Comprehensive proteomics approach in characterizing and quantifying allergenic proteins from northern shrimp: toward better occupational asthma prevention.

Occupational asthma is a major chronic health dilemma among workers involved in the seafood industry. Several proteins notoriously known to cause asthma have been reported in different seafood. This work involves the application of an allergenomics strategy to study the most potent allergens of northern shrimp. The proteins were extracted from shrimp tissue and profiled by gel electrophoresis. Allergenic proteins were identified based on their reactivity to patient sera and were structurally identified using tandem mass spectrometry. Northern shrimp tropomyosin, arginine kinase, and sarcoplasmic calcium-binding protein were found to be the most significant allergens. Multiple proteolytic enzymes enabled 100% coverage of the sequence of shrimp tropomyosin by tandem mass specrometry. Only partial sequence coverage was obtained, however, for the shrimp allergen arginine kinase. Signature peptides, for both tropomyosin and arginine kinase, were assigned and synthesized for use in developing the multiple reaction monitoring tandem mass spectrometric method. Subsequently, air samples were collected from a shrimp processing plant and two aerosolized proteins quantified using tandem mass specrometry. Allergens were detected in all areas of the plant, reaching levels as high as 375 and 480 ng/m(3) for tropomyosine and arginine kinase, respectively. Tropomyosine is much more abundant than arginine kinase in shrimp tissues, so the high levels of arginine kinase suggest it is more easily aerosolized. The present study shows that mass spectrometric analysis is a sensitive and accurate tool in identifying and quantifying aerosolized allergens.

Identification of physicochemical properties of Scylla paramamosain allergen, arginin kinase.

BACKGROUND: Arginine kinase (AK) is expressed in a wide variety of species, including human food sources (seafood) and pests (cockroaches and moths), and has been reported as a novel allergen. However, there has been little research on the allergenicity of AK in crustaceans. In this study the physicochemical properties of AK from mud crab (Scylla paramamosain) were investigated. RESULTS: Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and inhibition enzyme-linked immunosorbent assay revealed that purified AK was unstable in thermal processing and in acid buffer. Under simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions, purified AK was much more readily degraded by pepsin than by trypsin or chymotrypsin. The unpurified AK in crab myogen degraded more markedly than purified AK. In addition, in two-phase gastrointestinal digestion, AK was rapidly degraded by pepsin but resistant to trypsin and chymotrypsin digestion, while tropomyosin derived from mud crab was resistant to pepsin digestion but digested readily by trypsin or chymotrypsin. Further study of serum samples obtained from crab-allergic human patients indicated that the allergenicity of AK was markedly reduced by digestion with SGF but not SIF. CONCLUSION: AK is an important food allergen despite its unstable physicochemical properties of digestibility.

Purification, cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab allergen.

BACKGROUND: Although crustaceans have been reported to be one of the most common causes of IgE-mediated allergic reactions, there are no reports about the characterization and identification of arginine kinase (AK) from the mud crab (Scylla serrata) as allergen. In the present study, the purification, molecular cloning, expression and immunological analyses of the IgE allergen AK from the mud crab were investigated. RESULTS: The results showed that cloned DNA fragments of AK from the mud crab had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. Sequence alignment revealed that mud crab AK shares high homology with other crustacean species. Mud crab AK gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli BL 21. 2-D electrophoresis suggested that native AK (nAK) and recombinant AK (rAK) shared the same molecular weight of 40 kDa, and the pI is 6.5 and 6.3, respectively. The nAK and rAK were further confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Immunoblotting analysis and colloidal gold immunochromatographic assay (GICA) using sera from subjects with crustacean allergy confirmed that the nAK and rAK reacted positively with these sera, indicating AK is a specific allergen of mud crab. CONCLUSION: Both of purified nAK and rAK reacted positively with sera from subjects with crustacean allergy in immunoblotting and GICA analysis, indicating AK is a common allergen of mud crab. In vitro expressed AK is proposed as a source of the protein for immunological or clinical studies.

