Immunological and physical comparison of monomeric and dimeric phosphagen kinases: Some evolutionary implications

Biochimica et Biophysica Acta 1760 (2006) 364 – 371 http://www.elsevier.com/locate/bba

Immunological and physical comparison of monomeric and dimeric phosphagen kinases: Some evolutionary implications Brianne Wright-Weber a , Brenda C. Held a , Ashli Brown b , Steven H. Grossman a,⁎ a

b

University of South Florida, Department of Chemistry, 4202 East Fowler Avenue Tampa, FL 33620, USA USDA, Agriculture Research Station, Biological Control and Mass Rearing, Research Unit, PO Box 5367, 810 Highway 12 East, Mississippi State, MS 39762, USA Received 18 August 2005; received in revised form 7 November 2005; accepted 8 November 2005 Available online 13 December 2005

Abstract The antigenic and physical properties of several representative invertebrate phosphagen kinases have been examined in order to further characterize the relationship between taxonomic assignment, quaternary protein structure and evolution of this class of enzymes. Antibodies against dimeric arginine kinase from the sea cucumber cross-reacted with dimeric arginine kinase purified from sea urchin eggs, but failed to react with extracts from any species known to contain monomeric arginine kinase. However, strong immunoreactivity was observed when antibodies against purified dimeric arginine kinase were reacted with pure creatine kinase from the human muscle (CK-MM) and brain (CK-BB) as well as extracts from several species known to contain dimeric creatine kinase. Of particular interest with regard to evolution of the phosphagen kinases, we confirm the presence of creatine kinase activity in the very primitive sponge Tethya aurnatium and detect a reaction with antibodies against dimeric, but not monomeric, arginine kinase. This observation is consistent with recent studies of phosphagen kinase evolution. Substrate utilization was very specific with creatine kinase using only creatine. Arginine kinase catalyzed phosphorylation of arginine but enzymes from several species could also phosphorylate canavanine. No activities were detected with D-arginine. Isoelectric points, evaluated for several pure arginine kinases suggest that generally the monomeric forms are more acidic than the dimeric proteins. Heat inactivation of arginine kinase in several species indicated a wide range of stabilities, which did not appear to be correlated with quaternary structure, but rather distinguished by the organism's environment. On the other hand, homodimeric arginine kinase proteins from species inhabiting disparate environments are sufficiently homologous to form a catalytically active hybrid. © 2005 Elsevier B.V. All rights reserved. Keywords: Arginine kinase; Creatine kinase; Cross-reactivity; Physical property

1. Introduction Creatine kinase and arginine kinase are phosphagen: ATP phosphotransferases, found in vertebrate and invertebrate species respectively, and are important in muscle bioenergetics. While the cytoplasmic creatine kinases are nearly universally dimeric [1], both monomeric and dimeric forms of arginine kinase are well populated among various invertebrate species [2]. Comparisons of amino acid sequences as well as taxonomic and phylogenetic relationAbbreviations: AK1, Arginine kinase; ATP, arginine phosphotransferase (EC 2.7.3.3); CK, Creatine Kinase; ATP, creatine phosphotransferase (EC 2.7.3.2) ⁎ Corresponding author. Tel.: +1 813 974 3591; fax: +1 813 974 1733. E-mail address: [email protected] (S.H. Grossman). 0304-4165/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2005.11.005

ships suggest that the AK1 gene underwent two sets of gene duplication during evolution, one from a primordial monomer to a dimeric CK and a second from CK to dimeric AK [3,4]. Amino acid sequence analyses of numerous phosphagen kinases indicate that dimeric AK is more closely related to CK than it is to monomeric AK [4]. Indeed, bifunctional heterodimers containing a CK and AK subunit have been prepared [5]. Previous studies using heterologous antibodies against monomeric AK showed that dimeric CK would only react in the denatured state but not the native state [6]. More recent studies were unable to detect cross-reactivity between monomeric and dimeric forms of AK [7]. The antigenic relationship between dimeric AK, monomeric AK and dimeric CK is yet to be reported as evidence for the proposed evolutionary transitions.

