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.

Arginine kinase evolved twice: evidence that echinoderm arginine kinase originated from creatine kinase.

Arginine kinase (AK) was isolated from the longitudinal muscle of the sea cucumber Stichopus japonicus. Unlike the monomeric 40 kDa AKs from molluscs and arthropods, but like the cytoplasmic isoenzymes of vertebrate creatine kinase (CK), the Stichopus enzyme was dimeric. To explore the evolutionary origin of the dimeric AK, we determined its cDNA-derived amino acid sequence of 370 residues. A comparison of the sequence with those of other enzymes belonging to the phosphagen kinase family indicated that the entire amino acid sequence of Stichopus AK is apparently much more similar to vertebrate CKs than to all other AKs. A phylogenetic tree also strongly suggests that the Stichopus AK has evolved from CK. These results support the conclusion that AK evolved at least twice during the evolution of phosphagen kinases: first at an early stage of phosphagen kinase evolution (its descendants are molluscan and arthropod AKs) and secondly from CK later in metazoan evolution. A comparison of the amino acid sequence around the guanidino specificity (GS) region (which is a possible candidate for the guanidine substrate recognition site in the phosphagen kinase family) of the Stichopus enzyme with those of other phosphagen kinases showed that the GS region of the Stichopus enzyme was of the AK type: five amino acid deletions in the flexible loop region that might help to accommodate larger guanidine substrates in the active site. The presence of the AK-type deletions in the Stichopus AK, even though it seems that the enzyme's most immediate ancestor was probably CK, strongly suggests that the GS region has a role in substrate specificity. Stichopus AK and presumably other echinoderm AKs seem to have evolved from the CK gene; the sequence of GS region might have been replaced by the AK type via exon shuffling. The presence of an intron near the GS region in the Stichopus AK gene supports this hypothesis.

Evolution of phosphagen kinase VII. Isolation of glycocyamine kinase from the polychaete Neanthes diversicolor and the cDNA-derived amino acid sequences of alpha and beta chains.

Glycocyamine kinase (GK) was isolated from the marine polychaete Neanthes diversicolor by gel filtration, DEAE-cellulose chromatography, butyl-Toyopearl hydrophobic chromatography, and chromatofocusing. The GK was eluted as a single peak on the latter three chromatographies, and the molecular mass for the native GK was estimated to be about 80 kDa. The SDS-PAGE showed that the isolated GK consists of two distinct subunits in equal proportion, alpha and beta chains, with molecular masses of 42.2 and 43.8 kDa, respectively. The present results suggest that the Neanthes GK has a heterodimeric structure. The cDNAs for alpha and beta chains of Neanthes GK were amplified by PCR and their cDNA-derived amino acid sequences were determined. The alpha and beta chains are composed of 374 and 390 amino acids, and the molecular masses were calculated to be 42,392 and 43,966 Da, respectively, in good agreement with the apparent masses on SDS PAGE. The beta chain has a characteristic N-terminal extension of 15 amino acids, and all of the sequence differences between alpha and beta chains were restricted in the N-terminal region of 50 residues. The overall sequence identity was 92%. The occurrence of heterodimeric nature in Neanthes GK is of great interest from the evolutionary point of view, because the heterodimeric structure is only known for creatine kinase MB-isozyme specific for mammalian heart muscle among phosphagen kinases.

cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca(2+)-ATPase.

Sarcoplasmic reticulum (SR) Ca(2+)-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with approximately 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five 'stalk' domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca(2+)-ATPases is discussed.

Gene duplication and fusion have occurred frequently in the evolution of phosphagen kinases--a two-domain arginine kinase from the clam Pseudocardium sachalinensis.

In contrast to the 40 kDa arginine kinases from Molllusca and Arthropoda, the adductor muscle of the marine clam Pseudocardium sachalinensis contains an unusual arginine kinase consisting of an 86 kDa subunit. The cDNA encoding the 86 kDa arginine kinase was amplified by PCR and the cDNA-derived amino acid sequence of 724 residues was determined. The exact molecular mass for the protein was calculated to be 80941 Da. The amino acid sequence clearly indicates that Pseudocardium arginine kinase has a two-domain structure: the first domain residues 1-363 and the second domain 364-724. The two domains, which are separated by an intron of 176 bp in the gene, show 62% amino acid sequence identity. This two-domain arginine kinase from a mollusc represents yet another multiple-domain enzyme observed in the phosphagen kinase enzyme family. Two-domain and three-domain enzymes have been observed in three other diverse invertebrate groups. Thus, it is clear that gene duplication and subsequent fusion have occurred frequently, and likely independently, during the course of the evolution of this enzyme family. Comparison of the amino acid sequence in the GS region (a possible candidate for the guanidine substrate recognition site in the phosphagen kinase family) suggests that the first domain of Pseudocardium arginine kinase might not retain a complete enzyme activity, because the Asp-7 in the GS region, which is assumed to be involved in the recognition of the positive charge of arginine, was replaced by a Gly residue in the first domain.

