Molecular characterization and kinetic properties of a novel two-domain taurocyamine kinase from the lung fluke Paragonimus westermani

Taurocyamine kinase (TK) was previously reported to be restricted to certain marine annelids; however, the present study has proven otherwise. The lung fluke Paragonimuswestermani has a contiguous two-domain TK with a mass of 80 216 Da consisting of 713 amino acid residues sharing higher sequence identity with molluscan arginine kinase (AK). Both domains of P. westermani TK have significant activity for the substrate taurocyamine and exhibited synergism during substrate binding. Since TK plays a key role in energy metabolism and is not present in mammals, inhibitors against P. westermani TK could be effective novel chemotherapeutic agents and could be utilized for the development of specific diagnostic tools for the detection of paragonimiasis.     

Origin and properties of cytoplasmic and mitochondrial isoforms of taurocyamine kinase

Taurocyamine kinase (TK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase. TK is found only in certain marine annelids. In this study we used PCR to amplify two cDNAs coding for TKs from the polychaete Arenicola brasiliensis, cloned these cDNAs into the pMAL plasmid and expressed the TKs as fusion proteins with the maltose-binding protein. These are the first TK cDNA and deduced amino acid sequences to be reported. One of the two cDNA-derived amino acid sequences of TKs shows a high amino acid identity to lombricine kinase, another phosphagen kinase unique to annelids, and appears to be a cytoplasmic isoform. The other sequence appears to be a mitochondrial isoform; it has a long N-terminal extension that was judged to be a mitochondrial targeting peptide by several on-line programs and shows a higher similarity in amino acid sequence to mitochondrial creatine kinases from both vertebrates and invertebrates. The recombinant cytoplasmic TK showed activity for the substrates taurocyamine and lombricine (9% of that of taurocyamine). However, the mitochondrial TK showed activity for taurocyamine, lombricine (30% of that of taurocyamine) and glycocyamine (7% of that of taurocyamine). Neither TK catalyzed the phosphorylation of creatine. Comparison of the deduced amino acid sequences of mitochondrial CK and TK indicated that several key residues required for CK activity are lacking in the mitochondrial TK sequence. Homology models for both cytoplasmic and mitochondrial TK, constructed using CK templates, provided some insight into the structural correlation of differences in substrate specificity between the two TKs. A phylogenetic analysis using amino acid sequences from a broad spectrum of phosphagen kinases showed that annelid-specific phosphagen kinases (lombricine kinase, glycocyamine kinase and cytoplasmic and mitochondrial TKs) are grouped in one cluster, and form a sister-group with CK sequences from vertebrate and invertebrate groups. It appears that the annelid-specific phosphagen kinases, including cytoplasmic and mitochondrial TKs, evolved from a CK-like ancestor(s) early in the divergence of the protostome metazoans. Furthermore, our results suggest that the cytoplasmic and mitochondrial isoforms of TK evolved independently.     

Molecular and catalytic properties of an arginine kinase from the nematode Ascaris suum

We amplified the cDNA coding for arginine kinase (AK) from the parasitic nematode Ascaris suum, cloned it in pMAL plasmid and expressed the enzyme as a fusion protein with the maltose-binding protein. The whole cDNA was 1260 bp, encoding 400 amino acids, and the recombinant protein had a molecular mass of 45,341 Da. Ascaris suum recombinant AK showed significant activity and strong affinity ( K(m)(Arg) = 0.126 mM) for the substrate L-arginine. It also exhibited high catalytic efficiency ( k(ca)/K(m)(Arg) = 352) comparable with AKs from other organisms. Sequence analysis revealed high amino acid sequence identity between A. suum AK and other nematode AKs, all of which cluster in a phylogenetic tree. However, comparison of gene structures showed that A. suum AK gene intron/exon organization is quite distinct from that of other nematode AKs. Phosphagen kinases (PKs) from certain parasites have been shown to be potential novel drug targets or tools for detection of infection. The characterization of A. suum AK will be useful in the development of strategies for control not only of A. suum but also of related species infecting humans.     

