Inactivation of simian immunodeficiency virus by hydrostatic pressure.

The inactivation of the simian immunodeficiency viruses SIVmac251 and SIVagm by pressures of 150 and 250 MPa was determined. The extent of inactivation depended on the time that the virus was subjected to compression as well as the level of the pressure and at 150 Mpa reached 5 log10 dilution units after approximately 10 hr. The inactivations, which were uniformly carried out at room temperature, were independent of the concentration of the virus. Possible applications of pressure inactivation for molecular biological and clinical use are discussed.

Heterogeneity in molecular recognition by restriction endonucleases: osmotic and hydrostatic pressure effects on BamHI, Pvu II, and EcoRV specificity.

The cleavage specificity of the Pvu II and BamHI restriction endonucleases is found to be dramatically reduced at elevated osmotic pressure. Relaxation in specificity of these otherwise highly accurate and specific enzymes, previously termed "star activity," is uniquely correlated with osmotic pressure between 0 and 100 atmospheres. No other colligative solvent property exhibits a uniform correlation with star activity for all of the compounds tested. Application of hydrostatic pressure counteracts the effects of osmotic pressure and restores the natural selectivity of the enzymes for their canonical recognition sequences. These results indicate that water solvation plays an important role in the site-specific recognition of DNA by many restriction enzymes. Osmotic pressure did not induce an analogous effect on the specificity of the EcoRV endonuclease, implying that selective hydration effects do not participate in DNA recognition in this system. Hydrostatic pressure was found to have little effect on the star activity induced by changes in ionic strength, pH, or divalent cation, suggesting that distinct mechanisms may exist for these observed alterations in specificity. Recent evidence has indicated that BamHI and EcoRI share similar structural motifs, while Pvu II and EcoRV belong to a different structural family. Evidently, the use of hydration water to assist in site-specific recognition is a motif neither limited to nor defined by structural families.

Streaming potential measurements in αβγ-rat epithelial Na+ channel in planar lipid bilayers

Streaming potentials across cloned epithelial Na+ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (ΔP) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by ΔP when the Δπ was directed against ΔP. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na+] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na+ ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by ΔP can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity.

High pressure fosters protein refolding from aggregates at high concentrations

High hydrostatic pressures (1–2 kbar), combined with low, nondenaturing concentrations of guanidine hydrochloride (GdmHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone and lysozyme, and β-lactamase inclusion bodies. One hundred percent recovery of properly folded protein can be obtained by applying pressures of 2 kbar to suspensions containing aggregates of recombinant human growth hormone (up to 8.7 mg/ml) and 0.75 M GdmHCl. Covalently crosslinked, insoluble aggregates of lysozyme could be refolded to native, functional protein at a 70% yield, independent of protein concentration up to 2 mg/ml. Inclusion bodies containing β-lactamase could be refolded at high yields of active protein, even without added GdmHCl.