Evolution of phosphagen kinase V. cDNA-derived amino acid sequences of two molluscan arginine kinases from the chiton Liolophura japonica and the turbanshell Battilus cornutus.

The cDNAs of arginine kinases from the chiton Liolophura japonica (Polyplacophora) and the turbanshell Battilus cornutus (Gastropoda) were amplified by polymerase chain reaction (PCR), and the complete nucleotide sequences of 1669 and 1624 bp, respectively, were determined. The open reading frame for Liolophura arginine kinase is 1050 nucleotides in length and encodes a protein with 349 amino acid residues, and that for Battilus is 1077 nucleotides and 358 residues. The validity of the cDNA-derived amino acid sequence was supported by chemical sequencing of internal tryptic peptides. The molecular masses were calculated to be 39,057 and 39,795 Da, respectively. The amino acid sequence of Liolophura arginine kinase showed 65-68% identity with those of Battilus and Nordotis (abalone) arginine kinases, and the homology between Battilus and Nordotis was 79%. Molluscan arginine kinases also show lower, but significant homology (38-43%) with rabbit creatine kinase. The sequences of arginine kinases could be used as a molecular clock to elucidate the phylogeny of Mollusca, one of the most diverse animal phyla.

Evolution of phosphagen kinase. Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus.

Arginine kinase (AK) was isolated from the body wall muscle of the primitive sea anemone Anthopleura japonicus by Ultrogel AcA34 gel filtration, DEAE-32 chromatography and elution on a Cosmogel-SP column. The denatured molecular mass as determined with SDS/PAGE was 80 kDa, twice that of the usual AK subunit, indicating that this AK has an unusual two-domain structure. The native form was eluted on a Superose 12 column with the same retention time as that of rabbit homodimeric creatine kinase, indicating that Anthopleura AK is a monomer of 80 kDa. The isolated enzyme gave a specific activity of 100-120 micromol of Pi/min per mg of protein in the pH range 7.9-9.1 for the forward reaction. The enzyme is fully activated by Ca2+, as it is with Mg2+. The cDNA-derived amino acid sequence of 715 residues of Anthopleura AK was determined. The validity of the sequence was supported by chemical sequencing of internal tryptic peptides. A bridge intron of 686 bp, which separates the two domains of Anthopleura AK, is present between the second and third nucleotide in the codon of Ala-364. This is the first two-domain AK to be sequenced. Anthopleura AK shows 48-54% amino acid sequence identity with known invertebrate AKs, and also shows a lower, but significant, similarity (39-46%) to marine worm glycocyamine kinase and rabbit creatine kinase.

Evolution of phosphagen kinase. VI. Isolation, characterization and cDNA-derived amino acid sequence of lombricine kinase from the earthworm Eisenia foetida, and identification of a possible candidate for the guanidine substrate recognition site.

Lombricine kinase (LK) from the body wall muscle of the earthworm Eisenia foetida was purified to homogeneity. The enzyme was shown to be a dimer consisting of 40 kDa subunits. The cDNA-derived amino acid sequence of 370 residues of Eisenia LK was determined. The validity of the sequence was supported by chemical sequencing of internal tryptic peptides. This is the first reported lombricine kinase amino acid sequence. Alignment of Eisenia LK with those of creatine kinases (CKs), arginine kinases (AKs) and glycocyamine kinase (GK) suggested a region displaying remarkable amino acid deletions (referred to GS region), as a possible candidate for guanidine substrate recognition site. A phylogenetic analysis using amino acid sequences of all four phosphagen kinases indicates that CK, GK and LK probably evolved from a common immediate ancestor protein.

Two types of proton-modulated calcium binding in the sarcoplasmic reticulum Ca(2+)-ATPase. I. A model of two different conformations of chemically equivalent ATPase molecules.

Two pools of calcium binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase have been found at 0 degrees C: Half of the calcium sites are in a slow (t1/2 > or = 2 s)/rapid (t1/2 < 2 s) binding site dependent on pH, and the other half are in a slow binding state independent of pH (Nakamura, J. (1989) J. Biol. Chem. 264, 17029-17031). Herein, to clarify the molecular basis of the two calcium sites, the relation between calcium binding to the enzyme and phosphorylation of the enzyme was examined at pH 7.40, where the calcium sites are split into rapid and slow binding states. The enzyme was slowly or rapidly phosphorylated with ATP, accompanied by slow or rapid calcium binding with a stoichiometry of about 1:2. Analysis of the amino acid sequence of lysyl endopeptidase peptides of the ATPase preparation confirmed the homogeneity of the preparation, which was of fast twitch muscle type. These results suggest that each of the two pools of calcium sites belongs to one of the two different conformations of chemically equivalent ATPase molecules, which are in pH-dependent equilibrium between E1 (high affinity state for calcium) and E2 (low affinity state for calcium) and predominantly in E2 independent of pH, respectively, before calcium binding.

