Muscle Arginine Kinase Research Articles

Regulation of tail muscle arginine kinase by reversible phosphorylation in an anoxia-tolerant crayfish

Freshwater crayfish, Orconectes virilis, can experience periodic exposures to hypoxia or anoxia due to low water flow (in summer) or ice cover (in winter) in their natural habitat. Hypoxia/anoxia disrupts energy metabolism and triggers mechanisms that to support ATP levels while often also suppressing ATP use. Arginine kinase (AK) (E.C. 2.7.3.3) is a crucial enzyme involved in energy metabolism in muscle, gating the use of phosphagen stores to buffer ATP levels. The present study investigated AK from tail muscle of O. virilis identifying changes to kinetic properties, phosphorylation state and structural stability between the enzyme from aerobic control and 20h anoxic crayfish. Muscle AK from anoxia-exposed crayfish showed a significantly higher (by 59%) K (m) for L: -arginine and a lower I(50) value for urea than the aerobic form. Several lines of evidence indicated that AK was converted to a high phosphate form under anoxia: (a) aerobic and anoxic forms of AK showed well-separated elution peaks on DEAE ion exchange chromatography, (b) ProQ Diamond phosphoprotein staining showed a 64% higher bound phosphate content on anoxic AK compared with the aerobic form, and (c) treatment of anoxic AK with alkaline phosphatase reduced K (m) L: -arginine to aerobic levels whereas incubation of aerobic AK with protein kinase A catalytic subunit raised the K (m) to anoxic levels. The physiological consequence of anoxia-induced AK phosphorylation may be to suppress AK activity in the phosphagen-synthesizing direction and, together with reduced cellular pH and ATP levels, promote the phosphagen-catabolizing direction under anoxic conditions. This is first time that AK has been shown to be regulated by reversible phosphorylation.

Allergènes de poissons et crustacés

Concerning fish allergy, the major allergens are the β isoforms of muscle parvalbumins. Fish parvalbumins share among themselves important homologies in amino acid composition, which gives rise to extensive cross-sensitizations. Allergic cross-reactions with frog parvalbumin have been described. Due to the frequent sensitization to parvalbumins, the role of other allergens is masked. Allergy to fish gelatin has been reported. Additional muscle allergens have been incompletely described, but their exact role remains to be defined. With respect to shellfish allergy, muscle tropomyosin is the major allergen. Tropomyosins give rise to important cross-sensitization between shellfish, but also with gastropods, mites, cockroaches and other arthropods. The myosin light chain has been shown to be an allergen in 60% of white shrimp-allergic patients. A muscle arginine kinase has been recognized as an allergen in a third of patients sensitized to black tiger shrimp. A sarcoplasmic calcium-binding protein of the same tiger shrimp is recognized by IgE antibodies of 60% of shrimp-allergic patients.

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in arginine kinase complexes.

Two-dimensional proton transfer nuclear Overhauser spectroscopy (TRNOESY) studies of the conformations of the adenosine moieties in the nucleotides bound at the active site of the lobster (Homarus americanus) muscle arginine kinase are reported. TRNOESY measurements were made using a sample protocol chosen to minimize contributions from weak non-specific binding of the nucleotides to the observed NOE's. This was done by making the measurements as a function of ligand concentration while keeping the ligand to enzyme concentration ratio fixed at 10:1. The experiments were performed at 500 MHz and 10 degrees C for six different mixing times in the range 40-300 ms. The measurements were made on three complexes of the enzyme: E.MgATP, E.MgADP, and the long-lived transition-state-analog complex (E.MgADP.NO-3.arginine). All the complexes, including the transition-state-analog complex with an estimated lifetime of about 50 ms, satisfy the fast-exchange condition. The TRNOE buildup curves for all the nucleotide-proton pairs in each complex were analyzed using a complete relaxation matrix appropriate for fast exchange. The interproton distances obtained from the NOE analysis were used as constraints in obtaining an energy-minimized conformation on the basis of the program CHARMm. The glycosidic torsion angle (chi) for the adenosine moiety in all three complexes is about 50 degrees +/- 5 degrees. The glycosidic orientation agrees well with that determined for MgATP and MgADP complexes of creatine kinase (Murali et al., 1993), MgATP bound at the active and ancillary sites of pyruvate kinase (Jarori et al., 1994a), and PRPP synthetase (Jarori et al., 1994b).(ABSTRACT TRUNCATED AT 250 WORDS)