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In addition to immunological analysis, comparative studies of the fluorescence lifetime and anisotropy of monomeric AK and dimeric CK demonstrate significant localized differences in tertiary conformation, suggesting that in addition to primary structure, subunit/subunit interactions may be important in subunit conformation and exposure of epitopes [8]. These differences are also reflected by the observation that monomeric AK and dimeric CK significantly differ as substrates for proteolytic enzyme activity [9]. Characterization of structural and antigenic features which may distinguish elements of protein structural hierarchy would contribute to understanding the evolutionary relationships among the phosphagen kinases. In this study, we report results of antigenic cross-reactivity studies using antibodies against dimeric AK proteins and several additional physical structural comparisons. A strong consistency in the classification of cross-reactivity among the species investigated is demonstrated. 2. Materials and methods 2.1. Reagents, organisms and sequence comparisons All reagents used were obtained from Sigma Chemical Co. (St. Louis, MO) unless noted otherwise. Pure CK-MM and CK-BB were purchased from CalBiochem (San Diego, CA). Live sea urchins (Strongylocentrotus purpuratus) and sponges (Tethya aurnatium) were obtained from Marinus Scientific (Garden Grove, CA). Butterflies (Vanessa cardui) were purchased from Greathouse Butterflies, Inc. (Gainsville, FL). Local species were collected in the Tampa Bay area or purchased from Gulf Specimen Marine Laboratory, Inc. (Panacea, Fl). Species included Gryllus rubens. (Cricket), Periplaneta americana (Cockroach), Tenebrio molitor (Mealworm), Veronicella floridan, (Slug), Thyonella gemmata (Striped Sea Cucumber), Penaus aztecus (Shrimp), Paracheirodon innesi (Tetra), Xiphophorus maculates (Platy), Plecia nearctica (Love Bug), Anolis sagrei (Lizard), Xenopus laevis (Frog), Harmonia axyridis (Lady Bug), Pomacea bridgesii (Snail), Sabella melanostigma (Feather Duster), Chione cancellata (Clam), Clypeaster subdepressus (Sea Biscuit), Echinaster spinulosus (Starfish) Ophiothrix angulata (Brittle star) and Isostychopus badonotus (Giant Sea Cucumber). Sequences were obtained from the NCBI database and aligned using NCBI's Blast 2 sequences program.

2.2. Activity measurements Activity of AK was routinely determined using the enzyme-coupled method, production of NADH+ H+, [10] and a Beckman Model DU-640 spectrophotometer. The pH stat assay [11] was used in determining the substrate specificity of the phosphagen kinases. The pH stat assay mixture (maintained at pH 8.0 with NaOH) contained 5 mM phosphagen, 2.5 mM ATP, 3.8 mM magnesium acetate and 2.5 mM DTT, in a final volume of 2.0 mL. Protein concentration was determined using the dye binding method of Bradford [12].

2.3. Extracts for Western blot analysis High speed supernatant extracts were prepared by homogenizing 0.25 g of tissue in 1 mL of 0.05 M Tris/HCl, 10 mM 2-mercaptoethanol, 1 mM EDTA, 50 μM NaN3, and 25 μM phenylmethylsulfonyl fluoride, pH 8.0. Homogenization was followed by high speed centrifugation at 22,000 × g for 10 min at 4 °C. Phosphagen kinase activity of the supernatants was determined with arginine phosphate and creatine phosphate as substrate. Prior to Western blot analysis, samples were diluted and catalytic rates matched at 0.50 absorbance units per minute. Negative controls tested for Western blotting experiments included prebleed anti sera, hemoglobin, alcohol dehydrogenase, aldolase, lysozyme, and ribonucelase A.