Evolution of phosphagen kinase (III). Amino acid sequence of arginine kinase from the shrimp Penaeus japonicus.

The amino acid sequence of arginine kinase (AK) from the shrimp Penaeus japonicus has been determined chemically. It consists of 355 amino acid residues, and has a calculated molecular mass of 40,018 Da. The amino acid sequence of Penaeus AK showed 91% and 51% identity, respectively, with those of AKs from the lobster Homarus vulgaris and the abalone Nordotis madaka. It also showed significant homology (39-43%) with vertebrate or invertebrate creatine kinases and annelid glycocyamine kinase, suggesting that these enzymes evolved from a common origin.

Evolution of phosphagen kinase. Primary structure of glycocyamine kinase and arginine kinase from invertebrates.

Of the six phosphagen kinases found in animals, the primary structure is known only for creatine kinase. Here we report three cDNA-derived or chemically determined amino acid sequences of two kinds of phosphagen kinases: a glycocyamine kinase from the polychaete Neanthes diversicolor (Annelida) and arginine kinase from the abalone Nordotis madaka (Mollusca) and the shrimp Penaeus japonicus (Arthropoda). Like vertebrate creatine kinases are monomers. These enzymes consist of 350 to 390 amino acid residues, and have a calculated molecular mass of 39,900 to 44,500 Da. Neanthes glycocyamine kinase shows 50 to 58% sequence similarity with vertebrate and invertebrate creatine kinases, having the greatest similarity (57 to 58%) with vertebrate mitochondrial creatine kinase isoform. It shows lower, but significant similarity (37 to 39%) with invertebrate arginine kinases. The sequence similarity between Nordotis and Penaeus arginine kinases is 51%. A phylogenetic tree constructed from 14 amino acid sequences of phosphagen kinases showed that they can be separated into three major clusters corresponding to creatine kinase, glycocyamine kinase and arginine kinase. The cluster of glycocyamine kinase is apparently closer to that of creatine kinase than arginine kinase. The cluster of creatine kinase is composed of several subclusters, each corresponding to three vertebrate isoforms and the invertebrate enzyme.

Amino acid sequence of myoglobin from the chiton Liolophura japonica and a phylogenetic tree for molluscan globins.

Myoglobin was isolated from the radular muscle of the chiton Liolophura japonica, a primitive archigastropodic mollusc. Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved in Liolophura myoglobin. The autoxidation rate at physiological conditions indicated that Liolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence of Liolophura myoglobin shows low homology (11-21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26-29%) with monomeric myoglobins from the gastropodic molluscs Aplysia, Dolabella, and Bursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively. Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clams Anadara, Scapharca, and Barbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiontharboring clams Calyptogena and Lucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.

Primary structure of chain I of the heterodimeric hemoglobin from the blood clam Barbatia virescens.

The blood clam Barbatia virescens has a heterodimeric hemoglobin in erythrocytes. Interestingly, the congeneric clams B. reeveana and B. lima contain quite different hemoglobins: tetramer and polymeric hemoglobin consisting of unusual didomain chain. The complete amino acid sequence of chain I of B. virescens has been determined. The sequence was mainly determined from CNBr peptides and their subpeptides, and the alignment of the peptides was confirmed by sequencing of PCR-amplified cDNA for B. virescens chain I. The cDNA-derived amino acid sequence matched completely with the sequence proposed from protein sequencing. B. virescens chain I is composed of 156 amino acid residues, and the molecular mass was calculated to be 18,387 D, including a heme group. The sequence of B. virescens chain I showed 35-42% sequence identity with those of the related clam Anadara trapezia and the congeneric clam B. reeveana. An evolutionary tree for Anadara and Barbatia chains clearly indicates that all of the chains are evolved from one ancestral globin gene, and that the divergence of chains has occurred in each clam after the speciation. The evolutionary rate for clam hemoglobins was estimated to be about four times faster than that of vertebrate hemoglobin. We suggest that blood clam hemoglobin is a physiologically less important molecule when compared with vertebrate hemoglobins, and so it evolved rapidly and resulted in a remarkable diversity in quaternary and subunit structure within a relatively short period.

Application of a new slot comb to electrophoretic analysis.

