THE JOURNAL OF EXPERIMENTAL ZOOLOGY 281:73–79 (1998)

© 1998 WILEY-LISS, INC.

Immunogold Transmission Electron MicroscopicLocalization of Arginine Kinase in ArthropodMitochondria

A. PINEDA, JR., AND W.R. ELLINGTON*Department of Biological Science, Florida State University, Tallahassee,Florida 32306-4370

ABSTRACT Mitochondrial creatine kinase (MiCK) is widely distributed throughout the verte-brates and is also present in at least two invertebrate groups. MiCK is thought to play a pivotalrole in cellular energy transactions, although this view is the subject of considerable controversy.Arginine kinase (AK), another member of this enzyme family, is found in many invertebrate groups,yet mitochondrial AK (MiAK) appears to be restricted to crustaceans, a chelicerate, and possiblyinsects. The presence of MiAK has been validated by enzymatic and respiratory studies of mito-chondrial fractions from these animals. However, no direct evidence has been obtained to supportthe presence of MiAKs. In the present study, we have used blot-purified anti-AK antibodies tolocalize AK in the mitochondria of cardiac muscle of the blue crab Callinectes sapidus and thehorseshoe crab Limulus polyphemus. Immunogold transmission electron microscopy (TEM) showedthat AK was present in isolated mitochondria and in mitochondria in situ. Immunogold labelingwas generally restricted to the intermembrane space and what appears to be the outer portion ofthe inner membrane. The localization of MiAK is consistent with hypothesized access of the en-zyme to ATP transported out of the matrix by the adenine nucleotide translocase. J. Exp. Zool.281:73�79, 1998. © 1998 Wiley-Liss, Inc.

Creatine kinase (CK) is a member of a highlyconserved family of enzymes known as phospha-gen (guanidino) kinases, which play a central rolein energy homeostasis in cells capable of high andvariable metabolic outputs (muscle fibers, neu-rons, spermatozoa). In vertebrates, CK is foundas a number of isoenzymes, including two mito-chondrial forms of mitochondrial creatine kinase(MiCK): MiaCK (ubiquitous) and MibCK (sarco-meric). Both MiCKs exist primarily as octomersand appear to be localized in the intermembranespace in close proximity to the adenine nucleotidetranslocase (Wyss et al., ’92). The compartmen-talization of MiCK is thought to have importantconsequences with respect to the control of en-ergy production and high-energy phosphate trans-port (Wyss et al., ’92). MiCKs are not restrictedto vertebrates, since they have been found in thespermatozoa of several echinoderms (Tombes andShapiro, ’87; Wyss et al., ’95) and in polychaetes(Kamp et al., ’95).

Arginine kinase (AK), another member of thisenzyme family, is widely distributed throughoutthe higher and lower invertebrate groups (for re-views, see Van Thoai, ’68; Watts, ’71). Molluscsand arthropods exclusively use AK and its corre-

sponding phosphagen, arginine phosphate. How-ever, in molluscs AK activity is purely cytosolic,and no MiAK activity has been observed (Bal-lantyne et al., ’81; Mommsen and Hochachka, ’81;Ellington and Hines, ’91). In contrast, enzymaticand respiratory studies of mitochondrial fractionsfrom a variety of crustaceans have demonstratedsignificant mitochondrial AK (MiAK) activities(Chen and Lehninger, ’73; Skorkowski et al., ’76;Hird and McLean, ’83; Hird and Robin, ’85;Ellington and Hines, ’91). Work by Doumen andEllington (’90a) showed that mitochondria fromthe cardiac muscle of the horseshoe crab Limuluspolyphemus, a chelicerate arthropod, contain sig-nificant MiAK activity.

The situation in insects is somewhat uncertain.Flight muscle mitochondria from the blowflySarcophaga bullata (Ellington and Hines, ’91), themoth Manduca sexta (Ellington and Hines, ’91),and the locust Locusta migratoria (Schneider etal., ’89) lack MiAK activity. However, Munneke

Grant sponsor: National Science Foundation; Grant number: IBN96-31907.

