Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe.

Apolipoprotein A-I (apoA-I) has important physiologic roles in reverse cholesterol transport, as a component of HDL; however, apoA-I also exists in lipid-poor or lipid-free forms that are key intermediates in HDL metabolism and acceptors of lipids from cells. The aim of this study was to examine the structure and stability of the central and C-terminal regions of lipid-free apoA-I. To this end, five Cys mutants of proapoA-I were constructed and expressed in Escherichia coli: V119C, A124C, A154C, A190C, and A232C. These mutants were specifically labeled with 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AC) and were examined by CD spectroscopy and a variety of fluorescence methods. The results showed that the introduction of Cys residues and their covalent labeling with AC did not affect the overall structure and stability of apoA-I. However, AC fluorescence properties revealed that different segments of the central and C-terminal half of apoA-I have distinct folding and stability properties. From fluorescence energy transfer data, average distances between the N-terminal region containing Trp residues and the various AC locations were obtained. The current results, together with previously published observations, led to the construction of a three-dimensional model for the folding of lipid-free apoA-I.

A Vaccine to Coxsackievirus Prepared by High Pressure.

Effects of high hydrostatic pressure on infectious and immune activity of coxsackievirus group B (CVB) was described. CVB was completely inactivated at 230 MPa pressure and -16 degrees. The inactivated viruses remained highly immunogenic and induced CVB-specific serum antibody at a concentration as low as 1:1 500. 67% mice vaccinated with the pressurized viruses survived the challenge of fatal dose of CVB. These results indicated that the pressurized CVB induced immune reactions to protect mice from the viruses, and would be a potential vaccine candidate for human myocarditis.

Comparison of the Catalytic Domains of Collagenase-1 and Stromelysin-1.

The catalytic domains of two matrix metalloproteinases--collagenase-1 and stromelysin-1 have been studied by means of fluorescence spectroscopy and high hydrostatic pressure. The hydrophobic fluorescence probe ANS could bind to stromelysin-1, with a dissociation constant of 26.3 &mgr;mol/L, but could not bind to collagenase-1, indicating that there exists a hydrophobic site on the surface of stromelysin-1. Further study suggests that the hydrophobic site may not be the catalytic site. The biological activity of catalytic domains of collagenase-1 and stromelysin-1 showed obvious difference under high pressure the activity of collagenase-1 increased with elevating pressure, with an activation volume of D18.9 ml/mol however, the activity of stromelysin-1 did not change under high pressure. The results indicate that there are some obvious differences between the catalytic domain conformations of these two enzymes, though the crystal analysis indicated that they were very similar as reported before.

High Immunogenicity of the Pressure-inactivated Virus.

Effects of high hydrostatic pressure on the activity of infectious bursal disease virus (IBDV) was described. The infectivity of IBDV decreased with the increasing of pressure and pressurizing time and may be completely lost, indicated by the tests with chicken embryo fibrablast cell and young chicken as models. How-ever, pressure-inactivated IBDV still remained high immunogenicity and might induce high levels of protecting serum antibody. Fluorescence spectroscopy showed obvious differences between the structures of native and pressurized IBDV, similar with the dissociation-reassociation of oligomeric proteins under high pressure. The changes of pressurized IBDV were also directly observed with electron microscopy. These results suggest that pressure-inactivated IBDV may be used as a vaccine.

The Influence of NBS Modification of 241Trp on the Reconstitution of 33 kD Protein with PS and on the Recovery of Oxygen-evolving Activity.

The extrinsic 33 kD protein of photosystemII(PSII) plays an important role in the stabilizing of manganese cluster and maintaining high oxygen-evolving activity of PSII. In this research, (241)Trp, the only tryptophan in the 33 kD protein, was modified by N-Bromosuccinimide. The pH-dependence of modification suggests that this tryptophan is buried in the hydrophobic interior of the protein. The protein's capability of reconstitution to the PSII ecreased after modification, and no oxygen-evolving activity of PS was recovered after the reconstitution. Results suggest that (241)Trp of the 33 kD protein is essential for the binding of the protein to the PS and the normal oxygen-evolving activity.

Effect of High Hydrostatic Pressure on Activity of Restriction Endonucleases.

The effect of high hydrostatic pressure on the activities of type II restriction enzymes HindIII and XbaI in digesting plasmid pSPORT1 was studied. The endonuclease activity of HindIII and XbaI at 37 degrees were gradually inhibited by increasing pressure and completely inhibited at 200 and 180 MPa, respectively. No obvious irreversible effect was observed for HindIII after suffering high pressure, while a considerable irreversible inactivation was observed for XbaI. The standard molar volume changes for HindIII and XbaI estimated from the inhibition of endonuclease activity at different pressures were 213 and 103 ml/mol, respectively. It was also concluded that pressurization did not change the substrate sequence specificity of both HindIII and XbaI.