Horseshoe crab sperm contain a unique isoform of arginine kinase that is present in midpiece and flagellum

AbstractSpermatozoa of the horseshoe crab, Limulus polyphemus, contained high activities of arginine kinase (AK), the bulk of which was completely solubilized in buffer lacking detergent. Sperm AK, along with the fast isoform of muscle AK, were purified to homogeneity. Sperm AK had similar electrophoretic mobility on cellulose acetate to that of muscle AK. In contrast, sperm AK ran somewhat slower than muscle AK on SDS‐PAGE, indicating that this protein has a higher relative molecular mass (44 kD vs. 41 kD). These two forms also showed different chromatographic behavior on a reverse‐phase HPLC column. Peptide maps of cyanogen bromide cleavage fragments of sperm and muscle AK showed distinct differences. Collectively, these data suggest that the sperm of L. polyphemus contain a unique AK isoform. Polyclonal anti‐muscle AK antibodies, which showed strong reactivity against sperm AK, were used to localize AK in sperm. Both indirect immunofluorescence and immunogold transmission electron microscopy showed that AK was present in the midpiece in the vicinity of the mitochondria and also along the length of the flagellum. Given the polarized geometry of these cells, it appears that the AK reaction may play a role in high energy phosphate transport to dynein ATPases in the flagellum. © 1993 Wiley‐Liss, Inc.

Interaction in vitro of scallop muscle arginine kinase with filamentous actin.

Scallop muscle arginine kinase binds to F-actin from mollusc and rabbit muscle in vitro. One site of interaction appears to be located in residues 305-325 of a C-terminal fragment (residues 285-375) of actin. The binding is hindered in the presence of arginine, Mg(2+)-ADP and NO3-, which form a dead-end complex with the enzyme. F-actin inhibits the enzyme activity non-competitively with respect to Mg(2+)-ATP. As a function of arginine concentration, the inhibition is of the mixed type, where Km is affected more than Vmax.

Conformational heterogeneity of creatine kinase determined from phase resolved fluorometry

Fluorescence lifetimes of dimeric rabbit muscle creatine kinase specifically dansylated at both active sites and the homologous monomeric lobster muscle arginine kinase singly dansylated were determined using phase-modulation methods with global analysis of overdetermined data sets. For both proteins, the data is adequately described by three discrete exponential decays or a Lorentzian double distributed decay. Analogue phase resolved spectroscopy also reveals the presence of at least two distinct fluorophore domains for the dansyl moieties of creatine kinase. The model fluorophore, dansyllysine, exhibits a monoexponential decay with a value that is highly solvent dependent. Because the monomeric arginine kinase exhibits essentially the same decay law as doubly derivatized dimeric creatine kinase, it is proposed that the multiple lifetimes of creatine kinase reflect two or more isomeric dimeric states and not subunit asymmetry within a conformationally homogeneous dimeric population. Exposure of arginine kinase to 6 M guanidinium chloride results in a shift to shorter lifetimes and narrowing of the lifetime distributions. Creatine kinase displays a small narrowing of the distribution, but little change in fractional populations or lifetimes. These results suggest the presence of structural elements resistant to denaturation. The longest lifetime component in the triexponential discrete decay law of doubly dansylated creatine kinase is totally unquenched by acrylamide, whereas the two shorter lifetime components exhibit limited dynamic quenching. Steady-state quenching by acrylamide is significant and reveals a sharp distinction between accessible and non accessible dansyl groups. The major mechanism for interaction between the dansyl moieties and acrylamide is, atypically, static quenching. The results are consistent with two dansyl domains, one accessible and hydrophilic according to lifetime values and the other inaccessible and hydrophobic in solvent characteristics.Energy transfer between the dansyl group and the eight tryptophan residues of dimeric creatine kinase give similar results(~ 35%) from measurements of lifetimes, steady-state donor quenching and sensitized acceptor emission. The similarity suggests that the overall flexibility of the dimeric protein is limited. The occurrence of multiple conformers of muscle creatine kinase provides an explanation for several previous observations, most notably the structural origins for compartmentation of the muscle isozyme observed in the myofibril.