Protein extraction protocols for optimal proteome measurement and arginine kinase quantitation from cricket Acheta domesticus for food safety assessment.

Insects have been consumed by people for millennia and have recently been proposed as a complementary, sustainable source of protein to feed the world's growing population. Insects and crustaceans both belong to the arthropod family. Crustacean (shellfish) allergies are common and potentially severe; hence, the cross-reactivity of the immune system with insect proteins is a potential health concern. Herein, LC-MS/MS was used to explore the proteome of whole, roasted whole and roasted powdered cricket products. Eight protein extraction protocols were compared using the total number of protein and distinct peptide identifications. Within these data, 20 putative allergens were identified, of which three were arginine kinase (AK) proteoforms. Subsequently, a multiple reaction monitoring MS assay was developed for the AK proteoforms and applied to a subset of extracts. This targeted assay demonstrated that allergen abundance/detectability varies according to the extraction method as well as the food processing method.

Identification and characterization of Crassostrea angulata arginine kinase, a novel allergen that causes cross-reactivity among shellfish.

Oyster is a common food that causes allergy. However, little information is available about its allergens and cross-reactivity. In this study, arginine kinase (AK) was identified as a novel allergen in Crassostrea angulata. The primary sequence of AK was cloned which encoded 350 amino acids, and recombinant AK (rAK) was obtained. The immunodot results, secondary structure and digestive stability showed that native AK and rAK had similar IgG/IgE-binding activity and physicochemical properties. Serological analysis of 14 oyster-sensitive individuals demonstrated that AK exhibited cross-reactivity among oysters, shrimps, and crabs. Furthermore, nine epitopes in oyster AK were verified using inhibition dot blots and inhibition enzyme linked immunosorbent assay, six of which were similar to the epitopes of shrimp/crab AK. The most conserved epitopes were P5 (121-133) and P6 (133-146), which may be responsible for the cross-reactivity caused by AK. These findings will provide a deeper understanding of oyster allergens and cross-reactivity among shellfish.

Subcellular localization of Trypanosoma cruzi arginine kinase.

Phosphoarginine is a cell energy buffer molecule synthesized by the enzyme arginine kinase. In Trypanosoma cruzi, the aetiological agent of Chagas' disease, 2 different isoforms were identified by data mining, but only 1 was expressed during the parasite life cycle. The digitonin extraction pattern of arginine kinase differed from those obtained for reservosomes, glycosomes and mitochondrial markers, and similar to the cytosolic marker. Immunofluorescence analysis revealed that although arginine kinase is localized mainly in unknown punctuated structures and also in the cytosol, it did not co-localize with any of the subcelular markers. This punctuated pattern has previously been observed in many cytosolic proteins of trypanosomatids. The knowledge of the subcellular localization of phosphagen kinases is a crucial issue to understand their physiological role in protozoan parasites.

Sarcoplasmic calcium-binding protein: identification as a new allergen of the black tiger shrimp Penaeus monodon.

BACKGROUND: Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. METHODS: A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. RESULTS: The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. CONCLUSIONS: SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish.

Kinetic analysis of two purified forms of arginine kinase: absence of cooperativity in substrate binding of dimeric phosphagen kinase.

Arginine kinase from sea urchin eggs and sea cucumber muscle are dimeric enzymes, unlike the more widely distributed monomeric enzyme found in other invertebrates. Both purified enzymes exhibited features characteristic of the monomeric arginine kinases including pH optima, formation of a catalytic dead-end complex (enzyme-MgADP-arginine) and stabilization of this complex by monovalent anions. A complete analysis of initial velocity data, in both directions for each substrate, indicated that substrate binding cooperativity was either minimal or non-existent. Unlike many other multi-subunit enzymes, the significance of the dimeric state of the phosphagen kinases remains unclear. These present results would suggest that (a) cooperativity, or so-called synergism in substrate binding is not a characteristic of the dimeric state of the protein and (b) the functional significance of the dimeric state is not related to the ability of some of these enzymes to undergo cooperativity in substrate binding. The significance of the dimeric state for the creatine kinases and arginine kinases remains to be established.

Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis.

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. The gene encoding Locusta migratoria manilensis AK was cloned and expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 358 mg L(-1) of LB medium. Some parameters for the renatured and soluble forms of AK, including Km, Kd, specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest kcat and stronger synergistic substrate binding. The first report of a concise purification method enables the enzyme to be prepared in large quantities. This research should enable further detailed investigations of the enzymatic mechanism by site directed mutagenesis techniques.

Arginine kinase from Haemonchus contortus decreased the proliferation and increased the apoptosis of goat PBMCs in vitro.

BACKGROUND: Arginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates. Immunologically, AKs are important constituents of different body parts, involved in various biological and cellular functions, and considered as immune-modulator and effector for pro-inflammatory cytokines. However, immunoregulatory changes of host cells triggered by AK protein of Haemonchus contortus, a parasitic nematode of ruminants, are still unknown. The current study was focused on cloning and characterisation of Hc-AK, and its regulatory effects on cytokines level, cell migration, cell proliferation, nitric oxide production and apoptosis of goat peripheral blood mononuclear cells (PBMCs) were observed. METHODS: The full-length sequence of the Hc-AK gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sub-cloned into the prokaryotic expression vector pET-32a. The biochemical characteristics of recombinant protein Hc-AK, which was purified by affinity chromatography, were performed based on the enzymatic assay. Binding of rHc-AK with PBMCs was confirmed by immunofluorescence assay (IFA). Immunohistochemical analysis was used to detect localisation of Hc-AK within adult worms sections. The immunoregulatory effects of rHc-AK on cytokine secretions, cell proliferation, cell migration, nitric oxide production and apoptosis were determined by co-incubation of rHc-AK with goat PBMCs. RESULTS: The full-length ORF (1080 bp) of the Hc-AK gene was successfully cloned, and His-tagged AK protein was expressed in the Escherichia coli strain BL21. The recombinant protein of Hc-AK (rHc-AK) was about 58.5 kDa together with the fused vector protein of 18 kDa. The biochemical assay showed that the protein encoded by the Hc-ak exhibited enzymatic activity. Western blot analysis confirmed that the rHc-AK was recognised by the sera from rat (rat-antiHc-AK). The IFA results showed that rHc-AK could bind on the surface of goat PBMCs. Immunohistochemically, Hc-AK was localised at the inner and outer membrane as well as in the gut region of adult worms. The binding of rHc-AK to host cells increased the levels of IL-4, IL-10, IL-17, IFN-γ, nitric oxide (NO) production and cell apoptosis of goat PBMCs, whereas, TGF-ß1 levels, cell proliferation and PBMCs migration were significantly decreased in a dose dependent manner. CONCLUSIONS: Our findings suggested that rHc-AK is an important excretory and secretory (ES) protein involved in host immune responses and exhibit distinct immunomodulatory properties during interaction with goat PBMCs.

Purification and characterization of arginine kinase from locust.

L-Arginine kinase (AK; ATP:L-arginine N-phosphotransferase; EC 2.7.3.3) catalyzes the reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP thus buffering cellular ATP levels. AK was purified from the leg muscle of the locust Migratoria manilensis by Sephacryl S-200 HR gel filtration chromatography and DEAE Sepharose CL-6B fast flow anion exchange chromatography to an apparent homogeneity with a recovery of 80%. The enzyme behaved as monomeric protein with molecular mass of about 40 kD, and had a pH and temperature optimum of 8.6 and 30 degrees C, respectively, and a pI of about 6.3. The Michaelis constants for synthesis of PArg are 0.936 and 1.290 mM for L-arginine and ATP, respectively and k(cat)/K(m)(Arg) 174. The activity of AK required divalent cations such as Mg(2+) and Mn(2+). In the presence of Cu(2+) and Zn(2+), AK activity was greatly inhibited. The intrinsic protein fluorescence emission maximum at 330 nm using the excitation wavelength at 295 nm suggested that tryptophan residues are below the surface of the protein and not exposed to solvent.