*Correspondence to: W.R. Ellington, Department of Biological Sci-ence, Florida State University, Tallahassee, FL 32306-4370. E-mail:elling@bio.fsu.edu

Received 16 September 1997; Accepted 18 December 1997

74 A. PINEDA, JR., AND W.R. ELLINGTON

and Collier (’85, ’88) found AK activity in whatappeared to be mitochondrial fractions fromwhole-animal extracts of the fruit fly Drosophila.Recently, Wyss et al. (’95) failed to find MiAK inextracts of muscle from Drosophila and attributedthe results of Munneke and Collier (’85, ’88) tocontamination of the mitochondrial extracts. Fi-nally, Chamberlin et al. (’94) found appreciableMiAK activity in extracts of the posterior midgutepithelium of M. sexta.

All of the studies that found MiAK activitiesused extensively washed mitochondrial fractionsand subsequent spectrophotometric and respi-ratory assays for AK. Positive results could beviewed as suspect because of potential contami-nation of mitochondrial fractions by cytoplas-mic AK adsorbed onto cellular debris or trappedin vesicles formed during homogenization. Be-cause of this possibility, we have used animmunogold transmission electron microscopyapproach to localize AK in mitochondria from acrustacean and a chelicerate arthropod. Our re-sults show the presence of MiAK in both spe-cies. The MiAK seems to be primarily localizedin the intermembrane space.

MATERIALS AND METHODSAnimals and materials

Horseshoe crabs L. polyphemus were collectedat extreme high tides along the shores of BaldPoint, Franklin County, Florida. These animalswere maintained in running seawater at theFlorida State University (FSU) Marine Labora-tory at Turkey Point. Blue crabs Callinectessapidus were obtained from a local seafood retailerand were maintained in a recirculating seawatersystem on the FSU campus. Biochemicals wereobtained from Sigma Chemical Co. (St. Louis, MO)and Boehringer Mannheim (Indianapolis, IN). Allother chemicals were of reagent-grade quality. Theblot-purified anti-Limulus AK antibody prepara-tion was obtained as described in Strong andEllington (’93).

Isolation of MitochondriaLimulus polyphemus

Isolation of mitochondria from the cardiac tis-sue of the horseshoe crab was performed as de-scribed by Doumen and Ellington (’89). Specimenswere kept in ice for 1 h before being sacrificed.The isolation medium consisted of 600 mM su-crose, 150 mM KCl, 25 mM HEPES (pH 7.4), 2mM EGTA, and 1 mM EDTA.

Callinectes sapidusFor each mitochondrial preparation, four to five

animals were kept in a bucket of ice for 1 h torender them immobile. The hearts were dissectedout and minced with a single-edge razor. Theminced tissue was diluted in 20 volumes (wt:vol)of isolation medium (the same isolation mediumused for Limulus) and homogenized with fourpasses in a Potter-Elvehjem homogenizer using atight-fitting Teflon pestle. The homogenate wascentrifuged at 1,300g for 15 min and the result-ing supernatant was centrifuged at 11,000g for20 min. The final mitochondrial pellet was washedtwice with 15 ml isolation medium prior to fixa-tion. All manipulations were carried out at 4°C.

For the fractionation of C. sapidus heart intocytoplasmic and mitochondrial fractions, isolationof mitochondria was performed as in text preced-ing. The supernatant from the 11,000g spin wascollected as a source of cytoplasmic AK. Beta-mercaptoethanol was added to a final concentra-tion of 14 mM. The pellet was resuspended with15 ml of isolation medium and centrifuged at11,000g for 20 min. This step was done twice, andthe final mitochondrial pellet was resuspended in1% Triton X-100 with 14 mM of β-mercapto-ethanol. The activities of AK were assayed usingthe procedure described in text following.

Tissue preparationSmall strips (about 1 mm3) of heart tissue from

both L. polyphemus and C. sapidus were cut us-ing a single-edge razor. The strips were thenrinsed in isolation medium prior to fixation.