Purification and properties of two molecular forms of arginine kinase from the adductor muscle of the scallop, Pecten maximus

1. 1. Two molecular forms of arginine kinase, AK 1 and AK 2 have been purified from the adductor muscle of the scallop, Pecten maximus. AK 2 was retained on a DEAE-cellulose column at pH 7.5, but AK 1 was not. 2. 2. Both forms were monomeric (mol. wt. ≈42,000) and showed the same pH optimum (7.5–8.0) in the direction of phosphoarginine synthesis. 3. 3. AK 1 had slower electrophoretic mobility at pH 8.3 towards the anode, higher lysine content, lower glutamate content, lower K m for l-arginine and higher K m for Mg 2+-ATP than AK 2. Unlike AK 1, AK 2 was strongly inhibited at high concentrations of Mg 2+-ATP. 4. 4. Both molecular forms cross-reacted with antisera raised against native as well as performic acid-oxidized lobster muscle arginine kinase. However, AK 1 showed a greater affinity than AK 2 to anti-lobster arginine kinase antibodies, particularly to those raised against the native enzyme.

Phosphorus-31 and proton NMR studies of the structure of enzyme-bound substrate complexes of lobster muscle arginine kinase: relaxation measurements with manganese(II) and cobalt(II)

The paramagnetic effects of Mn(II) and Co(II) on the spin-lattice relaxation rates of 31P nuclei of ATP and ADP and of Mn(II) on the spin-lattice relaxation rate of the delta protons of arginine bound to arginine kinase from lobster tail muscle have been measured. Temperature variation of 31P relaxation rates in E.MnADP and E.MnATP yields activation energies (delta E) in the range 6-10 kcal/mol. Thus, the 31P relaxation rates in these complexes are exchange limited and cannot provide structural information. However, the relaxation rates in E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-2 kcal/mol; i.e., these rates depend upon 31P-Co(II) distances. These distances were calculated to be in the range 3.2-4.5 A, appropriate for direct coordination between Co(II) and the phosphoryl groups. The paramagnetic effect of Mn(II) on the 1H spin-lattice relaxation rate of the delta protons of arginine in the E.MnADP.Arg complex was also measured at three frequencies (viz., 200, 300, and 470 MHz). These 1H experiments were performed in the presence of sufficient excess of arginine to be observable over the protein background but with MnADP exclusively in the enzyme-bound form so that the enhancement in the relaxation rates of the delta protons of arginine arises entirely from the enzyme-bound complex. Both the observed frequency dependence of these rates and the delta E less than or equal to 1.0 +/- 0.3 kcal/mol indicate that this rate depends on the 1H-Mn(II) distances.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and distribution of arginine kinase in the tissues of the scorpion, Palamneus phipsoni

Arginine kinase in claw muscle extracts of the scorpion, Palamneus phipsoni, was characterized. The enzyme, with a pH optimum of 8.5 in the direction of phosphoarginine synthesis, showed activation by Mg2+, high specificity towards L-arginine as the guanidino substrate, slight inhibition by high concentrations of L-arginine and ATP, and a molecular weight of 33 500. On polyacrylamide gel electrophoresis at pH 8.3 the enzyme migrated to the anode as a single molecular species. In addition to the claw muscle, the enzyme activity was also found to be present in the heart, alimentary canal, hepatopancreas, and nervous system. In general, scorpion muscle arginine kinase appears to be similar in its properties to the enzyme from other arthropods.

