Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana)

Abstract

The isolation and characterization of homogeneous arginine kinase from the cockroach is reported. The purification protocol produces 6.6 mg of pure enzyme from 6.8 g of whole cockroach. The purified enzyme cross-reacts with a heterologous antibody and monoclonal antibody against arginine kinase from the shrimp. Both antibody preparations also cross-react with extracts from several species known to contain monomeric arginine kinase, but fail to react with extracts from organisms containing dimeric arginine kinase. Cockroach arginine kinase has a molecular mass of approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50 degrees C for 10 min) and has a pH optima of 8.5 and 6.5-7.5, for the forward and reverse reactions, respectively. Treatment with 5,5'dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and, interestingly, the reaction is biphasic. The Michaelis constants for the phosphagen substrates, arginine: 0.49 mM, phosphoarginine: 0.94 mM, and nucleotide substrates MgATP: 0.14 mM, MgADP: 0.09 mM, are in the range reported for other arginine kinases. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinase from the cockroach has 12% alpha-helical structure. The intrinsic protein fluorescence emission maximum at 340 nm suggests that tryptophan residues are below the surface of the protein and not exposed to solvent. Arginine kinase from the cockroach and shrimp are known to be deleterious immunogens towards humans. The availability of pure protein, its characterization and potential regulation of activity, will be useful in developing agents to control the cockroach population and its destructive role in agriculture and human health.

Gene profiling and characterization of arginine kinase-1 (MrAK-1) from freshwater giant prawn (Macrobrachium rosenbergii)

“…5 A and B). This optimum levels are quite different from the optimum pH (7.5e8.0) reported by Qing-Yun et al [41] for monomeric AK from scallop, but very similar to that of greasyback shrimp M. ensis [16], white shrimp L. vannameii [17] and American cockroach [42].…”

Arginine kinase: Differentiation of gene expression and protein activity in the red imported fire ant, Solenopsis invicta

“…Total protein concentration was measured using a Beckman DU-600 spectrophotometer and a BioRad protein assay kit at 595 nm, with bovine serum albumin as the standard (Bradford, 1976). The AK activity assay was conducted using a modification of the procedures described in "Enzymatic Assay of AK" available on the following website: http://www.sigmaaldrich.com/ img/assets/18220/Arginine_Kinase.pdf and by Brown et al (2004). Briefly, a 1 ml reaction mix was prepared by combining 178 mM glycine, 0.33 mM 2-mercaptoethanol, 13 mM magnesium sulfate, 133 mM potassium chloride, 20 mM phosphor(enol)pyruvate, 6.7 mM adenosine 5′-triphosphate, 0.13 mM β-nicotinamide adenine dinucleotide, 2 U pyruvate kinase, 3 U lactic dehydrogenase, 17 mM Larginine in a final concentration.…”

“…Total protein was isolated from whole bodies of 15 alate female, 15 alate male, 15 3rd + 4th instars, 40 workers, 15 worker pupae and 15 alate pupae and 60 head, 20 thorax and 20 abdomen of alate females, alate males, and workers according to Brown et al (2004). Briefly, each fire ant sample was homogenized in ice-cold glycine buffer (250 mM glycine [pH 8.6], 10 mM 2-mercaptoethanol, 1 mM EDTA, 0.1 mM DTT, and 1 mM PMSP).…”

Mechanism of inhibition of arginine kinase by flavonoids consistent with thermodynamics of docking simulation

“…* target for the control of cockroach proliferation [6,7]. The screening of substrate analogs as potential enzyme inhibitors for AK from Trypanosoma cruzi had also been investigated [4].…”