Arginine kinase isoforms in the closest protozoan relative of metazoans

The genome of the choanoflagellate Monosiga brevicollis contains at least three genes for the phosphoryl transfer enzyme, arginine kinase (AK; EC 2.7.3.3). Bioinformatic analyses of the deduced amino acid sequences of the proteins coded for by two of these genes showed that one of these AKs is cytoplasmic (denoted AK1) while the other appears to have an N-terminal mitochondrial targeting peptide (denoted AK2). Cloning and expression of the cDNA for AK1 yielded considerable soluble AK activity. Three AK2 constructs were expressed - one corresponding to the full length protein and two corresponding to truncated versions in which the signal peptide had been deleted. Expression of the former construct yielded minimal soluble activity. In contrast, significant AK activity was found in both truncated constructs confirming the importance of removal of the targeting peptide for proper folding and catalytic activity. Both AK1 and AK2 are functional oligomers unlike typical AKs which are monomeric. A phylogenetic analysis showed that these choanoflagellate AKs group more closely with a supercluster consisting of cytoplasmic and mitochondrial CKs and invertebrate AKs that evolved secondarily from a CK-like ancestor. Reaction-diffusion constraints in choanoflagellates are likely mitigated by the presence of AK isoforms which facilitate energy transport in these highly polarized cells.     

Amino acid residues 62 and 193 play the key role in regulating the synergism of substrate binding in oyster arginine kinase

The purpose of this study is to clarify the amino acid residues responsible for the synergism in substrate binding of arginine kinase (AK), a key enzyme in invertebrate energy metabolism. AKs contain a pair of highly conserved amino acids (D62 and R193) that form an ion pair, and replacement of these residues can cause a pronounced loss of activity. Interestingly, in the oyster Crassostrea AK, these residues are replaced by an N and a K, respectively. Despite this replacement, the enzyme retains high activity and moderate synergism in substrate binding (Kd/Km=2.3). We replaced the N62 by G or D and the K193 by G or R in Crassostrea AK, and also constructed the double mutants of N62G/K193G and N62D/K193R. All of the mutants retained 50-90% of the wild-type activity. In N62G and N62D mutants, the Kmarg for arginine binding was comparable to that of wild-type enzyme, but the Kdarg was increased 2-5-fold, resulting in a strong synergism (Kd/Km=4.9-11.3). On the other hand, in K193G and K193R mutants, the Kmarg was increased 4-fold, and synergism was lost almost completely (Kd/Km=1.0-1.4). The N62G/K193G double mutant showed similar characteristics to the K193G and K193R mutants. Another double mutant, N62D/K193R, similar to the amino acid pair in the wild-type enzyme, had characteristics similar to those of the wild-type enzyme. These results indicate that the amino acid residues 62 and 193 play the key role in mediating the synergism in substrate binding of oyster arginine kinase.     

Expression of horseshoe crab arginine kinase in Escherichia coli and site-directed mutations of the reactive cysteine peptide

Arginine kinase (AK) from the horseshoe crab Limulus polyphemus was expressed in Escherichia coli. The bulk of expressed protein resided in insoluble inclusion bodies. However, approximately 3 mg enzyme protein/l culture was present as active soluble AK. The AK-containing expression vector construct was subjected to site-directed mutagenesis via a polymerase chain reaction-based megaprimer protocol. The AK reactive cysteine peptide was engineered so that it was identical to the corresponding peptide sequence of creatine kinase, another member of the guanidino kinase enzyme family. The resulting expressed protein had a considerably reduced specific activity but was still specific for arginine/arginine phosphate. No catalytic activity was observed with other guanidine substrates (creatine, glycocyamine, taurocyamine, lombricine). The reactive cysteine peptide, characteristic of all guanidino kinases, very likely plays a minimal role in determining guanidine specificity.     

The interaction between residues 62 and 193 play a key role in activity and structural stability of arginine kinase

The purpose of this study is to clarify that the amino acid residues (Asp62 and Arg193) are responsible for the activity and stability of arginine kinase (AK). The amino acid residues Asp62 (D62) and Arg193 (R193) are strictly conserved in monomeric AKs and form an ion pair in the transition state analogue complex. In this research, we replaced D62 with glutamate (E) or glycine (G) and R193 with lysine (K) or glycine (G). The mutants of D62E and R193K retained almost 90% of the wild-type activity, whereas D62G and R193G had a pronounced loss in activity. A detailed comparison was made between the physic-chemical properties and conformational changes of wild-type AK and the mutants by means of ultraviolet (UV) difference and fluorescence spectra. The results indicated that the conformation of all of the mutants had been changed and the stability in a urea solution was also reduced. We speculated that the hydrogen bond and electrostatic interactions formed between residues 62 and 193 play a key role in stabilizing the structure and mediating the synergism in substrate binding of arginine kinase from greasyback shrimp (Metapenaeus ensis).     