The use of arginine analogues for investigating the functional organization of the arginine-binding site in lobster muscle arginine kinase. Role of the ‘essential’ thiol group

1. The nature of arginine binding to lobster arginine kinase and the extent of its possible involvement with the ;essential' thiol group of the enzyme has been investigated with some inhibitory analogues of arginine. 2. Most of the analogues inhibit competitively, although mixed inhibition may occur if the alpha-carboxy group or alpha-amino group is absent. 3. The K(i) values indicate that strength of binding depends on the length of the carbon chain (l-isoleucine>l-valine>l- alpha-aminobutyrate>l-alanine) and the integrity of the substituents on the alpha-carbon atom (l-arginine>agmatine and l-ornithine>putrescine). The guanidino group probably contributes little to substrate binding, but a positive charge near the delta-nitrogen atom appears to be important (l-ornithine>l -citrulline>l-alpha-aminobutyrate). A cyclic analogue, 2-carboxymethyl-3-oxo-2,3,5,6,7,8-hexahydro-1H-imidazo [1,2-a][1,3]diazepine-8-carboxylic acid, has a low K(i) value similar to that of an equivalent straight-chain form, suggesting that arginine probably binds in a folded configuration. 4. The aliphatic l-amino acids give enzyme difference spectra similar to that with l-arginine and the integrity of the alpha-carboxy and alpha-amino groups appears to be a minimal but not sufficient requirement for this, as l-ornithine gives an atypical difference spectrum. A difference spectrum is interpreted as indicating an enzyme conformational change. No difference spectrum was observed with methylguanidine. 5. The ability of aliphatic alpha-l-amino acids to protect against inhibition by 5,5'-dithiobis-(2-nitrobenzoic acid) is proportional to the number of atoms in the carbon chain and inversely proportional to K(i). Ornithine gives greater protection than citrulline; analogues lacking the alpha-amino groups also protect. Agmatine, lacking the alpha-carboxy group, did not protect. 6. It is concluded that it is unlikely that the ;essential' thiol group in the enzyme interacts with any part of the arginine molecule during catalysis except, possibly, the alpha-carboxyl group.

Photoaffinity Labelling of Arginine Kinase and Creatine Kinase with a gamma-P-Substituted Arylazido Analogue of ATP

1. An ATP analogue with a photoactivated azide group attached to the gamma-phosphate via an amide bond, ATP gamma-p-azidoanilide, appeared to have potential use as a photoaffinity label for the nucleotide-binding regions of ATP: guanidine phosphotransferases. Upon photolysis in the presence of lobster muscle arginine kinase and rabbit muscle creatine kinase, the analogue is converted to a potent inhibito of these two kinases. This photo-dependent inhibition is specific as it cannot be induced by azidoaniline, a mixture of azidoaniline and ATP or by ATP gamma-p-aminoanilide. Preirradiated under suitable conditions, the photoanalogue still shows a transitory inhibitory effect which, however, slowly vanishes with time (t0.5 = 3 h). 2. The photoinhibition is significantly decreased by the presence of ATP or ADP but is completely prevented by the addition of a mixture of nucleotide and guanidine substrates. Differential spectroscopy and affinity chromatography on Sepharose-ATP demonstrated the inability of photoinactivated arginine kinase and creatine kinase to recognize their nucleotide substrates. 3. Experiments with [14C]ATP gamma-p-azidoanilide indicated that photolysis is associated with an irreversible and stoichiometric binding of the ATP analogue to the enzymes. Autoradiographs made with the peptide maps corresponding to the tryptic digests of each 14C-labelled photomodified enzyme showed an unexpected highly specific labelling of the proteins. 4. Thiiol titrations of the kinases which have been subjected to various photolysis conditions led to the conclusion that the arylnitrene moiety of the photoanalogue is covalently attached to the single reactive cysteinyl side chain present in the active-site region of the two homologous kinases. This amino acid residue appears, therefore, to be located near the phosphate chain binding subsite occupied by the ATP analogue and probably also by the natural nucleotide substrates.

Effect of substrate — binding on the immunologic reactivity of lobster — muscle arginine kinase: A comparison with rabbit - muscle creatine kinase

The effects of substrate-binding upon the immunologic reactivity of rabbit creatine kinase and lobster arginine kinase have been investigated. The separate binding of the guanidine or the nucleotide substrate to creatine kinase yields no alteration of antigenicity and a substantial effect is only observed when all the loci at the active center of the enzyme, including that for the transferable phosphoryl group, are occupied. In contrast, the antigenic reactivity of arginine kinase is affected by the separate binding of either the guanidine or the nucleotide substrate, and the simultaneous binding of the two substrates results in a cumulative effect, which is irrespective of the phosphorylated or non-phosphorylated form of the complex. These results support the existence of substrate-induced conformational changes demonstrated by other methods, and they reveal appreciable differences in their effect on the antigenic reactivity of the two enzymes.

