtilis with the nonionic detergent Triton X-100. J.Bacteriol. 138:370-376.

14. Hederstedt, L., and L. Rutberg. 1981. Succinate dehy-drogenase-a comparative review. Microbiol. Rev.45:542-555.

15. Kaback, H. R. 1971. Bacterial membranes. Methods En-zymol. 22:99-120.

16. Laemmli, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

17. Linder, R., M. R. J. Salton, and J. J. Pollock. 1975.Affinity chromatography of succinate dehydrogenasefrom the membranes of Micrococcus lysodeikticus. Prep.Biochem. 5:349-357.

18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

19. Lukoyanova, M. A., and S. D. Taptykova. 1968. Cyto-chromes of Micrococcus lysodeikticus. Biokhimiya33:888-894.

20. Mitchell, P. 1962. Metabolism, transport, and morphogen-esis: which drives which? J. Gen. Microbiol. 29:25-37.

21. Owen, P., and C. Condon. 1982. The succinate dehydroge-nase of Escherichia coli: subunit composition of the

Triton X-100-solubilized antigen. FEMS Microbiol. Lett.14:223-227.

22. Owen, P., and H. Doherty. 1979. Immunochemical analy-sis of Triton X-100-insoluble residues from Micrococcuslysodeikticus membranes. J. Bacteriol. 140:881-887.

23. Owen, P., and J. H. Freer. 1970. Factors influencing theactivity of succinate dehydrogenase in membrane prepa-rations from Micrococcus lysodeikticus. Biochem. J.120:237-243.

24. Owen, P., K. A. Graeme-Cook, B. A. Crowe, and C.Condon. 1982. Bacterial membranes: preparative tech-niques and criteria of purity. p. B407b/1-B407b/69. InT. R. Hesketh, H. L. Kornberg, J. C. Metcalfe, D. H.Northcote, C. I. Pogson, and K. F. Tipton (ed.), Tech-niques in lipid and membrane biochemistry, part 1. Else-vier/North-Holland Publishing Co., Shannon, Ireland.

25. Owen, P., and H. R. Kaback. 1979. Immunochemicalanalysis of membrane vesicles from Escherichia coli.Biochemistry 18:1422-1426.

26. Owen, P., and M. R. J. Salton. 1975. Isolation andcharacterization of a mannan from mesosomal membranevesicles of Micrococcus lysodeikticus. Biochim. Biophys.Acta 406:214-234.

27. Owen, P., and M. R. J. Salton. 1975. Antigenic andenzymatic architecture of Micrococcus lysodeikticusmembranes established by crossed immunoelectrophore-sis. Proc. NatI. Acad. Sci. U.S.A. 72:3711-3715.

28. Owen, P., and M. R. J. Salton. 1977. Membrane asymme-try and expression of cell surface antigens of Micrococcuslysodeikticus established by crossed immunoelectropho-resis. J. Bacteriol. 132:974-985.

29. Pollock, J. L., R. Linder, and M. R. J. Salton. 1971.Characterization of the membrane-bound succinic dehy-drogenase of Micrococcus lysodeikticus. J. Bacteriol.107:230-238.

30. Prager, E. M., and A. C. Wilson. 1971. The dependence ofimmunological cross-reactivity upon sequence resem-blance among lysozymes. 1. Micro-complement fixationstudies. J. Biol. Chem. 246:5978-5989.

31. Prager, E. M., and A. C. Wilson. 1971. The dependence ofimmunological cross-reactivity upon sequence resem-blance among lysozymes. 11. Comparison of precipitinand micro-complement fixation results. J. Biol. Chem.246:7010-7017.

32. Switalski, L. M., 0. Schwan, C. J. Smyth, and T. Wad-strom. 1978. Peptocoagulase: clotting factor produced bybovine strains of Peptococcus indolicus. J. Clin. Micro-biol. 7:361-367.

33. Turgenbaeva, D. A., E. F. Kharat'yan, I. G. Zhukova,M. A. Lukoyanova, and D. N. Ostrovskii. 1979. Triton X-100-solubilized cytochrome b556 complexes from Micro-coccus lysodeikticus membranes. Biokhimiya 44:729-737.

34. Weeke, B. 1973. General remarks on principles, equip-ment, reagents and procedures. Scand. J. Immunol.2:(Suppl 1):15-35.

35. Weiss, H., and H. J. Kolb. 1979. Isolation of mitochondrialsuccinate: ubiquinone reductase, cytochrome c reductaseand cytochrome c oxidase from Neurospora crassa usingnonionic detergent. Eur. J. Biochem. 99:139-149.

36. Yu, C.-A., and L. Yu. 1980. Isolation and properties of amitochondrial protein that converts succinate dehydroge-nase into succinate-ubiquinone oxidoreductase. Biochem-istry 19:3579-3585.

37. Yu, C.-A., and L. Yu. 1981. Ubiquinone-binding proteins.Biochim. Biophys. Acta 639:99-128.

VOL. 153, 1983

on July 12, 2018 by guesthttp://jb.asm

.org/D

ownloaded from