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3,3',5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.

Two orders of saturable binding sites for L-triiodothyronine were found on washed rat erythrocyte membranes. The high affinity, low capacity site showed values of Kd 0.19 X 10(-10) M. This value was in the range of concentration of free L-triiodothyronine found in the plasma and was several orders of magnitude higher than the Kd values previously reported for other L-triiodothyronine membrane-binding systems. The binding site also showed a high stereospecificity for L-triiodothyronine. D-3,5,3'-triiodothyronine and L-thyroxine were less potent (about 1000-fold) than L-triiodothyronine in competing for these sites. L-3,3,5'-triiodothyronine and triiodothyroacetic acid were inactive. The physiological relevance of this site is considered.

Electrophoretic patterns of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from rat erythrocyte were studied. The enzyme was solubilized by the following treatments: a) Triton X-100, b) sodium deoxycholate, or c) ultrasonic irradiation. When the erythrocyte membrane was solubilized by Triton X-100 at concentrations higher than 0.3%, by 10 mM sodium deoxycholate, or by ultrasonic irradiation for more than 5 min, a single band of acetylcholinesterase activity appeared in the gel. Two bands of activity were stained in the gel when the membrane was solubilized by Triton X-100 at concentrations between 0.1--0.2%, or by ultrasound for 5 min. Electrophoretic patterns of acetylcholinesterase from rats fed a fat-sufficient diet were similar to those for the enzyme from animals fed a fat-free diet. The recombination of lipids with the enzyme eluted from the gels confirmed the "phenotypic allosteric desensitization phenomenon".

The influence of cholesterol on the membrane-bound acetylcholinesterase and (Ca2+ + Mg2+)-ATPase was studied in erythrocytes of five groups of male rats fed different fat-supplemented diets. Two groups of rats were fed essential fatty acid (EFA) sufficient diets with 5% lard or corn oil as the dietary fat, and two groups were fed EFA-deficient diets: a basic, fat-free diet and the same diet supplemented with 5% hydrogenated beef fat. One additional group of rats was fed a stock diet. The kinetic changes recorded were in the degree of the cooperativity of the inhibition by F- of the acetylcholinesterase and the activation by Ca2+, and by Mg2+ of the (Ca2+ + Mg2+)-ATPase. The kinetic behavior of the enzymes was only modified by cholesterol feeding when they were bound to a membrane with a high fatty acid fluidity (e.g. derived from rats fed the corn oil-supplemented diet). The enzymes from a membrane with a low fatty acid fluidity (e.g. derived from rats fed a lard-supplemented diet) were not altered by cholesterol feeding. The changes were noticeable after 24 hours of cholesterol feeding. It is suggested that the in vivo cholesterol sites are involved in a regulatory mechanism for mammalian membrane-bound enzymes.