Isolement du peptide à tyrosine essentielle de l'arginine kinase de homard

The essential tyrosine residue of Lobster muscle arginine kinase, which is part of an antigenic determinant, has been modified by tetranitromethane. Cleavage of the S-carboxymethylated nitrated enzyme with cyanogen bromide gives rise to eight peptides, one of which containing the labelled essential tyrosyl group. Ion exchange chromatography on sulfoethyl-Sephadex C-25 in urea medium has been used with success for isolation and purification of the nitrated peptide. From its amino acid composition and end groups structure this peptide is the N-terminal fragment of the protein.

Preliminary X-ray diffraction studies on lobster ATP: l-Arginine phosphotransferase

Lobster muscle arginine kinase crystals have been grown by a procedure of gaseous diffusion. The resulting crystals are monoclinic. space group P2 1 with unit cell dimensions of a = 75.9 A ̊ , b = 88.3 A ̊ , c = 58.2 A ̊ , β = 102.0 ° . Considerations of cell volume and protein molecular weight indicated the presence of two molecules in the asymmetric unit. A search for heavy-atom derivatives is in progress.

Unspecific Arginine Kinase of Molecular Weight 150000. Amino Acid Composition, Subunit Structure and Number of Substrate Binding Sites

The amino acid composition of unspecific arginine kinase of molecular weight 150 000 of Sabella pavonina muscle has been determined. If was found to be very similar to that of the phosphagen kinases previously studied. The subunit structure of the enzyme has been investigated by physical and chemical means. The data obtained from ultracentrifugation studies in 6 M guanidine hydrochloride and from molecular sieving and disc electrophoresis in 8 M urea, as well as the tryptic peptide mapping, suggest that Sabella muscle kinase is composed of four non-covalently linked polypeptide chains, with similar molecular weights. The number of binding sites for the nucleotide substrate ADP-Mg2+ has been estimated, using differential spectrophotometry and gel filtration on Sephadex columns. By both methods it was demonstrated that the enzyme contains two catalytic sites per protein molecule of molecular weight 150 000. Thus, arginine kinase from Sabella muscle, of molecular weight 150 000, consists of four similar polypeptide chains, but possesses only two substrate binding sites per tetrameric molecule.

Comparative study of two ATP: l-arginine phosphotrans-ferases of molecular weight 84 000

Solen ensisensis muscle arginine kinase (ATP : l-arginine phosphotransferase, EC 2.7.3.3.) was isolated in an homogeneous state. Its molecular weight was found to be about 80 000. The properties of this enzyme were compared with those of arginine kinase from Sipunculus nudus, an enzyme which also has a molecular weight of about 80 000 Both enzymes have several reactive thiol groups (8 thiol groups in the Solen kinase and 12 in the Sipunculus enzyme were titrateable with 5,5′- dithio-bis-(2- nitrobenzoic ) acid and histidine residues (both enzymes have 6 reactive histidine residues). These kinases were, therefore, highly susceptible to oxidation. Both enzymes show the same pH optimum and absolute specificity towards the guanidine substrate, l-arginine. The reaction kinetics of both enzymes are of the sequential type. In the presence of α-aminoacids or Mg 2+-ADP, similar spectral effects were obtained. The enzymes differ in their enzymic activities and in their rate of recovery following urea denaturation. The most important difference that appeared to be a special feature of the Sipunculus enzyme is that the spectrum of the Mg 2+-ADP-enzyme complex is strongly intensified by l-arginine.

