We prepared clathrin-coated vesicles from bovine forebrain utilizing sucrose or deuterium oxide-Ficoll density gradient centrifugation followed by permeation chromatography. Homogeneity was monitored by electron microscopy (EM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). EM revealed that the predominant (up to 98% of the total) organelles were coated vesicles and empty hexagonal baskets. Diameters of the coated vesicles ranged from 37 to 120 nm with a mean of 65.2 +/- 2.2. Upon SDS-PAGE of the coated vesicle fraction, the most prominent band appeared at 180,000 daltons. There were also three additional bands at 100,000, 50,000 and 35,000 daltons, giving the overall pattern characteristic of coated vesicles. Both 0.5 nM tritiated naltrexone and etorphine displayed specific binding to coated vesicles. Naltrexone binding in coated vesicles from gradient fractions was increased 2.5-fold over the original 100,000 X g pellet. An additional 4-fold enrichment in specific binding was observed after permeation chromatography which was concomitant with an increase in the volume density of coated vesicles in electron micrographs. Naltrexone binding was stereospecific and etorphine binding was inhibited by 100 mM NaCl (40%). Both naltrexone and etorphine binding were inhibited by 50 microM guanyl-5'-yl imidodiphosphate (40 to 50%). In summary, purified bovine brain-coated vesicles contained high affinity stereospecific opiate alkaloid-binding sites with characteristic opioid binding properties.

Ground-water monitoring regulations on the state and Federal level establish general performance standards for the construction of monitoring wells. Of the many drilling techniques available for the construction of monitoring and observation wells, dry techniques are recognized as superior at sites of contamination. The dry technique of modified reverse-circulation air rotary drilling (RARD) is viable and a more advantageous alternative than many traditional techniques. It requires no liquid drilling fluids or muds, and therefore no foreign fluids are added to the aquifer. Furthermore, composite soil and water samples are collected during installation specifically and exclusively from the bit depth and are uncontaminated by drilling fluids. The method utilizes compressed air to clear the borehole and a tube within a tube drill pipe system to maintain the borehole. This allows the well assembly, filter pack, and borehole sealant to be properly placed thereby satisfying the strictest regulatory requirements for short and long-term monitoring. The method accommodates various field testing such as the measurement of in situ hydraulic conductivity and the collection of soil cores. To insure proper construction of monitoring wells, procedures and material requirements must be designed and fully specified.