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2.4. Purification The purification of AK from the eggs of the purple sea urchin, Strongylocentrotus purpuratus, is a modification of the method developed by Ratto and Christen [13] for the purification of AK from the sea urchin eggs of Paracentrotus lividus. Unfertilized eggs from the purple sea urchin were obtained by injection of 0.5 M KCl. The eggs were washed with sea water and centrifuged three times. Washed eggs were then stored at −80 °C. Packed sea urchin eggs were thawed and homogenized in 0.025 M potassium HEPES, 25 mM magnesium acetate, 10 mM 2-mercaptoethanol, 5 mM magnesium chloride, 5% glycerol, 0.2 mM ADP, 1 mM EDTA, protease inhibitor cocktail tablets from Roche (Indianapolis, IN), pH 7.0, with 5 mL of buffer for 1 mL of packed cells. The resulting suspension was then centrifuged at 81,000×g for 1 h at 6 °C and the collected supernatant subjected to fractionation with ethanol. A first ethanol precipitation of 27% was performed and the majority of AK activity was found in the supernatant. A second ethanol precipitation of 62% yielded the majority of AK activity in the pellet. The 62% pellet was solubilized in 10 mL of 0.05 M Tris/HCl, 10 mM 2-mercaptoethanol, 5 mM magnesium chloride, 0.2 mM ADP, 1 mM EDTA, pH 7.5. The solution was then applied to an ACA 34 (Ciphergen, Freemont, CA) gel filtration column (3.8 × 76 cm) and eluted with the same buffer. Fractions containing 16 mL were collected. Active fractions were combined and applied to a DEAE Sepharose Fast Flow (Pharmacia, Piscataway, NJ) ion-exchange column (1.3 × 12 cm) and washed with 0.02 M sodium phosphate buffer, 10 mM 2-mercaptoethanol, 5 mM magnesium chloride, 0.2 mM ADP, 1 mM EDTA, pH 7.5. The protein was eluted using a 0.02 M to 0.2 M sodium phosphate gradient, pH 7.5. Fractions containing 10 mL were collected, assayed for activity and the most active fractions were combined and concentrated using an Amicon ultra filtration cell (Millipore Corp. Bedford, MA) equipped with an YM-30 membrane. Arginine kinase from Isostychopus badonotus (Giant Sea Cucumber) was purified by the same procedure described for Caudina arenicola [5].

2.5. Preparation of antibodies Polyclonal antibodies against purified sea cucumber AK were developed by ProSci, Inc. (Poway, CA). Polyclonal antibody fractions from serum were purified and concentrated by 33% ammonia sulfate precipitation. The precipitate was then solubilized and dialyzed extensively against 0.05 M sodium phosphate, pH 7.5 containing 0.9% NaCl.

2.6. Western blotting Western blotting was performed with polyacrylamide gels (12.5% running, 4% stacking) [14] in the presence of SDS. Five microliters of each sample was loaded on the gel and subjected to electrophoresis. Detection of AK was determined by the method of Dunbar [15]. Unbound sites were blocked in a 5% (w/v) non-fat dry milk/Tris buffered saline solution. Primary antibody dilution was 1:2,000 and secondary antibody dilution (goat anti-rabbit IgG conjugated with alkaline phosphatase) was 1:20,000.

2.7. Hybridization of AK subunits and detection with polyacrylamide gels Purified preparations of sea cucumber (13 μg) and sea urchin egg AK (36 μg) were mixed in a total volume 1 mL and then diluted with 6 M guanidine hydrochloride (in 0.05 M Tris/HCl, 2 mM DTT, 1 mM EDTA, 50 μg NaN3, pH 8.0 buffer) to a final concentration of 3 M guanidine hydrochloride. After 30 min at 4 °C, the sample was renatured using exhaustive dialysis against the same buffer. Samples were subjected to nondenaturing electrophoresis, without SDS, and stained for catalytic activity. The assay reagent contained 30 mM arginine phosphate, 2 mM ADP, 20 mM glucose, 10 mM magnesium acetate, 2 mM NADP, hexokinase (2 U/mL), glucose-6-phosphate dehydrogenase (1.6 units/mL), 22 μM phenazine methosulfate, and 0.36 mM tetranitroblue tetrazolium in a total volume of 25 mL of 1 M Tris/Acetate, pH 7.5.