Effects of cadmium on the cuttlefish Sepia pharaonis' arginine kinase: unfolding kinetics integrated with computational simulations.

Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd(2+) on the cuttlefish Sepia pharaonis' arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd(2+) effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd(2+) induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd(2+) induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd(2+) showed that Cd(2+) partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd(2+)-induced inactivation of SPAK. These results demonstrate the effect of Cd(2+) on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd(2+) in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.

O-Crystallin, arginine kinase and ferritin from the octopus lens.

Three proteins have been identified in the eye lens of the octopus, Octopus dofleini. A 22 kDa protein comprising 3-5% of the soluble protein of the lens is 35-43% identical to a family of phosphatidylethanolamine-binding proteins of vertebrates. Other members of this family include the immunodominant antigen of the filarial parasite, Onchocerca volvulus, putative odorant-binding proteins of Drosophila and a protein with unknown function of Caenorhabditis elegans. We have called this protein O-crystallin on the basis of its abundance in the transparent lens. O-Crystallin mRNA was detected only in the lens. Two tryptic peptides of another octopus lens protein, less abundant than O-crystallin, showed 80% identity to arginine kinase of invertebrates, a relative of creatine kinase of vertebrates. Finally, ferritin cDNA was isolated as an abundant cDNA from the octopus lens library. Northern blots showed that ferritin mRNA is not lens-specific.

The Inhibitory Effects of Cu(2+) on Exopalaemon carinicauda Arginine Kinase via Inhibition Kinetics and Molecular Dynamics Simulations.

We studied the Cu(2+)-mediated inhibition and aggregation of Exopalaemon carinicauda arginine kinase (ECAK). We found that Cu(2+) significantly inactivated ECAK activity and double-reciprocal kinetics demonstrated that Cu(2+) induced noncompetitive inhibition of arginine and ATP (IC50 = 2.27 ± 0.16 µM; K i for arginine = 13.53 ± 3.76; K i for ATP = 4.02 ± 0.56). Spectrofluorometry results showed that Cu(2+) induced ECAK tertiary structural changes including the exposure of hydrophobic surfaces that directly induced ECAK aggregation. The addition of osmolytes such as glycine and proline successfully blocked ECAK aggregation induced by Cu(2+) and recovered ECAK activity. We built a 3D structure for ECAK using the ECAK ORF gene sequence. Molecular dynamics (MD) and docking simulations between ECAK and Cu(2+) were conducted to elucidate the binding mechanisms. The results showed that Cu(2+) blocked the entrance to the ATP active site; these results are consistent with the experimental result that Cu(2+) induced ECAK inactivation. Since arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrates, our study can provide new information about the effect of Cu(2+) on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding to better understand the role of the invertebrate ECAK metabolic enzyme in marine environments.

Malaysian cockle (Anadara granosa) allergy: Identification of IgE-binding proteins and effects of different cooking methods.

The purpose of this study was to evaluate the effect of different cooking methods on the allergenicity of cockle and to identify proteins most frequently bound by IgE antibodies using a proteomics approach. Raw, boiled, fried and roasted extracts of the cockle were prepared. The protein profiles of the extracts were obtained by separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis (2-DE). IgE-immunoblotting was then performed with the use of individual sera from patients with cockle allergy and the major IgE-binding proteins were analyzed by mass-spectrometry. SDS-PAGE of raw extract showed 13 protein bands. Smaller numbers of protein bands were detected in the boiled, fried and roasted extracts. The 2-DE gel profile of the raw extract further separated the protein bands to ~50 protein spots with molecular masses between 13 to 180 kDa and isoelectric point (pI) values ranging from 3 to 10. Immunoblotting of raw extract exhibited 11 IgE-binding proteins with two proteins of 36 and 40 kDa as the major IgE-binding proteins, while the boiled extract revealed 3 IgE-binding proteins. Fried and roasted extracts only showed a single IgE-binding protein at 36 kDa. 2-DE immunoblotting of raw extract demonstrated 5 to 20 IgE reactive spots. Mass spectrometry analysis led to identification of 2 important allergens, tropomyosin (36 kDa) and arginine kinase (40 kDa). Heated extracts showed a reduction in the number of IgE-reactive bands compared with raw extract, which suggest that thermal treatment can be used as a tool in attempting to reduce cockle allergenicity. The degree of allergenicity of cockle was demonstrated in the order raw > boiled > fried ≈ roasted. Two important allergens reacting with more than 50% of patients' sera identified using mass spectrometric approaches were tropomyosin and arginine kinase. Thus, allergens found in this study would help in component based diagnosis, management of cockle allergic patients and to the standardisation of allergenic test products as tools in molecular allergology.

