Expression, purification, and reconstitution of receptor for pituitary adenylate cyclase-activating polypeptide. large-scale purification of a functionally active G protein-coupled receptor produced in Sf9 insect cells

Human pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was expressed in Sf9 insect cells and Chinese hamster ovary (CHO) cells. The recombinant receptor in Sf9 cell membranes had low affinity for 125I-PACAP27 (Kd = 155.3 pM) and was insensitive to guanosine 5'-O-3-thiotriphosphate (GTPgammaS), whereas the receptor in CHO membranes had a high affinity (Kd = 44.4 pM) and was GTPgammaS sensitive. The receptor in Sf9 membranes was converted to a high affinity state (Kd = 20-40 pM) following solubilization with digitonin. A large quantity (2 mg from 8 liters of insect cells) of the purified PACAP receptors (Bmax = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (Kd = 17.3 pM) using biotinylated ligand affinity chromatography. The apparent molecular weight of the purified receptor (Mr = 48,000) was smaller than that of the receptor from CHO cells (Mr = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (Kd = 182.6 pM) upon reconstitution into lipid vesicles, however, the receptor reconstituted with Gs protein had a high affinity (Kd = 40.2 pM) and was GTPgammaS sensitive. [35S]GTPgammaS binding to the reconstituted Gs protein was enhanced by PACAP27 and PACAP38 (EC50 = 42.5 and 9.4 pM, respectively) but not by antagonist PACAP(6-38), indicating that the purified receptor was functionally active.     

High resolution imaging and interpretation of regular and irregular structures in n-niobium pentoxide crystal

High resolution structure images of crystal wedges of n-Nb₂O₅ were obtained with the Kyoto 500 kV electron microscope. The two-dimensional arrangement of contrasty dots in the image taken at a very thin area of the fragment was found to coincide with the positions of Nb ions in the a-c projection. The variations in the image feature due to the thickness increase and defocusing showed a fairly close correlation with those of a multislice image simulation. As an application of HREM observation to the study of irregular structure, the sporadic appearance of cross-type images was detected and was interpreted in terms of the existence of interstitial ions. The reliability of intuitive interpretation of images was also discussed on the basis of multislice simulations.     

Molecular dynamics study on evaporation process of adherent molecules on surface by high temperature gas

Surface degreasing method with premixed flame is proposed as the removal method of adherent impurities on materials. Effects of adherent molecular thickness and surface potential energy on evaporation rate of adherent molecules and molecular evaporationmechanism were investigated and discussed in the present study. Evaporation processes of adherent molecules on surface molecules were simulated by the molecular dynamics method to understand thermal phenomena on evaporation processes of adherent molecules by using high temperature gas like burnt gas. The calculation system was composed of a high temperature gas region, an adherent molecular region and a surface molecular region. Both the thickness of adherent molecules and potential parameters affceted the evaporation rate of adherent molecules and evaporation mechanism in molecular scale.     

The 11 A resolution projection map of Na+/K+-ATPase calculated by application of single particle analysis to two-dimensional crystal images

Two-dimensional (2D) crystals of Na+/K+-ATPase were vitrified and observed with an electron cryo-microscope that allowed specimen observation at liquid helium temperature. Images showing 19 A spots on the optical diffraction patterns were digitized and processed by a crystallographic method. After optimization of parameters for the image processing, the locally averaged images revealed that each 2D crystal contained heterogeneous molecules. Single particle analysis was therefore applied to the 2D crystal images to select homogeneous molecule images, and averaging the selected images significantly improved map quality. Five major peak densities were clearly resolved in the new projection map, while two were found previously.     

Structural genomics of membrane proteins

A program on the structural genomics of membrane proteins has started at the BIRC, AIST, involving other academic institutions and industrial companies. Emphasis is being put on the development of techniques for the structural determination of membrane proteins of biological importance and ligand-receptor interactions by means of electron microscopy, X-ray diffraction, NMR, and computer simulation. Most efforts at the present stage, however, are being directed to finding suitable expression and purification systems and crystallization conditions for such proteins. The program is expected to be linked with the human full-length cDNA project and should lead to medical and industrial uses.     

The effect of cast iron graphites on friction and wear performance I: Graphite film formation on grey cast iron surfaces

Cast iron is assessed as a self-lubricating metal-base composite material. The formation of cast iron graphite films and the effective surface treatment of grey cast iron are discussed. It was found that the friction and wear behaviour of cast iron are influenced by the formation of a graphite surface layer. When cast iron surfaces are etched with Nital, they are found to be covered with a graphite film during the sliding friction process. This contributes to the protection of metallic contact points. The formation of graphite films during testing is assumed to be due to the non-elastic deformation of surface graphite. The effects of applied load, sliding velocity and lubricant are also discussed. The coefficient of friction increases with applied load and sliding velocity, and therefore lubrication becomes more important. These phenomena are discussed in terms of the formation of graphite films during the friction process.     

A study of cast alloys with partially dispersed graphite II: The process of partial dispersion with uncoated flake graphites

The mechanism of partial dispersion of uncoated flake graphite particles was investigated and compared with that of carbon microballoons (carbospheres) investigated earlier. Two types of flake graphite particles and the same base metals as previously used were investigated. The dispersive power of uncoated flake graphites was relatively small compared with that of spherical carbon balloons. For dispersion a temperature of 50 – 100 °C above the melting point with G = 1500 (where G is a non-dimensional gravity variable) is required with pure aluminium and its alloys. The variation in surface roughness of cast aluminium alloys containing flake graphites is due mainly to the magnitude of the centrifugal forces; the roughness decreases with increase in the acceleration (expressed in terms of G). The dispersive width of the graphitic layers and the clearance ratio are related; the width increases with ratio up to D/d = 2.5.