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2.8. Isoelectric focusing

3. Results

cucumber antibody against dimeric AK. On the other hand, positive results using the Western blotting technique were detected with each phosphagen kinase known to be dimeric. As expected, dimeric AK from the sea cucumber and sea urchin eggs (both Echinoderms) were positive against anti-AK prepared from AK purified sea cucumber. The clam extract also exhibited AK activity and positive reactivity with anti-sea cucumber AK antibodies, although it is not clear if this AK is monomeric or dimeric. The extract from sabellid worm (an Annelid with a tetrameric AK), though containing significant AK activity, did not react with antibodies against dimeric AK. Both gastropod species, for which the phosphagen kinase structure is not known, possessed AK activity; however, in contrast to the extract from the snail, the slug extract did not react with anti-dimer AK antibodies. Extracts of the vertebrate species examined possessed only CK activity and produced a positive reaction in Western blot analysis with anti-dimer AK antibodies. It is noteworthy that both human CK isozymes were purified samples.

3.1. Purification of sea urchin egg AK

3.4. Substrate specificity

Table 1 summarizes the stepwise results of the isolation from approximately 13 mL of packed eggs from gravid purple sea urchins. The overall purification resulted in a 94-fold enrichment, a 33% yield and 1.2 mg of pure enzyme.

Substrate specificity comparisons show CK is absolutely specific for creatine while monomeric and dimeric arginine kinases react primarily with arginine but also react with canavanine. With canavanine as substrate, AK protein from sea cucumber, sea urchin and cockroach [17] exhibits 15%, 27%, and 30% respectively of the rates compared to Larginine. None of the AK preparations examined exhibited any activity with D-arginine, L-ornithine, glycocyamine or aminoguanidine.

Isoelectric focusing was carried out using a Rotofor Preparative IEF Cell (BioRad Corp., Richmond, CA). The sample (50 mL) contained 2 units (1 unit = 1 μmol NADH+ H+ produced/min) of activity in 0.2 mM DTT and 1% ampholytes, pH 3–10. The preparation was electrofocused for 6 h at 6 °C. Two mL fractions were collected and assayed by the spectrophotometric, enzymecoupled procedure. The pH of each fraction was measured with a Radiometer IE 7.5 mm × 103 mm electrode.

2.9. Circular dichroism Circular dichroic spectra in the far ultraviolet were performed with an Aviv Model 215 spectropolarimeter (Lakewood, NJ) or a Jasco J-500 spectropolarimeter (Easton, MD). Protein samples of monomeric AK (0.25 mg/mL), dimeric AK (0.07 mg/mL), and CK (0.15 mg/mL) were equilibrated in a 0.002 M sodium phosphate, 1 mM 2-mercapthoethanol, pH 8.0 buffer. Alpha helical content was obtained from calculations using equation fn = θ222 + 2340/30,300 [16].

3.2. Molecular weight of native AK from sea urchin eggs Molecular weight of pure AK from sea urchin eggs was determined using calibrated gel filtration for the native enzyme and calibrated SDS-PAGE for the denatured protein (Fig. 1A, B). Analysis of a purified denatured sample exhibited a single protein staining band at a molecular mass corresponding to 43,000 Da (Fig. 1C). In the native state, AK eluted with a molecular mass of 89,000 Da. The profiles shown in the figures indicate that AK from sea urchin eggs is a dimer. 3.3. Antigenic cross-reactivity Results of antigenic reactivity with antibodies against dimeric AK and detection of activity are summarized in Table 2. For each of the species tested in which AK is known to be monomeric, no cross-reactivity was observed with the sea Table 1 Purification of arginine kinase from sea urchin eggs Procedure

