Cloning and sequence analysis of the cDNA for arginine kinase of lobster muscle. (65/75)

Arginine kinase belongs to an evolutionary conserved family of ATP:guanidino phosphotransferases, whose members play an important role in energy metabolism. In this work, a lambda gt11 lobster muscle library was constructed and screened by using both polyclonal antibodies and two synthetic oligonucleotides. The complete amino acid sequence of arginine kinase (ATP:L-arginine N-phosphotransferase, EC 2.7.3.3) from lobster muscle was determined by cloning and sequencing the DNA complementary to its mRNA. The identity of the clone was confirmed by comparing the amino acid sequence deduced by nucleotide sequence analysis with previously published partial sequences of amino- and carboxyl-terminal regions of the enzyme and some of its fragments (Regnouf, F., Kassab, R., Debuire, B., Richard, C., and Han K. K. (1981) Int. J. Peptide Protein Res. 17, 143-155). The nucleotide sequence of the cDNA was found to contain an open reading frame encoding 355 amino acid residues with a calculated molecular mass of 39,830 daltons. This enzyme exhibits a significant sequence identity to those of representative members of the guanidino kinase family, including creatine kinase. This report represents the first molecular cloning and sequencing of an ATP-guanidino phosphotransferase which is not a creatine kinase isoform.  (+info)

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: a model for studying phosphagen kinases. (66/75)

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.  (+info)

Mitotic phosphorylation of the lamin B receptor by a serine/arginine kinase and p34(cdc2). (67/75)

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that is modified at interphase by a nuclear envelope-bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH2-terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, we performed phosphopeptide mapping of in vitro and in vivo 32P-labeled LBR and analyzed a series of recombinant proteins and synthetic peptides. Our results show that LBR undergoes two types of mitotic phosphorylation mediated by the RS and the p34(cdc2) protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser76, Ser78, Ser80, Ser82, Ser84), whereas p34(cdc2) mainly phosphorylates Ser71. These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsible for nuclear envelope assembly and disassembly.  (+info)

Conformational changes of arginine kinase induced by photochemical release of nucleotides from caged nucleotides--an infrared difference-spectroscopy investigation. (68/75)

The conformations of arginine kinase (AK) in AK x Mg x ADP, AK x Mg x ATP, AK x Mg x ADP x NO3-, AK x Mg x ADP x Arg and AK x Mg x ADP x NO3- x Arg complexes were investigated by measuring their reaction-induced infrared difference spectra (RIDS). The photochemical release of ATP from ATP[Et(PhNO2)] and of ADP from ADP[Et(PhNO2)] produced distinct RIDS of AK complexes, suggesting that binding of ADP and ATP promoted different structural alterations of the enzyme active-site. Small infrared changes in the amide-I region were observed, indicating that about 5-10 amino acid residues were involved in the nucleotide-binding site. These infrared changes were due to the structural alteration of the peptide backbone caused by the nucleotide-binding and to the coupling effects between the nucleotide-binding site and the other substrate (Arg or NO3-)-binding site. ATP binding to AK (as well as ADP-binding to AK in the presence of NO3-) induced protonation of a carboxylate group of Asp or Glu, as evidenced by the appearance of the 1733-cm(-1) band, which was not observed with the AK x Mg x ADP, AK x Mg x ADP x Arg and AK x Mg x ADP x NO3- x Arg complexes. The RIDS of the AK x Mg x ADP x NO3- x Arg complex showed new infrared bands at 1622 cm(-1) (negative) and at 1613 cm(-1) (positive), which were not seen in the RIDS of other complexes (without NO3- or/and Arg). In the transition-state-analog complex of AK, no protonation of the carboxylate residue (Asp or Glu) was observed, and the binding site of NO3- or the gamma-phosphate group of nucleotide was altered.  (+info)

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. (69/75)

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.  (+info)

Evolution of phosphagen kinase. Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus. (70/75)

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.  (+info)

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. (71/75)

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.  (+info)

Arginine kinase expression and localization in growth cone migration. (72/75)

Migrating neuronal growth cones exert traction forces that are generated by ATP-driven F-actin/myosin interactions. Sustained generation of these forces may require an energy supply mediated by the guanidino kinases, creatine kinase and arginine kinase. We cloned and sequenced grasshopper arginine kinase and examined its expression during embryogenesis and its subcellular localization in vivo and in vitro. During the first half of embryogenesis, arginine kinase is expressed selectively in a small percentage of ectodermal cells (dorsal closure cells), in a small percentage of mesodermal cells (muscle pioneers), and throughout the developing CNS. Most of these cell types are motile, including nascent neurons, muscle pioneers, dorsal closure cells, and many CNS glia. Neuroblasts also strongly express arginine kinase; they are nonmotile but are undergoing repeated rounds of (ATP-dependent) mitosis. Arginine kinase is colocalized with F-actin in a narrow band along the leading edges of lamellipodia of migrating glia. In neurons undergoing axonogenesis, arginine kinase is concentrated in growth cones and extends to the tips of filopodia. The amount of arginine kinase varies widely between growth cones, even between different growth cones of the same neuron. Energy for growth cone migration appears to be mobilized by (1) selective expression of arginine kinase by neurons, (2) localization of arginine kinase within growth cones, and (3) concentration of arginine kinase within specific growth cones, depending on the traction forces being generated. Mobilization of guanidino kinases may participate in the selective growth of specific growth cones.  (+info)