Isolation of arginine kinase from Apis cerana cerana and its possible involvement in response to adverse stress.

Arginine kinases (AK) in invertebrates play the same role as creatine kinases in vertebrates. Both proteins are important for energy metabolism, and previous studies on AK focused on this attribute. In this study, the arginine kinase gene was isolated from Apis cerana cerana and was named AccAK. A 5'-flanking region was also cloned and shown to contain abundant putative binding sites for transcription factors related to development and response to adverse stress. We imitated several abiotic and biotic stresses suffered by A. cerana cerana during their life, including heavy metals, pesticides, herbicides, heat, cold, oxidants, antioxidants, ecdysone, and Ascosphaera apis and then studied the expression patterns of AccAK after these treatments. AccAK was upregulated under all conditions, and, in some conditions, this response was very pronounced. Western blot and AccAK enzyme activity assays confirmed the results. In addition, a disc diffusion assay showed that overexpression of AccAK reduced the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, our results indicated that AccAK may be involved of great significance in response to adverse abiotic and biotic stresses.

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: a model for studying phosphagen kinases.

Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated from E. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 A resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cryo temperatures have all yielded significant improvements in diffraction.

Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences.

Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a Km value of 0.6 mM for arginine and a Vmax value of 13 micromole Pi min(-1) mg protein(-1) for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1,608 and 1,239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66-73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63-71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55-57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.

Gene structure of two-domain arginine kinases from Anthopleura japonicus and Pseudocardium sachalinensis.

Unusual two-domain arginine kinases (AKs) arose independently at least two times during molecular evolution of phosphagen kinases: AKs from the primitive sea anemone Anthopleurura japonicus and from the clam Pseudocardium sachalinensis. To elucidate its unusual evolution, the structures of Anthopleura and Pseudocardium AK genes have been determined. The Anthopleura gene consisted of 4 exons and 3 introns: two domains are linked by a bridge intron, and each domain contains one intron in different positions. On the other hand, the Pseudocardium gene consisted of 10 exons and 9 introns: two domains are also linked by a bridge intron, and domains 1 and 2 contains 3 and 5 introns, respectively, of which 3 introns are located in exactly same positions. Since the two domains of Pseudocardium AK are estimated to have diverged about 290 million years ago, the 3 introns have been conserved at least for this long. Comparison of intron positions in Anthopleura, Pseudocardium and C. elegans AK genes indicates that there is no intron conserved through the three AK lineages, in sharp contrast to relatively conservative intron positions in creatine kinase (CK) gene family.

A dimeric creatine kinase from a sponge: implications in terms of phosphagen kinase evolution.

This study demonstrates conclusively that tissues of the sponge Tethya aurantia contain significant creatine kinase (CK) activity. This CK was purified and analyzed with respect to a number of physico-chemical properties. Size exclusion chromatography and denaturing gel electrophoresis analyses showed that this enzyme is dimeric. The sequences of several Lys-C endoproteinase peptides from Tethya CK are consistent with this enzyme being a member of the phosphagen kinase family and a true CK. CK in higher organisms exists in a variety of quaternary structure forms--dimer, octamer and large monomer consisting of a three contiguous CK domains. The present results indicate that CK evolved very early in metazoan evolution and that the dimeric structure preceded other subunit association forms.