Total Total Total Specific volume protein activity activity

Homogenization 38 Centrifugation 32 Ethanol 10 Precipitation Gel filtration 126 (ACA 34) DEAE 30 Sepharose

Fold Percent enrichment yield

3.5. Isoelectric point Isoelectric points, measured over the pH 3 to 10 range, using preparative procedures, were at pH 5.8 for AK from cockroach and at pH 6.0 for AK from sea urchin egg (Fig. 2). Owing to the denaturation of AK at its isolectric point only about 2% of the initial activity was recovered. Creatine kinase MM from monkey exhibited distinct pI values at pH 6.8 and 7.1 [18]. 3.6. Heat inactivation Arginine kinase and creatine kinase activities were affected differently when heated at increasing temperatures each for 10 min (Fig. 3). Arginine kinase from the sea urchin lost approximately 50% activity when heated at 26 °C while AK from the cockroach [7], sea cucumber and butterfly, showed approximately 50% activity loss at 50 °C, 46 °C and 48 °C, respectively. Monkey CK (isozyme CK-MM) appeared the most stable to thermal inactivation with approximately 50% activity still available at 61 °C [18].

349 131 18.3

82.8 80.1 28.4

0.24 0.61 1.6

– 2.5 6.4

6.3

37.2

5.9

24

45

3.7. Preparation and detection of a heterodimer AK

1.2

27.1

22.6

94

33

Dimeric AK from the sea cucumber and dimeric AK from sea urchin eggs were mixed and denatured with guanidine

100 97 34

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Fig. 1. Molecular weight determination of AK from sea urchin eggs. (A) Calibration curve of SDS-PAGE with molecular weight standards (lysozyme 18.8 kDa, soybean trypsin inhibitor 32.3 kDa, carbonic anhydrase 42.2 kDa, bovine serum albumin 87 kDa, beta-galactosidase 131 kDa, myosin 210 kDa) and sea urchin egg AK. Approximately 1 μg of AK was applied to the gel. (B) Calibration curve of Ultrogel AcA 34 column (35 cm × 1.8 cm) with molecular weight standards (carbonic anhydrase 29 kDa, cockroach arginine kinase 42.5 kDa, rabbit creatine kinase 81.5 kDa, and alkaline phosphatase 140 kDa) and sea urchin egg AK. Approximately 0.77 units of activity (1 unit = 1 μmol of NADPH+ H+ produced per minute) was applied to the gel filtration column, previously equilibrated and subsequently eluted with 0.05 M Tris/HCl, 1 mM EDTA, 10 mM 2-mercaptoethanol, pH 8.0 buffer. (C) Figure showing SDS-PAGE results. Lane 1: molecular weight markers, lane 2: crude homogenate, lane 3: purified sea urchin AK, lane 4: purified bovine serum albumin.

hydrochloride. After removal of denaturant, a third activity staining band was detected approximately half-way between the two parent homodimers (Fig. 4). Attempts to prepare a hybrid between dimeric AK from the sea cucumber and monomeric AK from the cockroach were unsuccessful. 3.8. Alpha helical content Circular dichroic spectrum display characteristic minima at 222 and 208 nm for purified samples of AK from the sea cucumber. Calculations [16] show that this dimeric phosphagen kinase has approximately 9% alpha helical content. Studies also showed a 12% α-helical content for monomeric cockroach AK [7] and 19% for dimeric rabbit muscle CK. The results with CKMM agree with previous evaluation of the muscle CK protein (17.8%), which included reported values of 21% for CK-BB and 24% CK-MB [19]. 4. Discussion 4.1. Purification and molecular weight The purifications to homogeneity of AK from sea urchin (Strongylocentrotus purpuratus) eggs and the sea cucumber (Isostychopus badonotus) supplement the list of previously