Arginine kinase from the beetle Cissites cephalotes (Olivier). Molecular cloning, phylogenetic analysis and enzymatic properties

Here, we report the PCR amplification and cloning of a cDNA for arginine kinase (AK) from the beetle Cissites cephalotes (Olivier). The cDNA is 1210bp and has an open reading frame of 1125bp and 5' and 3'-untranslated regions of 30 and 55bp, respectively. The open reading frame encodes a 374 amino acid protein with most of the residues considered necessary for AK function: five residues predicted to interact with the substrate arginine (S77, Y82, E239, C285 and E328), and five residues predicted to interact with the substrate ADP (R138, R140, R243, R294 and R323). A phylogenetic tree of arthropod AKs indicated clearly that insect AKs can be separated into typical AKs from various insect species (group 1) and putative AK sequences deduced from genomic sequences (group 2). Cissites AK clustered in group 2 and provides the first evidence that a group-2 gene is indeed expressed in insects. Moreover, we expressed Cissites AK protein in Escherichia coli as a fusion with maltose-binding protein, and kinetic constants (K(m), K(d), V(max) and k(cat)) were determined for the forward reaction. Comparison of kinetic constants with those of AKs from other sources (insects, mollusks and echinoderms) indicated that insect AKs from Cissites and Periplaneta have two very unique features, the lowest k(cat) (and k(cat)/K(m)(arg)) among AKs, and a lack of synergistic substrate binding (K(d)/K(m) approximately 1).     

Role of amino-acid residue 95 in substrate specificity of phosphagen kinases

The purpose of this study is to elucidate the mechanisms of guanidine substrate specificity in phosphagen kinases, including creatine kinase (CK), glycocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and arginine kinase (AK). Among these enzymes, LK is unique in that it shows considerable enzyme activity for taurocyamine in addition to its original target substrate, lombricine. We earlier proposed several candidate amino acids associated with guanidine substrate recognition. Here, we focus on amino-acid residue 95, which is strictly conserved in phosphagen kinases: Arg in CK, Ile in GK, Lys in LK and Tyr in AK. This residue is not directly associated with substrate binding in CK and AK crystal structures, but it is located close to the binding site of the guanidine substrate. We replaced amino acid 95 Lys in LK isolated from earthworm Eisenia foetida with two amino acids, Arg or Tyr, expressed the modified enzymes in Escherichia coli as a fusion protein with maltose-binding protein, and determined the kinetic parameters. The K95R mutant enzyme showed a stronger affinity for both lombricine (Km=0.74 mM and kcat/Km=19.34 s(-1) mM(-1)) and taurocyamine (Km=2.67 and kcat/Km=2.81), compared with those of the wild-type enzyme (Km=5.33 and kcat/Km=3.37 for lombricine, and Km=15.31 and kcat/ Km=0.48for taurocyamine). Enzyme activity of the other mutant, K95Y, was dramatically altered. The affinity for taurocyamine (Km=1.93 and kcat/Km=6.41) was enhanced remarkably and that for lombricine (Km=14.2 and kcat/Km=0.72) was largely decreased, indicating that this mutant functions as a taurocyamine kinase. This mutant also had a lower but significant enzyme activity for the substrate arginine (Km=33.28 and kcat/Km=0.01). These results suggest that Eisenia LK is an inherently flexible enzyme and that substrate specificity is strongly controlled by the amino-acid residue at position 95.     

Two-domain arginine kinases from the clams Solen strictus and Corbicula japonica: exceptional amino acid replacement of the functionally important D(62) by G

Arginine kinases (AKs) isolated from the adductor muscle of the clams Solen strictus and Corbicula japonica have relative molecular masses of 80 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in contrast to the 40 kDa AKs found in Mollusca and Arthropoda. The cDNAs encoding Solen and Corbicula AKs have open reading frames of 2175 nucleotides (724 amino acid protein) and 2172 nucleotides (723 amino acid protein), respectively. The amino acid sequence clearly indicates that Solen and Corbicula AKs have a two-domain structure: the first-domain includes residues 1-363 and the second-domain includes residue 364 to the end. There is approximately 60% inter-domain amino acid identity. It is clear that gene-duplication and subsequent fusion occurred in the immediate ancestor of the clams Solen, Corbicula, and Pseudocardium. During substrate binding, it is proposed that AK undergoes a substrate-induced conformational change and that the hydrogen bond between D(62) and R(193) stabilizes the substrate-bound structure. However, in Solen and Corbicula two-domain AKs, D(62) is replaced by a G, and R(193) by A, S, or D. Consequently, the two-domain AKs can not form the stabilizing hydrogen bond. Nevertheless, the enzyme activity of Corbicula AK is comparable to those of other molluscan 40 kDa AKs. We assumed that the substrate-bound structure of the two-domain AK is stabilized not by the hydrogen bond between D(62) and R(193) but by the bond between H(60) and D(197), characteristic of the unusual two-domain AKs. This explains why D(62) and R(193), which remain highly conserved in other AKs, have undergone amino acid replacements in Solen and Corbicula AKs.     