Sample preparation for electron microscopyTissue samples and final mitochondrial pellets

of both L. polyphemus and C. sapidus were re-suspended in fixative consisting of 4% paraform-aldehyde and 0.5% glutaraldehyde in 0.1 Msodium cacodylate buffer (pH 7.4) and fixed for 2h at 4°C. The samples were washed three timesat 30 min each with 0.1 M sodium cacodylatebuffer (pH 7.4) containing 600 mM sucrose. Oneset of samples was then postfixed with 1% osmiumtetroxide for 1 h while the other set remained inthe washing solution. The samples were againwashed three times at 15 min each and were car-ried through dehydration in graded ethyl alcohol(30%–100%). Infiltration was carried out using LRWhite (London Resin Company, England) and100% ethanol at 2:1 ratio for 1 h in a rotary mixer.Subsequent infiltration was done using pure LR

MITOCHONDRIAL ARGININE KINASE 75

White, incubating the samples twice for 1 h, andthen allowing them to go overnight. The sampleswere transferred into gelatin capsules with LRWhite and subjected to 10-psi vacuum for 1 h. Po-lymerization was carried out at 50°C for 24 h invacuum. Silver-gold sections were prepared usingReichert-Jung Ultracut E microtome. The sectionswere then mounted on 300-mesh nickel grids.

The grids were blocked with 2% BSA in PBS (7mM Na2HPO4; 2.6 mM NaH2PO4, pH 7.4; 138 mMNaCl) for 2 h in a shaker. The grids were thenwashed in filtered PBS and incubated in blot-pu-rified rabbit anti-AK for 24 h. Labeling was doneby incubating the grids in 10 nm gold-conjugatedantirabbit IgG for 2 h at a dilution of 1:100 withPBS. The grids were washed with PBS, air-dried,and stained with uranyl acetate for 10 min and leadcitrate for 5 min. Samples were viewed using aJEOL 1200EX transmission electron microscope.

SDS-PAGE/Western blotSDS-PAGE was carried out in a Hoefer minislab

apparatus following the discontinuous Laemmliprocedure (Laemmli, ’70). Western blot was thenperformed as described by Ellington (’91) using aBIORAD Mini Transblot device. Proteins werestained with Ponceau S red. Blot-purified rabbitanti-Limulus AK antibody was used to incubatethe blot, which was followed by incubation withgoat anti-rabbit IgG conjugated with alkalinephosphatase.

Enzyme assaysActivity of arginine kinase was measured by

standard spectrophotometric assay in a Gilfordmodel 262 spectrophotometer at 340 nm at 25°C.The assay contained 70 mM Tris/HCl (pH 8.0), 6mM MgCl2, 5 mM ATP, 1.25 mM PEP, 0.25 mMNADH, 4.3 mM imidazole/HCl (pH 7.0), 5.5 unitsof lactate dehydrogenase (L-LDH), and 2 units ofpyruvate kinase. The reaction was started by theaddition of 30 mM L-arginine.

RESULTSFractionation of C. sapidus heart into cytoplas-

mic and mitochondrial fractions showed AK activi-ties of 318.17 ± 28.17 µmoles · min–1 · g wet wt–1

(mean ± SD, n = 6) and 3.65 ± 1.14 µmoles · min–1 ·g wet wt–1, respectively. MiAK represents 1.12 ±0.27% (mean ± SD, n = 6) of total AK activity inthis species. This is comparable to the results ob-tained by Doumen and Ellington (’90a), who re-ported that in L. polyphemus heart, 1% of the totalAK activity is mitochondrial activity. These data

were obtained from extensively washed mitochon-drial fractions. Contamination by cytoplasmic AKis unlikely since activity of lactate dehydrogenase,a cytoplasmic enzyme, was totally absent from themitochondrial extracts.

To validate the specificity of the blot-purifiedanti–L. polyphemus AK antisera, Western blotswere performed using mitochondrial extracts fromboth species. A single immunologically reactiveband was observed in extracts from C. sapidusmitochondria (Fig. 1). This protein’s mobility wasconsistent with a relative molecular mass (Mr)typical of arthropod AKs. Similarly, the antiserareacted strongly against two bands in L. poly-phemus mitochondrial extracts (see Fig. 1) thatcorrespond to the slow and fast MiAKs first ob-served by Doumen and Ellington (’90a). No non-specific interactions were observed. Thus theblot-purified antiserum preparation was deemedacceptable for immunogold localization studies.