isolated AK enzymes, both dimeric and monomeric. The percent activity yield is somewhat greater than reported by Ratto and Christen [13] for 5 × 107 eggs from sea urchin (Paracentrotus lividus). The molecular weight and subunit composition is consistent with determinations of AK from other echinoderms {Caudina arenicola, [5], Stichopus japonicus [4] and Holothuria forskali [20]. 4.2. Cross-reactivity All of the arthropod species tested, known to contain monomeric AK did not react with antibodies against dimeric AK. Positive reactions were reported in a previous study involving cross-reactivity of these species with antibody against monomeric AK from the shrimp [7]. While all of the species known to contain dimeric AK reacted with antibodies against dimeric AK from the sea cucumber, strong positive reactions were also obtained with both purified homodimeric isozymes of CK as well as extracts from several species known to contain CK. The sequence homology and now the epitope reactivity strongly support the close evolutionary relationship between dimeric AK and CK. It is interesting, however, that no reaction is detected between anti-dimer AK antibodies and monomeric AK from any species. The positive response for AK from the clam is noteworthy since the clam

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Table 2 Catalytic activity and immunological cross-reactivity of phosphagen kinases with dimeric arginine kinase polyclonal antibody Species

Monomeric Gryllus sp. (Cricket) Periplaneta americana (Cockroach) Tenebrio molitor (Mealworm) Penaeus aztecus (Shrimp) Plecia nearctica (Love Bug) Harmonia axyridis (Lady Bug) Chione cancellata (Clam) a Echinaster spinulosus (Starfish) b Ophiothrix angulata (Brittle Star)b Dimeric Thyonella gemmata (Striped Sea Cucumber) Strongylocentrotus p. (Sea Urchin Egg) Clypeaster subdepressus (Sea Biscuit) Tethya aurantia (Sponge) Paracheirodan innesi (Fish) Xiphophorus maculates (Fish) Anolis sagrei (Lizard) Xenopus laevis (Frog) Homosapien (CK-BB Human) Homosapien (CK-MM Human) Monomeric and trimeric Strongylocentrotus p. (Sea Urchin Sperm) Tetrameric Sabella melanostigma (Feather Duster) Undetermined Veronicella floridan (Slug) Pomacea bridgesii (Snail) a b

Enzymatic activity

Cross reactivity

AK

CK

±

+ +

− −

− −

+

−

−

+ +

− −

− −

+

−

−

+ −

− +

+ −

−

+

−

+

−

+

+

−

+

+

−

+

− − − − − −

+ + + + + +

+ + + + + +

−

+

+

−

+

−

+

−

−

+ +

− −

− +

Fig. 2. Isoelectric focusing of AK from sea urchin eggs. Experimental details are described in the text. Approximately 2 units of activity in 50 mL of 0.2 mM DTT containing 1% ampholytes (pH 3–10) were injected into the electrofocusing chamber. (Δ) pH; (●) activity.

phosphagen kinase [23]. The finding that this primitive species contains a dimeric CK that cross-reacts with antidimeric AK suggests a much earlier expression of a dimeric phosphagen kinase able to phosphorylate creatine. 4.3. Isoelectric point Comparison of the isoelectric points show that the monomeric form is generally more acidic than the two dimeric forms investigated. The pI value of 5.8 for monomeric cockroach AK compares closely with values of 5.3 for monomeric squid muscle enzyme [24] and monomeric lobster muscle enzyme of 5.4 [25]. Previously, we determined the pI of a different dimeric sea cucumber (Caudina arenicola) to be 7.8 [5] and here show a pI value of 6.7 for dimeric arginine kinase from the sea urchin. Experimentally determined isoelectric points of native proteins

Two domain monomer with molecular weight of 80 kDa [21]. Monomer with molecular weight of 150 kDa [3].