Kinetic properties and structural characteristics of an unusual two-domain arginine kinase of the clam Corbicula japonica

Arginine kinase (AK) from the clam Corbicula japonica is a unique enzyme in that it has an unusual two-domain structure with molecular mass of 80 kDa. It lacks two functionally important amino acid residues, Asp-62 and Arg-193, which are conserved in other 40 kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. K m arg and Vmax values for the recombinant two-domain AK were determined. These values were close to those of usual 40 kDa AKs, although Corbicula AK lacks the functionally important Asp-62 and Arg-193. Domain 2 of Corbicula AK was separated from the two-domain enzyme and was expressed in Escherichia coli. Domain 2 still exhibited activity. However, kinetic parameters for domain 2 appeared to be slightly, but significantly, different from those of two-domain AK. Thus, it is likely that the formation of the contiguous dimer alters the kinetic properties of its constituent domains significantly. Comparison of K d arg and K m arg for two-domain AK and its domain 2 showed that the affinity of the enzyme for arginine is greater in the presence of substrate ATP than in its absence. Presumably this difference is correlated with the large structural differences in the enzyme in the presence or absence of substrate, namely open and closed structures. We expressed three mutants of Corbicula AK domain 2 (His-60 to Gly or Arg, Asp-197 to Gly), and determined their K m arg and Vmax values. The affinity for the substrate arginine in mutant enzymes was reduced considerably, accompanied by a decrease in Vmax. These results suggest that His-60 and Asp-197 affect the substrate binding system, and are consistent with the hypothesis that a hydrogen bond is formed between His-60 and Asp-197 in Corbicula AK as a substitute for the Asp-62 and Arg-193 bond in normal AKs.     

A Novel Arginine Kinase with Substrate Specificity Towards <Emphasis Type="SmallCaps">d</Emphasis>-arginine

We determined the cDNA-derived amino acid sequences of two arginine kinases (AK1, AK2) from the annelid Sabellastarte indica, cloned the cDNAs into pMAL plasmid and expressed them in E. coli. The phylogenetic analyses suggested that Sabellastarte AKs have evolved from a CK-related gene, not from the usual AK gene. The recombinant Sabellastarte AK1 showed a broad specificity towards various guanidine compounds, while the Sabellastarte AK2 mainly showed stronger activity for both d- and l-arginine, a very unique substrate specificity not seen before in usual AKs. We isolated guanidino compounds from the body wall musculature of Sabellastarte, and found that the major compound is d-arginine with a concentration of 4.85 ± 0.51 mmol/kg. From these results, we suggest strongly that in Sabellastarte, d-arginine is the major phosphagen substrate and that the AK2 with substrate specificity towards d-arginine, catalyzes the phosphorylation of d-arginine.     

Enzymatically inactive adenylate kinase 4 interacts with mitochondrial ADP/ATP translocase

Adenylate kinase 4 (AK4) is a unique member with no enzymatic activity in vitro in the adenylate kinase (AK) family although it shares high sequence homology with other AKs. It remains unclear what physiological function AK4 might play or why it is enzymatically inactive. In this study, we showed increased AK4 protein levels in cultured cells exposed to hypoxia and in an animal model of the neurodegenerative disease amyotrophic lateral sclerosis. We also showed that short hairpin RNA (shRNA)-mediated knockdown of AK4 in HEK293 cells with high levels of endogenous AK4 resulted in reduced cell proliferation and increased cell death. Furthermore, we found that AK4 over-expression in the neuronal cell line SH-SY5Y with low endogenous levels of AK4 protected cells from H₂O₂ induced cell death. Proteomic studies revealed that the mitochondrial ADP/ATP translocases (ANTs) interacted with AK4 and higher amount of ANT was co-precipitated with AK4 when cells were exposed to H₂O₂ treatment. In addition, structural analysis revealed that, while AK4 retains the capability of binding nucleotides, AK4 has a glutamine residue instead of a key arginine residue in the active site well conserved in other AKs. Mutation of the glutamine residue to arginine (Q159R) restored the adenylate kinase activity with GTP as substrate. Collectively, these results indicate that the enzymatically inactive AK4 is a stress responsive protein critical to cell survival and proliferation. It is likely that the interaction with the mitochondrial inner membrane protein ANT is important for AK4 to exert the protective benefits to cells under stress.     

