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Working with pig brain striatum in which A1 and A2 adenosine receptor subtypes coexist, we describe an uncomplicated method for unequivocally obtaining the equilibrium parameters (KD and binding capacity) of A1 receptor without interference from ligand binding to A2 receptor. Also, the equilibrium parameter estimation method we propose avoids the experimental determination of nonspecific binding by the inclusion of the corresponding unknown parameter in the function. This not only saves time but also avoids the use of expensive radioligands in saturation experiments. The method is suitable for any system with two different receptor subtypes for the same physiological ligand, and good estimates of the equilibrium parameters corresponding to the subtype displaying the higher affinity for the ligand can be obtained.

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2',3'-cyclic nucleotide 3'-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5'-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH:cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5'-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and 5'-nucleotidase (5'N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5'N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5'N activity maxima suggest that myelin may be a predominant locus of 5'N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5'N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5'N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (LUbrol WX) on 5'N activity were studied, and the results also suggest the intrinsic nature of 5'N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5'N activity, but not for the solubilization of this enzyme.