Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving a2-adrenergic receptors. In order to facilitate purification of these inhibitory receptors we have prepared a highly effective biospecific affinity adsorbent. The immobilized ligand SKF 101253 is a 3-benzazepine with a2-adrenergic antagonist activity. SKF 101253 is coupled to Sepharose CL-4B by using a bifunctional reagent (1,4-butanediol diglycidyl ether) which also provides a hydrophilic spacer moiety between the ligand and the gel matrix. Membranes from human platelets, containing a12-ad- To understand fully the coupling of stimulatory and inhibitory receptors to adenylate cyclase it will be important to purify these receptors. Progress in this regard has been made for the adenylate cyclase-stimulatory f3-adrenergic receptor (2); however, purification of the adenylate cyclase-inhibitory a2-adrenergic receptor has not been achieved. A formidable problem in the purification of membrane-bound receptors is their miniscule quantity relative to the complete chemical content of the cell. An additional difficulty is the need for solubilization of the receptor from the membrane. Solubilization of membranebound proteins is usually incomplete, and it may lead to receptor inactivation which exacerbates the problem of the low initial concentration of receptor. Furthermore, due to the similarity of physiochemical properties among detergent-solubilized proteins, solubilization vitiates the use of many of the traditional methods for the purification of macromolecules.Affinity chromatography, a technique that exploits the specificity and affinity of ligand-receptor interactions, can over-

No abstract

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30 degrees C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg(2+), the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg(2+) concentration in excess of 5mm and increased linearly with increasing Mg(2+) concentrations up to 20mm. At a Mg(2+) concentration of 5mm, GTP and either a 40-70%-saturated-(NH(4))(2)SO(4) fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.

The characterization and identification of “unknowns” is an often-used laboratory activity in microbiology courses. As course enrollments become large, the logistics of providing unknown culture mixes, tubed and plated culture media, plus incubator and refrigerator space become quite daunting. A few years ago we discontinued the three-week Unknowns lab exercise when enrollment reached 240 or so students. In its place we have developed an hour long, midterm lab practical that incorporates smear preparation and the Gram stain, biochemical media, rapid tests, and a streak plate. This easy-to-grade laboratory assessment tool has taken the place of the more traditional station-to-station lab practical, and has worked well for the instructors responsible for administering practicals to 480 students in 13 lab sections over a two-day period

Flagellar preparations from several serotypes of Salmonella and one strain of Escherichia coli were tested for their ability to cross-repolymerize. Repolymerization was found not to be dependent on the antigenic determinants of the flagella.