Fig. 1. Composite Western blot of AKs from Limuluspolyphemus and Callinectes sapidus. (A) Ponceau S red stain-ing of blot. Lane 1 contained the molecular mass (Mr) mark-ers β-galactosidase (116 kDa), phosphorylase (97 kDa),fructose-6-phosphate kinase (84 kDa), bovine serum albumin(66 kDa), glutamate dehydrogenase (55 kDa), ovalbumin (45kDa), and glyceraldehyde-3-phosphate dehydrogenase (36kDa). Lane 2 contained highly purified cytoplasmic AK fromL. polyphemus. Lane 3 contained a detergent extract of C.sapidus mitochondria. (B) Immunoblot stain. Lane 1 con-tained the same extract as used in lane 3 of A. Lane 2 con-tained purified cytoplasmic AK from L. polyphemus. Lane 3had a detergent extract of mitochondria from L. polyphemus.

76 A. PINEDA, JR., AND W.R. ELLINGTON

Immunogold localization of MiAK using a 10-nm gold-conjugated goat antirabbit IgG revealedthe presence of MiAK in both isolated mitochon-dria (Fig. 2) and in situ preparations of cardiacmuscles from L. polyphemus (Fig. 3) and C.sapidus (Fig. 4). In electron micrographs of iso-lated mitochondria, there was minimal labelingof nonmitochondrial contaminants (see Fig. 2). Tis-sue sections that had been postfixed with 1% os-mium tetroxide (see Figs. 3A and 4A) showedrelatively well defined membrane structures com-pared to the nonosmicated samples. Sections thatwere not treated with osmium tetroxide had rela-tively higher density of immunogold label com-pared with the osmicated samples. It can be seenthat AK is indeed present in the mitochondria ofboth species, as evidenced by the distribution ofimmunogold label. It appears that MiAK is pri-marily localized in the intermembrane space(lighter regions). A fraction of the label seems tobe attached to the outer portion of the inner mi-tochondrial membrane. When immunogold distri-bution was counted in mitochondria, over 80% ofthe label was observed to be localized in the in-termembrane space compartment (Table 1). Con-trol sections that were not incubated with the

primary antibody did not show any labeling (datanot shown).

DISCUSSIONThe presence of MiAK in arthropods has been

documented via enzyme and respiratory assaysby various investigators (Chen and Lehninger, ’73;Munneke and Collier, ’88; Doumen and Ellington,’89, ’90a; Ellington and Hines, ’91; Chamberlin etal., ’94). A significant activity of MiAK was ob-served in horseshoe crab heart (Doumen andEllington, ’90a) and in the posterior midgut of to-bacco hornworm (Chamberlin et al., ’94). MiAKactivity from whole-animal extracts of the fruitfly Drosophila was likewise noted by Munnekeand Collier (’88). This finding, however, has beendisputed because Wyss et al. (’95) failed to repro-duce the same result, attributing the results ofMunneke and Collier (’88) to cytoplasmic contami-nation of the mitochondrial fraction. It is possiblethat positive results could have been broughtabout by vesiculation or adsorption of cytoplas-mic AK onto membrane fragments in the mito-chondrial extracts. This observation raised someuncertainty as to the presence of mitochondrialAK in other arthropods, since the potential pres-

Fig. 2. Electron micrographs of isolated mitochondria from L. polyphemus (A) and C.sapidus (B). Bar corresponds to 0.4 µm.

MITOCHONDRIAL ARGININE KINASE 77

Fig. 3. Electron micrographs of mitochondria from L. polyphemus in situ. Tissue waspostfixed with osmium tetroxide in A and not postfixed in B. Bar corresponds to 0.4 µm.

Fig. 4. Electron micrographs of mitochondria from C. sapidus in situ. Tissue was postfixedwith osmium tetroxide in A and not postfixed in B. Bar corresponds to 0.4 µm.