protein is reported to be a monomer but of 80 kDa molecular size [21]; however, the bivalves exhibit several different types of subunit assembly for AK. If, as noted, the clam enzyme evolved from gene duplication and fusion, it may have at this point developed the epitopes which are common to the modern dimeric AK enzyme from the Echinoderms and vertebrate CK. One the other hand, the large, multidomain monomer of CK found in sea urchin sperm, does not crossreact with anti-dimeric AK. Most notable is the detection of CK activity in the orange puff ball sponge [22,23] and its antigenic reaction with antibodies against dimeric AK particularly in view of the evolutionary position of this organism with respect to the proposed development of the

Fig. 3. Heat Inactivation. Thermal stability of the phosphagen kinases were determined in preparations obtained from crude, whole organism homogenates or partially purified samples. Each extract was incubated at the indicated temperatures for 10 min, rapidly cooled on ice, and assayed using the spectrophotometric, enzyme-coupled assay (production of NADH+ H+). (●) CK, monkey; (♦) AK, cockroach; (■) AK, sea cucumber; (●) AK, sea urchin eggs; (▴) AK butterfly.

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Fig. 4. Hybridization of dimeric AK. Hybridization procedures are described in the text. The 10% separating and 4% stacking polyacrylamide gel was prepared with SDS omitted and developed at 4 °C. AK activity was detected after incubating the gel in assay reagent for 4 h at 4 °C. After development, the gel was destained in 70% methanol–5% acetic acid solution for 30 min. (A) Native sea cucumber AK; (B) native sea urchin egg AK; (C) mixed native sea cucumber and sea urchin egg AK; (D) denatured/renatured mix of sea cucumber AK and sea urchin egg AK; (E) denatured/renatured sea urchin egg AK; (F) denatured/renatured sea cucumber AK.

are not only due to amino acid composition but also to the degree of exposure of charged amino acid side chains as well as the interaction with each other and the solvent in the tertiary structure. For example among the isozymes of CK, though 80% sequence homologous [26], the brain isozyme and muscle isozyme exhibit isoelectric points of 4.8 and 6.9, respectively [18]. This is attributed to several additional basic amino acids in the muscle form but also possible conformational differences. Interestingly, comparison of the sum of the acidic and basic amino acids for monomeric AK from the moth [27] and dimeric AK from the sea cucumber [5], are nearly identical, even though the sequences are only about 40% homologous. This difference in pI apparently reflects differences in conformation, which is consistent with the observed non-cross reactivity of antigenic sites. 4.4. Heat inactivation Among the samples of AK and CK examined for thermal stability, the AK preparation from the sea urchin was significantly more sensitive to heat inactivation. Indeed, among all the phosphagen kinases so far examined, this enzyme appears to be most sensitive to temperature. Although there is a high degree of homology between CK and dimeric AK from the sea urchin, it appears that some structural distinctions are sufficient to result in substantial differences in stability. In the case of the cold water sea urchin (Strongylocentrotus purpuratus), this sensitivity may have evolved from environmental demands. On the other hand, AK from the terrestrial insects (monomeric AK), cockroach and butterfly, as well as the warm water sea cucumber (dimeric AK) exhibit similar temperature inactivation profiles. This suggests that quaternary state is not a determinate in thermal stability. While more organisms need to be examined, the preliminary conclusion is that environment has been an important factor in the evolution of thermal stability of the arginine kinases. 4.5. Hybridization The ability of dimeric AK and CK to form heterofunctional dimers has been reported previously [5]. It is well known that CK isozymes form a homofunctional heterodimer both in vivo and in vitro [28]. We have now demonstrated the ability of two

