Xicheng Wang

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 mgL(-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.

Urea Induced Inactivation and Unfolding of Arginine Kinase from the Sea Cucumber Stichopus japonicus

Urea titration was used to study the inactivation and unfolding equilibrium of arginine kinase (AK) from the sea cucumber Stichopus japonicus. Both fluorescence spectral and circular dichroism spectral data indicated that an unfolding intermediate of AK existed in the presence of 1.0 to 2.0 M urea. This was further supported by the results of size exclusion chromatography. The spectral data suggested that this unfolding intermediate shared many structural characteristics with the native form of AK including its secondary structure, tertiary structure, as well as its quaternary structure. Furthermore, according to the residual activity curve, this unfolding intermediate form still retained its catalytic function although its activity was lower than that of native AK. Taken together, the results of our study give direct evidence that an intermediate with partial activity exists in unfolding equilibrium states of AK during titration with urea.

Effects of N-Terminal Deletion Mutation on Rabbit Muscle Lactate Dehydrogenase

Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues. The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b. The mutant enzymes were expressed in E. coli BL21/DE3 and were purified. Then their characteristics and stabilities were studied. The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions. The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers. The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl. The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded. Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH. These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme.

Evidence for proximal cysteine and lysine residues at or near the ative site of arginine kinase of stichopus japonicus

Inactivation of arginine kinase (AK) of Stichopus japonicus by o-phthalaldehyde (OPTA) was investigated. The modified enzyme showed an absorption peak at 337 nm and a fluorescent emission peak at 410 nm, which are characteristic of an isoindole derivative formed by OPTA binding to a thiol and an amine group in proximity within the enzyme. Loss of enzymatic activity was concomitant with an increase in fluorescence intensity at 410 nm. Stoichiometry studies by Tsou’s method showed that among the cysteine residues available for OPTA modification in the enzyme, only one was essential for the enzyme activity. This cysteine residue is located in a highly hydrophobic environment, presumably near ATP and ADP binding region. This conclusion was verified by 5,5′-dithiobis(2-nitrobenzoic acid) modification. In addition, these results were supported by means of electrophoresis and ultraviolet, fluorescence, circular dichroism spectroscopy and fast performance liquid chromatography. Sequence comparison suggested that this essential cysteine residue maybe the conservative Cys274.

Multiple effects of chemical reagent on enzyme: o-phthalaldehyde-induced inactivation, dissociation and partial unfolding of lactate dehydrogenase from pig heart

The effects of o-phthalaldehyde (OPTA) on lactate dehydrogenase (LDH) have been studied by following changes in enzymatic activity, aggregation state and conformation. Treatment with OPTA resulted in pseudo first-order inactivation of LDH over a wide concentration range of the inhibitor, and the second-order rate constant was estimated to be 1.52M(-1)s(-1). The loss of enzyme activity was concomitant with the increases in absorbance at 337nm and fluorescence intensity at 405nm. Complete loss of enzyme activity was accompanied by the formation of approximately 4mol isoindole derivatives per mole LDH subunit. Cross-linking experiments verified enzyme dissociation during OPTA modification, which could be attributed to the modification of both thiol groups and lysine residues. Circular dichroism (CD) spectra showed that the secondary structure of the OPTA-modified enzyme decreased correspondingly. Comparison of the inactivation with the conformational changes of the enzyme suggests that the active site of the enzyme exhibits greater conformational flexibility than the enzyme molecule as a whole. It is concluded that OPTA modification has multiple effects on LDH, including its inactivation, dissociation and partial unfolding.

Nb-doped BaTiO3-(Bi0.5Na0.5)TiO3 ceramics with core-shell structure for high-temperature dielectric applications

Nb-doped 0.90BaTiO3-0.10(Bi0.5Na0.5)TiO3 temperature-insensitive ceramics with novel core-shell structure were sintered at low temperature by the conventional solid-state reaction method. The beneficial role of Nb in facilitating the formation of core-shell structure because of chemical inhomogeneity is verified, which is responsible for the weak temperature dependence of dielectric properties. Temperature dependence of permittivity measured at different frequencies shows high frequency dispersion at low temperature, while without relaxor characteristic at high temperature. The Vogel–Fulcher model was adopted to study the relaxor behaviour of Nb-doped 0.90BaTiO3-0.10(Bi0.5Na0.5)TiO3 ceramics at low temperature. The samples with an addition of 1.5 mol% Nb2O5 provide a temperature coefficient of capacitance meeting the requirements of the X9R characteristic, and result exhibits an optimum dielectric behaviour of εr ∼1900, tanδ ∼1.8% at room temperature, making the material a promising candidate for high temperature applications.

Fabrication of nanosized 3Y-TZP ceramic dental restoration frameworks on gypsum models by aqueous electrophoretic deposition

Abstract The feasibility of using electrophoretic deposition (EPD) as a technique to form nanosized zirconia dental restoration frameworks was evaluated and optimised. By using constant low temperature ultrasonic agitation and magnetic stirring, a 3Y-TZP nanopowder suspension with 75 wt-% solid content was obtained as the EPD starting suspension. High density [80% theoretical density (TD)] nanosized 3Y-TZP green bodies were fabricated by aqueous EPD with an input voltage of 30–40 V. High density (99˙9%TD) pure nanosized 3Y-TZP ceramic dental restoration frameworks were successfully achieved after 3 h of sintering at 1280°C. The crystalline structure of the framework was pure tetragonal phase zirconia, and the microstructure was very homogeneous with an average grain size of 230 nm. These results suggest that pure nanozirconia dental restoration frameworks could be near shape manufactured by aqueous EPD.

Two fused proteins combining Stichopus japonicus arginine kinase and rabbit muscle creatine kinase

Two fused proteins of dimeric arginine kinase (AK) from sea cucumber and dimeric creatine kinase (CK) from rabbit muscle, named AK-CK and CK-AK, were obtained through the expression of fused AK and CK genes. Both AK-CK and CK-AK had about 50% AK activity and about 2-fold Km values for arginine of native AK, as well as about 50% CK activity and about 2-fold Km values for creatine of native CK. This indicated that both AK and CK moieties are fully active in the two fused proteins. The structures of AK, CK, AK-CK, and CK-AK were compared by collecting data of far-UV circular dichroism, intrinsic fluorescence, 1-anilinonaphthalene-8-sulfonate binding fluorescence, and size-exclusion chromatography. The results indicated that dimeric AK and CK differed in the maximum emission wavelength, the exposure extent of hydrophobic surfaces, and molecular size, though they have a close evolutionary relationship. The structure and thermodynamic stability of AK, CK, AK-CK, and CK-AK were compared by guanidine hydrochloride (GdnHCl) titration. Dimeric AK was more dependent on the cooperation of two subunits than CK according to the analysis of residual AK or CK activity with GdnHCl concentration increase. Additionally, AK and CK had different denaturation curves induced by GdnHCl, but almost the same thermodynamic stability. The two fused proteins, AK-CK and CK-AK, had similar secondary structure, tertiary structure, molecular size, structure, and thermodynamic stability, which indicated that the expression order of AK and CK genes might have little effect on the characteristics of the fused proteins and might further verify the close relationship of dimeric AK and CK.