Characterization Of Arginine Kinase Research Articles

Molecular Characterization of the Allergenic Arginine Kinase from the Edible Insect Hermetia illucens (Black Soldier Fly).

Arginine kinase (AK) is an important enzyme for energy metabolism of invertebrate cells by participating in the maintenance of constant levels of ATP. However, AK is also recognized as a major allergen in insects and crustaceans capable of cross-reactivity with sera of patients sensitized to orthologous proteins. In the perspective of introducing insects or their derivatives in the human diet in Western world, it is of primary importance to evaluate possible risks for allergic consumers. This work reports the identification and characterization of AK from Hermetia illucens commonly known as the black soldier fly, a promising insect for human consumption. To evaluate allergenicity of AK from H. illucens, putative linear and conformational epitopes are identified by bioinformatics analyses, and Dot-Blot assays are carried out by using sera of patients allergic to shrimp or mites to validate the cross-reactivity. Gastrointestinal digestion reduces significantly the linear epitopes resulting in lower allergenicity, while the secondary structure is altered at increasing temperatures supporting the possible loss or reduction of conformational epitopes. The results indicate that the possible allergenicity of AK should be taken in consideration when dealing with novel foods containing H. illucens or its derivatives.

Characterization of Arginine Kinase in the Barnacle Amphibalanus Amphitrite and Its Role in the Larval Settlement.

Energy metabolism is a key process in larval settlement of barnacles, but the underlying molecular mechanisms remain ambiguous. Arginine kinase (AK) mainly participates in energy metabolism in invertebrates. So far, its roles in barnacles have not been studied. In the present study, we raised an antibody against AK from Amphibalanus amphitrite Darwin to characterize the roles of AK in the larval settlement process. Among the developmental stages, AK was highly expressed during the cypris stage. Along with the aging process in cyprids, the level of AK decreased. The immunostaining results showed that AK was localized to muscular tissues in cyprids, including antennules, antennular muscles, and thoracic limbs. The larval settlement rate decreased and larval movement was inhibited in response to treatments with high concentrations of AK inhibitors (rutin and quercetin). These results demonstrated that AK was involved in the larval settlement of A. amphitrite through mediating energy supply in muscle tissues. Moreover, further analysis indicated that both the p38 MAPK and NO/cGMP pathways positively mediated the expression of AK in cyprids.

Characterization of arginine kinase from the caenogastropod Semisulcospira libertina, an intermediate host of Paragonimus westermani

Arginine kinase (AK) belongs to the phosphagen kinase (PK) family of enzymes that have a significant role in the maintenance of energy homeostasis. In this study, cDNA of Semisulcospira libertina AK was determined, cloned and the recombinant protein was expressed as fusion protein with maltose-binding protein. The protein has a theoretical molecular mass of 39,412 Da and an estimated isoelectric point (p I) of 6.37. The recombinant enzyme showed high affinity and significant activity only for the substrate L-arginine (Km ¼ 0.53 mM; Vmax ¼ 61.30 mmol/min mg protein). Phylogenetic analyses showed that S. libertina AK clusters with gastropod and cephalopod AKs. Comparison of gene structure showed that all intron positions of S. libertina AK are shared with molluscan AKs. Certain intron positions are also shared with trematode TK and sipunculid HTK. Semisulcospira libertina AK can be utilized in the control of paragonimiasis since it is absent in mammalian hosts.

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

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

Purification and Characterization of Arginine Kinase from Locust

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

Expression, purification, and characterization of arginine kinase from the sea cucumber Stichopus japonicus

The arginine kinase gene of sea cucumber Stichopus japonicus was cloned and inserted into the prokaryotic expression plasmid pET-21b. The protein was expressed in a soluble and functional form in Escherichia coli and purified by Blue Sepharose CL-6B, DEAE-32, and Sephadex G-100 chromotography with a final yield of 83 mg L −1 of LB medium. The specific activity, electrophoretic mobility, and isoelectric focusing were all identical with those of arginine kinase that was purified from sea cucumber muscle. The fluorescence emission spectrum of arginine kinase had a maximum fluorescence at a wavelength of 330 nm upon excitation at 295 nm. These results are the first report of this purified protein.

Purification and characterization of arginine kinase from sea-urchin eggs.