different dimeric AK proteins to form a functional hybrid with each other, with one homodimer from the gamete of one species and the other homodimer for the muscle of another species. These studies show that dimeric phosphagen kinases possess conserved structural features required for subunit association, catalytic activity and formation of appropriate quaternary structure. 4.6. Evolution Based on a review of numerous organisms, Kerkut [29] presciently concluded that there was no clear distinction between vertebrates and invertebrates with regard to the distribution of phosphagen substrates. Furthermore, since related genera within a class differ in phosphagens used, it is unlikely that distribution of these substrates can characterize phylogenetic relationships. However, Watts [30] used the distribution of the phosphagen kinases to construct an evolutionary tree, with monomeric AK being the primary source for subsequent forms of AK and CK. From the characterization of distributions, activities and subunit structures for the phosphagen kinases, Moreland et al. [31] proposed two possible pathways: (1) Monomeric AK → Dimeric AK → Dimeric CK; (2) Monomeric AK → Dimeric CK → Dimeric AK. Subsequent investigations have resulted in proposed models of phosphagen kinase evolution and accompanying taxonomy [3,4,32,33]. Recently, Peroviæ-Ottstadt [34] reported the detection of AK in the sponge Suberites domuncula and incorporated this observation in developing a generalized evolution pattern in which the known distribution of arginine, creatine, and glycocyamine kinases can be traced back to a primitive sponge. Support for the proposal that dimeric CK preceded dimeric AK is obtained from the following observations. First, it has been shown that the sequence homology between dimeric AK and dimeric CK is much greater than between dimeric AK and monomeric AK [4]. The present study supports this idea by demonstrating a strong and consistent reaction between antibodies against dimeric AK and the two cytoplasmic CK homodimers, but no antigenic response against any of monomeric AK proteins tested. Furthermore, no positive

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Western reactions have been detected between anti-monomeric AK and dimeric AK or CK. Second, the active site region is the only deletion/insertion region with activity conserved specificity (using monomeric sequences to identify this region) and the deletion size of dimeric AK is closer to that of dimeric CK than monomeric AK [4]. Third, the unusual detection of dimeric CK in the sponge, a very primitive organism, suggests that dimeric CK arose from a very primitive phosphagen kinase, long before dimeric AK evolved in the echinoderms and dimeric CK appeared in the vertebrates [23]. The close structural and conformational relationship between dimeric CK in the sponge and dimeric AK in the sea cucumber is further supported here by antigen cross-reactivity experiments. Fourth, Suzuki et. al [35] have recently detailed and refined the evolutionary sequence in the creatine kinase isozymes with newly determined sequences for cytoplasmic, mitochondrial, and flagellar CK from several organisms. Comparison of these sequences, show the presence of the CK gene even prior to divergence of protostomes and deuterostomes. Results strongly support the proposal that AK evolved from CK since the gene for sea cucumber (Stichopus) AK is nearly identical to that of cytoplasmic CK. It has been suggested that the conversion of AK to CK resulted from the wide role of arginine in metabolism and regulation (histones for example) whereas creatine is only involved in the CK reaction [30]. If CK thus arose for specialized needs, the question remains why the CK gene (which presumably arose from an AK gene) again reverted to express a protein which would still be competitive for the available arginine. It should be noted that the evolution of dimeric CK, if from early monomeric AK, required two mutations: substrate specificity and ability for subunit association. If ability to dimerize preceded the change in substrate specificity, then one need not assume that AK evolved twice. However, if cytoplasmic CK, which is known today to be nearly always dimeric (the monomeric CK in the Echinoderm Ophiuroids, Echinoids and Asteroids, are high molecular weight forms containing three identical sequence repeats [3]) evolved from a monomeric AK or other phosphagen kinase, then at one time, there may have occurred a monomeric CK. Present day evidence for this is that for a typical vertebrate dimeric CK, Sepharose-bound monomeric derivatives are catalytically active [36], and it now appears proven [37] that the subunits of CK are catalytically active [38,39], independent of subunit/ subunit association or interaction. It remains unclear, however, if subunit/subunit interaction is significant in the known regulatory properties of the creatine kinases.

Acknowledgements This work was supported, in part, by a grant (S.G.) from the US Department of Agriculture (2001-02817). We thank Dr. Ted Gauthier for his assistance with the circular dichroism measurements and Mr. Kristopher Wentzel for butterfly AK heat inactivation data.

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