Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31(WSV340/WSSV396), an envelope protein of white spot syndrome virus(WSSV), contains an Arg-Gly-Asp(RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into p ET30a(+), expressed in Escherichia coli strain BL21 and purifi ed with immobilized metal ion affi nity chromatography. Four gill cellular proteins of shrimp( Fenneropenaeus c hinensis) were pulled down by an affi nity column coupled with recombinant VP31(r VP31), and the amino acid sequences were identifi ed with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase(AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's(r AK) binding activity with r VP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the fi rst evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

Improved Activity Assay Method for Arginine Kinase Based on a Ternary Heteropolyacid System

This paper presents a new system for the activity assay of arginine kinase (AK), based on the spectrophotometric determination of an ascorbic acid reduced blue ternary heteropolyacid composed of bismuth, molybdate and the released phosphate from N phospho L arginine (PArg) formed in the forward catalysis reaction. The assay conditions, including the formulation of the phosphate determination reagent (PDR), the assay timing, and the linear activity range of the enzyme concentration, have been tested and optimized. For these conditions, the ternary heteropolyacid color is completely developed within 1 min and is stable for at least 15 min, with an absorbance maximum at 700 nm and a molar extinction coefficient of 15.97 (mmol/L) -1 ·cm -1 for the phosphate. Standard curves for phosphate show a good linearity of 0.999. Compared with previous activity assay methods for AK, this system exhibits superior sensitivity, reproducibility, and adaptability to various conditions in enzymological studies. This method also reduces the assay time and avoids the use of some expensive instruments and reagents.

Activity and Structure Changes of Arginine Kinase from Shrimp Feneropenaeus chinensis Muscle in Trifluoroethanol Solutions

Trifluoroethanol has often been used in protein folding studies. The changes in activity and unfolding of arginine kinase from shrimp Feneropenaeus chinensis muscle during denaturation in different concentrations of trifuoroethanol were investigated using far ultraviolet circular dichroism and fluorescence emission spectra. Arginine kinase was inactivated in trifluoroethanol solutions. The tertiary and secondary structures of arginine kinase were also destroyed in the trifluoroethanol solutions. The unfolding and inactivation courses were measured and compared. Inactivation occurred prior to unfolding, which suggests that the arginine kinase active site is more easily damaged by the denaturant than the enzyme as a whole. The result also indicates that the arginine kinase active site is situated in a limited and flexible region of the enzyme molecule.

Characteristics of Two Intermediates Trapped in the Unfolding Pathway of Arginine Kinase Induced by Guanidinium Chloride

Equilibrium guanidinium chloride （GdmCl）-induced unfolding of arginine kinase （AK） was investigated by enzymatic activity, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid （ANS） fluorescence, circular dichroism （CD） spectrum, and size-exclusion chromatography. The measurements showed that AK unfolded through two equilibrium intermediates： the molten globule state and the partly folded state. Both intermediates have no enzyme activity. The molten globule state exists at 0.4-0.8 mol/L GdmCi, perhaps after the N-terminal domain has unfolded but the C-terminal domain is still intact. The partly folded state occurs at 1.1-1.5 mol/L GdmCI with a hydrodynamic volume no more than 1.6-fold larger than the native state and a pronounced far UV-CD signal. Its ANS fluorescence intensity is about 50% of the molten globule state. This partly folded state shares similarities with the “burst” kinetic intermediate of protein folding.