78 A. PINEDA, JR., AND W.R. ELLINGTON

ence of cytoplasmic contaminants in mitochondrialfractions has not been totally excluded.

In the present study, transmission electron mi-crographs of isolated mitochondria and tissuepreparations from the hearts of L. polyphemus andC. sapidus clearly demonstrate the presence ofMiAK (see Figs. 2–4). Nonspecific interaction canbe ruled out because the results of the Westernblot clearly indicated that the blot-purified anti-AK antisera react specifically with AK from bothL. polyphemus and C. sapidus mitochondria. Thelabel appears to be primarily localized in the in-termembrane space (lighter areas). Some of theseimmunogold particles seem to be closely associ-ated with the outer portion of the inner mitochon-drial membrane.

MiAK is present as fast and slow electrophoreticforms in L. polyphemus (Doumen and Ellington,’90a). It was reported that 50% of MiAK could bereleased by treatment of the mitochondria withhypotonic solutions, which would release thesoluble fraction of MiAK. The remaining fractionof MiAK, which corresponds to the slow MiAKform, could only be released by treatment withdetergent. This second fraction of MiAK is believedto associate with the inner mitochondrial mem-brane by hydrophobic interaction, which is dis-rupted by nonionic detergents (Doumen andEllington, ’90a). This hydrophobic nature of mito-chondrial phosphagen kinase in invertebrates wasalso observed in muscle of the crab, Cancerpagurus (Hird and Robin, ’85), Drosophila (Mun-neke and Collier, ’88), and the sea urchin spermMiCK, which can only be detached from the mem-branes by a relatively high concentration of de-tergents (Tombes and Shapiro, ’87). In contrastto the invertebrate mitochondrial phosphagen ki-nase, the interaction of the vertebrate MiCK withmitochondrial membrane is ionic in nature andcan be disrupted by a high salt concentration.With its basic isoelectric point, the positively

charged MiCK interacts electrostatically with thenegatively charged phospholipids of the innermembrane (Wyss et al., ’92).

The localization of MiAK primarily in the in-termembrane compartment is consistent with theobserved localization of the vertebrate MiCK.Studies have shown that MiCK is associated withthe outer face of the inner membrane (Scholte etal., ’73; Jacobus and Lehninger, ’73), in close prox-imity to the adenine nucleotide translocase (ANT).Some suggest that this localization of MiCK seemsto give it a privileged access to ATP transportedby the ANT (Wyss et al., ’92). The fact that, inintact mitochondria, MiCK has a lower Km forATP produced in the matrix than exogenouslyadded ATP was taken as an argument that MiCKhas privileged access to intramitochondrially syn-thesized ATP because of the interaction of MiCKwith ANT (Wyss et al., ’92). Immunogold labelingof MiCK has shown that MiCK is localized in theintermembrane space (Schlegel et al., ’88; Wegmannet al., ’91). Doumen and Ellington (’90b) did not ob-serve any functional coupling between MiAK andANT, as evidenced by the absence of kinetic differ-ences in the forward reaction of AK in the presenceor absence of oxidative phosphorylation. It was sug-gested that the functional significance of MiAKin this system is enhancement of the ADP gradi-ent, resulting in increased inward flux of ADP intothe ANT (Doumen and Ellington, ’90b).

The present study conclusively demonstratesthat AK is localized in mitochondria of L. poly-phemus and C. sapidus. The bulk of MiAK activityis found in the intermembrane space compartment,where it could potentially influence adenine nucle-otide ratios and high-energy phosphate flux. Cur-rent efforts center on elucidating the deducedamino acid sequence of the detergent-soluble formof MiAK in L. polyphemus in order to gain an un-derstanding of the nature of the interaction of thisprotein with the inner membrane and also to de-velop some insight into the mechanisms of mito-chondrial targeting, insertion, and processing.

ACKNOWLEDGMENTSResearch was supported by a grant from the Na-

tional Science Foundation (IBN 96-31907) to W.R.E.

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TABLE 1. Distribution of imunogold particles inmitochondria from the cardiac muscle of C. sapidus

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MITOCHONDRIAL ARGININE KINASE 79

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