Characterization of the Active Site Structures of Arginine Kinase-Substrate Complexes: WATER PROTON MAGNETIC RELAXATION RATES AND ELECTRON PARAMAGNETIC RESONANCE SPECTRA OF MANGANOUS-ENZYME COMPLEXES WITH SUBSTRATES AND OF A TRANSITION STATE ANALOG

Abstract The apparent number, n, of water ligands coordinated to bound Mn(II) in the ternary arginine kinase-MnADP or ATP complexes, in the abortive quaternary complex E-MnADP-arginine and in the transition state analog complex formed by addition of nitrate to the quaternary complex was estimated from the frequency dependence of the longitudinal proton relaxation rate of water (PRR) of lobster (Homarus americanus) muscle arginine kinase complexes. The apparent number of residual water ligands becomes progressively smaller as successive sites on the enzyme become occupied and is approximately 2 for the ternary nucleotide complexes, 1 for the abortive quaternary complex and considerably less than 1 for the transition state analog complex. The low values of n may be ascribed to the successive substitution of water ligands by protein ligands as each substrate and anion is successively bound to the enzyme or alternatively to an apparent rather than real disappearance of water ligands. The latter explanation implies that conformational changes induced by successive occupation of the binding sites on the enzyme are of such a nature that the rate of exchange of Mn(II)-bound water with solvent water becomes so slow for some Mn(II) water ligands that only a fraction of the total Mn(II)-bound water exchanges sufficiently rapidly to contribute to the PRR of bulk solvent. From the frequency dependence of the PRR of the ternary and quaternary complexes, it has been established that T1e, the electron spin relaxation time, is the dominant term in the correlation time which modulates the interaction between the Mn(II) electron spin and the water proton nuclear spin. Values of the electron spin relaxation times, T1e, estimated from PRR experiments are consistent with the lower limits of T1e obtained from line widths of the EPR spectra of the same complexes. No change is observed in the position of the peaks in the EPR spectra in proceeding from the binary MnADP complex to the ternary E-MnADP to the abortive quaternary E-MnADP-arginine or from MnATP to E-MnATP, and it may be inferred that there has been no gross modification in the symmetry of the electronic environment of Mn(II). Small changes in the line widths of the EPR spectra for these complexes do indicate that the accessibility of the Mn(II) liganded structure to collision with solvent molecules has been modified consistent with changes in the number of exchangeable water protons determined from PRR. Addition of nitrate to the abortive quaternary complex to form the transition state analog, as in the case of rabbit muscle creatine kinase, induces significant changes in the active site structure of arginine kinase as reflected in the altered EPR line shape. In this case altered line positions of the EPR spectrum also are observed. The latter indicates an asymmetry of the electronic environment of Mn(II) which may result from a substitution of ligands. The corresponding transition state analog for creatine kinase exhibits a much more anisotropic EPR spectrum. The EPR spectra are much more sensitive than PRR values to subtle changes in active site structure, e.g. spectral changes induced by d-arginine differ markedly from those produced by l-arginine in the presence of nitrate. Thiocyanate, nitrite, and formate at relatively high concentrations also modified the active site structure of arginine kinase, as evidenced by the alterations in the EPR spectrum of the quaternary complex upon addition of these anions. Modifications of the EPR spectra caused by these anions were qualitatively similar to those of nitrate but produced smaller changes in peak positions.

Unspecific arginine kinase of molecular weight 150000.

Arginine kinase has been purified from body-wall muscle of a marine polychaetous annelid, Sabella pavonina. The preparation is homogeneous to analytical ultracentrifugation and disc electrophoresis on polyacrylamide gel. The enzyme is labile and undergoes a rapid decrease of its specific activity, even in the presence of protecting agents. The molecular weight of the native enzyme is 150 000 ± 5000, the sedimentation coefficient s°20,b being found to be equal to 7.5. Unlike the enzyme from lobster muscle (mol. wt ≃ 43000) and that from Sipunculus nudus muscle (mol. wt ≃ 83000), the phosphokinase from Sabella pavonina muscle is not strictly specific towards l-arginine. The enzyme shows no optical isomer specificity and is slightly active on a number of analogues of l-arginine, the integrity of the guanidine and carboxylic groups of which is indispensable to their activity. The enzyme is strictly specific for the nucleotidic substrate ATP. Partial isotopic exchange and kinetic studies suggest that the reaction mechanism is somewhat different from that described for sea-water crustacean muscle arginine kinase and more similar to that reported for other phosphagen phosphokinases.