Arginine kinase from the Tardigrade, Macrobiotus occidentalis: molecular cloning, phylogenetic analysis and enzymatic properties

Arginine kinase (AK), which catalyzes the reversible transfer of phosphate from ATP to arginine to yield phosphoarginine and ADP, is widely distributed throughout the invertebrates. We determined the cDNA sequence of AK from the tardigrade (water bear) Macrobiotus occidentalis, cloned the sequence into pET30b plasmid, and expressed it in Escherichia coli as a 6x His-tag—fused protein. The cDNA is 1377 bp, has an open reading frame of 1080 bp, and has 5′- and 3′-untranslated regions of 116 and 297 bp, respectively. The open reading frame encodes a 359-amino acid protein containing the 12 residues considered necessary for substrate binding in Limulus AK. This is the first AK sequence from a tardigrade. From fragmented and non-annotated sequences available from DNA databases, we assembled 46 complete AK sequences: 26 from arthropods (including 19 from Insecta), 11 from nematodes, 4 from mollusks, 2 from cnidarians and 2 from onychophorans. No onychophoran sequences have been reported previously. The phylogenetic trees of 104 AKs indicated clearly that Macrobiotus AK (from the phylum Tardigrada) shows close affinity with Epiperipatus and Euperipatoides AKs (from the phylum Onychophora), and therefore forms a sister group with the arthropod AKs. Recombinant 6x His-tagged Macrobiotus AK was successfully expressed as a soluble protein, and the kinetic constants (K(m), K(d), V(ma) and k(cat)) were determined for the forward reaction. Comparison of these kinetic constants with those of AKs from other sources (arthropods, mollusks and nematodes) indicated that Macrobiotus AK is unique in that it has the highest values for k(cat) and K(d)K(m) (indicative of synergistic substrate binding) of all characterized AKs.     

Mycobacterial adenosine kinase is not a typical adenosine kinase

Adenosine kinase (AK) is only found in eukaryotes. Recently, a Mycobacterium tuberculosis (MTub) protein exhibiting greater sequence similarity to ribokinases (RK) was identified as AK. We have expressed AKs from MTub, human and Chinese hamster (CH) cells in Escherichia coli and also AK from human and MTub in AK-deficient CH cells. While both E. coli and CH cells expressing mammalian AKs efficiently metabolized various adenosine analogs, those expressing MTub-AK were completely inactive. The AK activity of the MTub protein was very low (50-fold lower than E. coli RK) and it was not stimulated by phosphate or inhibited by several AK inhibitors. These results raise questions over MTub protein’s true function and whether it functions as AK in cells.     

The Early Evolution of the Phosphagen Kinases—Insights from Choanoflagellate and Poriferan Arginine Kinases

Arginine kinase (AK) is a member of a large family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases. AKs are present in certain protozoans, sponges, cnidarians, and both lophotrochozoan and ecdysozoan protostomes. Another phosphagen kinase, creatine kinase (CK), is found in sponges, cnidarians, and both deuterostome and protostome groups but does not appear to be present in protozoans. To probe the early evolution of phosphagen kinases, we have amplified the cDNAs for AKs from three choanoflagellates and from the hexactinellid sponge Aphrocallistes beatrix and the demosponges Suberites fuscus and Microciona prolifera. Phylogenetic analysis using maximum likelihood of these choanoflagellate and sponge AKs with other AK sequences revealed that the AK from the choanoflagellate Monosiga brevicollis clusters with the AK from the glass sponge Aphrocallistes and is part of a larger cluster containing AKs from the demosponges Suberites and Microciona as well as basal and protostome invertebrates. In contrast, AKs from Codonosiga gracilis and Monosiga ovata form a distinct cluster apart from all other AK sequences. tBLASTn searches of the recently released M. brevicollis genome database showed that this species has three unique AK genes—one virtually identical to the M. brevicollis cDNA and the other two showing great similarity to C. gracilis and M. ovata AKs. Three distinct AK genes are likely present in choanoflagellates. Two of these AKs display extensive similarity to both CKs and an AK from sponges. Previous work has shown CK evolved from an AK-like ancestor prior to the divergence of sponges. The present results provide evidence suggesting that the initial gene duplication event(s) leading to the CK lineage may have occurred before the divergence of the choanoflagellate and animal lineages.     

Identification and characterization of a unique adenosine kinase from Mycobacterium tuberculosis

Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.     

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 K_m value of 0.6 mM for arginine and a V_max value of 13 μmole 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 1608 and 1239 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.