In most invertebrates, creatine kinase is replaced by arginine kinase, which catalyzes reversibly the transfer of a phosphate group between adenosine triphosphate and arginine. In sea-urchin larvae, arginine kinase only is expressed whereas in adult sea-urchins both arginine kinase and creatine kinase can be found in the same tissue. In order to study their developmental regulation and properties, we have purified arginine kinase to homogeneity from the eggs of the sea-urchin Paracentrotus lividus. The purification involves ethanol and ammonium sulfate precipitations, followed by an anion-exchange chromatography, an affinity chromatography and a gel filtration. A 500-fold increase in specific activity leads to a specific activity of 360 IU/mg protein at 25 degrees C. Arginine kinase (pI = 5.7) is rapidly and irreversibly inactivated at 45 degrees C. Amino acid composition and Km values (2.08 mM for phospho-L-arginine and 1.25 mM for ADP) are also given. Determination of molecular mass by gel filtration and separation by SDS/polyacrylamide gel electrophoresis indicate that the enzyme is an 81-kDa dimer of two subunits of 42 kDa.

Purification and characterization of arginine kinase from the sea cucumber Caudina arenicola

1. 1. Arginine kinase from Caudina arenicola, a Pacific sea cucumber, has been purified from muscle to apparent homogeneity and shown to be a dimeric protein of mol. wt 80,000. 2. 2. The isoelectric point of arginine kinase from Caudina arenicola occurs at pH 7.8, unusually high compared to the monomeric arginine kinase from lobster muscle, but similar to muscle type creatine kinase from vertebrate. The enzyme is also unusually unstable to heat, losing half its activity after exposure to 40°C for about 10 min. The pH optimum of arginine kinase is approximately 7.9. 3. 3. Kinetic analysis reveals Michaelis constants of 1.3 mM for MgATP and 0.5 mM for arginine. The dissociation constant for arginine from the binary enzyme—substrate complex is four times greater than for the ternary complex containing MgATP. Dissociation constants for MgATP from the binary and ternary complexes do not differ significantly. Inhibition of arginine kinase by MgADP is potentiated by nitrate anion, presumably by formation of a transition state analog in which nitrate stabilizes the dead-complex between enzyme, MgADP and arginine. 4. 4. Arginine kinase from Caudina arenicola and creatine kinase from rabbit brain will hybridize in vitro to form a bifunction dimer.

Purification and characterization of arginine kinase from scallop smooth muscle.

Purification and characterization of arginine kinase from scallop smooth muscle.

Purification and characterization of arginine kinase from the mantle muscle of the squid, Symplectoteuthis oualaniensis: Role of the phosphagen/phosphagen kinase system in a highly aerobic muscle

Arginine kinase (EC 2.7.3.3) from squid mantle muscle was purified to homogeneity as judged by isoelectric focusing and electrophoresis (sodium dodecyl sulfate, starch). The enzyme is a monomer of molecular weight 55,000 ± 6000 and occurs in mantle muscle in a single electrophoretic form with a specific activity of 310 units/g wet wt at 25 °C. The pH optima are 8.2 and 6.9 in the arginine phosphate-synthesizing and ATP-synthesizing directions, respectively. The divalent cation requirement of the enzyme is satisfied by Mg 2+ or Mn 2+ but added Ca 2+, Zn 2+, Cu 2+, and Fe 2+ are inhibitory. Monovalent ions ( NH 4 +, K +, Na +, Cl −) inhibit the enzyme. Michaelis constants for ATP and arginine (both measured at pH 7.6) and for ADP and arginine phosphate (pH 7.2) are 0.8, 1.0, 0.2, and 3.5 m m, respectively, the K m 's for any one substrate being independent of the concentration of its cosubstrate. Of over 40 metabolites tested for in vitro effects on the purified enzyme, only NADH, within its physiological range, affected enzyme activity. NADH is a noncompetitive inhibitor with a K i of 0.15 m m. Like other enzymes of energy metabolism in squid mantle muscle, arginine kinase is tightly regulated, its activity being closely integrated with that of glycolysis and the Krebs cycle by NADH control. A theory is proposed, based on the data obtained, that the arginine kinase/arginine phosphate system in this highly aerobic muscle serves to reversibly regulate glycolytic flux and that its role in “energy storage” is very limited.