In the present experiment, a new slot comb was designed in order to form a wide and sloped sample well on the stacking gel of electrophoresis. Using this slot comb, a gradient of the reagent layer of interest can be easily formed transversely on the gel that is perpendicular to the direction of electrophoresis. Thus, the protein sample overlaid on the agent migrates across the gradient layer during electrophoresis to produce a continuous electrophoretic band reflecting the interaction between the protein and reagent. This new slot comb (tentatively called slope comb) was applied to the following two experiments. In the first experiment, in combination with this comb and a reducing agent, 2-mercaptoethanol, the reducing steps of cross-linked axonemal proteins with o-iodosobenzoic acid (OIBA) were analyzed electrophoretically, enabling visualization of the reducing pattern of each axonemal protein in a single experiment. The results obtained indicate that alpha and beta tubulins are cross-linked differently by OIBA. In the second experiment, the formation of the Ca2+ gradient layer using this slope comb could electrophoretically differentiate the Ca2+ sensitivity of three Ca(2+)-binding proteins.

Amino acid sequence of myoglobin from the mollusc Bursatella leachii.

The complete amino acid sequence of myoglobin from the triturative stomach of gastropodic mollusc Bursatella leachii has been determined. It is composed of 146 amino acid residues, is acetylated at the N-terminus, and contains a single histidine residue at position 95 which corresponds to the heme-binding proximal histidine. The E7 distal histidine, which is conserved widely in myoglobins and hemoglobins, is replaced by valine in Bursatella myoglobin. The amino acid sequence of Bursatella myoglobin shows strong homology (73-84%) with those of Aplysia and Dolabella myoglobins.

A calcium-dependent galactose-binding lectin from the tunicate Polyandrocarpa misakiensis. Isolation, characterization, and amino acid sequence.

A lectin was isolated from the homogenate of the tunicate Polyandrocarpa misakiensis by heat treatment, ammonium sulfate fractionation, gel filtration, and high-performance ion-exchange chromatography. Analytical gel filtration on Superose 12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the lectin is a monomeric protein with a molecular mass of approximately 15 kDa. The lectin bound to an immobilized D-galactose column in the presence of calcium ion with a threshold of 500 microM and eluted completely with 5 mM EDTA. It did not bind to an immobilized D-mannose or N-acetyl-D-galactosamine column. Thus, Polyandrocarpa lectin was found to be a calcium-dependent galactose-binding lectin. The complete amino acid sequence of Polyandrocarpa lectin was determined by automated or manual Edman sequencing of the peptides derived by digestion with trypsin, endoproteinase Asp-N, Staphylococcus aureus V8 protease, and pepsin. It is composed of 125 residues, contains no carbohydrate group, and has a calculated molecular mass of 14,034 Da. The lectin contains four half-cystines, and Cys-21 and Cys-119 and also Cys-96 and Cys-111 form intrachain disulfide bridges, respectively. The amino acid sequence of Polyandrocarpa lectin shows about 20-30% homology with those of fly, barnacle, sea urchin, and several vertebrate lectins that belong to C-type lectin (Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9560). Although the physiological role of Polyandrocarpa lectin is not clear, preliminary experiments suggest that the lectin may be related to defense mechanisms because it has a strong antibacterial activity.

The ear-shell (Sulculus diversicolor aquatilis) myoglobin is composed of an unusual 39 kDA polypeptide chain.

An unusual myoglobin was isolated from the buccal mass of the ear-shell Sulculus diversicolor aquatilis. The myoglobin consists of a 39 kDa polypeptide chain which is about double the size of the usual myoglobin subunit, contains one heme per molecule, and has an unusual spectral property ion the oxy-form. On the basis of these properties and partial amino acid sequencing, we propose that Sulculus myoglobin has a didomain structure, and that one of the two domains does not function as an oxygen-binding domain. So far, a myoglobin of this type has not been described in molluscs.

Amino acid sequence of polypeptide chain IIB of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus.

The giant extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus consists of two types of subunits: a "monomeric" chain (chain I) and a disulfide-bonded trimer of chains IIA, IIB, and IIC. The complete amino acid sequence of chain IIB was determined. This chain has 148 amino acid residues and a molecular weight of 17,236 including a heme group. Of the residues in chain IIB, 74 (50%) and 34 (30%) were found to be identical with those in the corresponding positions in Tylorrhynchus chains IIC and I, respectively (Suzuki, T., Furukohri, T., and Gotoh, T. (1985) J. Biol. Chem. 260, 3145-3154). Marked differences were found between the chains of Tylorrhynchus and Lumbricus in the COOH-terminal regions. Significant differences were predicted between the monomeric chain I and the "trimeric" chains (IIB and IIC) in the hydropathy profiles and alpha-helical contents.