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Binding sites for the dipeptide L-carnosine (beta-alanyl-L-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[3H]-carnosine was saturable, reversible and stereospecific and had a Kd of about 770 nM. The stereospecific binding of L-carnosine represented about 30% of the total binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibited by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interferred with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. Binding sites for 6 other radiolabeled receptor ligands were also detected in bulb membranes. Peripheral deafferentation of the olfactory bulbs by intranasal irrigation with ZnSO4 led to a loss greater than 90% of the L-[3H]carnosine binding in 4--5 days with much smaller losses in binding of the other 6 ligands over a 180-day observation period. This initial loss of carnosine binding after denervation was due to a loss of binding site stereo-specificity followed by a loss of binding sites. The characteristics of the carnosine binding site in olfactory bulb fulfil 6 of the 7 criteria considered relevant for a functional receptor.
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Pig NADPH-adrenodoxin reductase was crystallized from pig adrenocortical mitochondria and its physicochemical properties were investigated. Pig NADPH-adrenodoxin reductase is a typical flavoprotein. Its optical absorption spectrum showed peaks at 272, 377, and 450 nm in the oxidized form. The adrenodoxin reductase contained one FAD per mol. The molecular weight was 49,000. The isoelectric points of the adrenodoxin reductase and its complex with adrenodoxin were 5.3 and 4.6, respectively. Pig NADPH-adrenodoxin reductase, unlike bovine NADPH-adrenodoxin reductase, was found to be free of carbohydrate. The fluorescences of tryptophanyl residues and FAD of the adrenodoxin reductase were quenched by holo- and apo-adrenodoxins. The NADPH-binding site of the adrenodoxin reductase was examined by photooxidation and selective chemical modifications with diethyl pyrocarbonate and sulfhydryl reagents. The results indicate that a histidyl and a cysteinyl residue of the adrenodoxin reductase are essential for the NADPH-binding site. The circular dichroism spectrum of the adrenodoxin reductase showed negative ellipticity in the visible region. Spur formation was observed between pig and bovine NADPH-adrenodoxin reductases against the antibody to bovine NADPH-adrenodoxin reductase in Ouchterlony double-diffusion agar plates. The antibody did not interact with spinach ferredoxin-NADP+ reductase.
X-ray crystallagraphic studies studies indicate that there are at least four site-specifically bound hydrated Mg2+ ions, [Mg(H2O)n]2+, in yeast tRNAPhe. The size and the octahedral coordination geometry, rather than the charge, of [Mg(H2O)N]2+ appear to be the primary reasons for the specificity of magnesium ions in site-binding and in the stabilization of the tertiary structure of tRNA.
The presence of sites specifically binding natural glucocortocoids in plasma membrane (PM) preparations (PM0, density=1.13-1.16; PM1, density=1.16-1.18) from rat liver was elucidated by equilibrium dialysis as well as by centrifugal methods. Equilibrium dialysis showed the presence of binding sites having a higher affinity for [3-H]cortisol (Kd=1.4 times 10- minus 9M at 4C) in PM0, and that of the binding sites having a lower affinity for [3H] cortisol (Kd=1.3 times 10- minus 8M at 4C) in PM1, while centrifugal analysis showed the presence of higher affinity binding sites (Kd=1.5-1.9 times 10- minus 97 at 0 C) in both PM0 and PM1, and also of intermediate affinity binding sites (Kd=4.1 times 10- minus 9M at 0 C) in PM1. The discrepancy in the cortisol binding parameters obtained by the two different methods seems to be due mainly to the lability of some binding sites, especially the PM1. The glucocorticoid-binding sites in the plasma membranes of rat liver appear to have the highest affinity of corticosterone, followed by cortisol and cortisone. A synthetic glucocorticoid [3H]-dexamethasone, did not show any specific binding to the liver plasma membranes. Neither dexamethasone nor nonglucocorticoids such as estradiol given simultaneously affected [3H] cortisol binding to the plasma membranes.
The nature of the intermediate-affinity (n2) Mn(II) binding sites in glutamine synthetase [EC 6.3.1.2] has been studied as a function of adenylylation in a variety of enzyme-metal complexes by EPR. In the absence of nucleotide the n2 Mn(II) environment is nearly isotropic, the Mn(II) bonds are highly ionic, and the interaction distance R greater than or equal to 12-14 A. Nucleotide binding at the n2 Mn(II) site renders the n2 Mn(II) signal unobservable and causes a reduction in signal amplitude (approximately 30%) and line broadening (approximately 6 G) at the high-affinity (n1) Mn(II) site. This behavior indicates that nucleotide binding induces a conformational change in the enzyme which brings the previously distant n1 and n2 sites into closer proximity (R less than or equal to 8-11 A), possibly for the purpose of activating the nucleotide for direct phosphoryl transfer to L-glutamate. In line with this suggestion, the broad, unresolved resonances in complexes containing both L-methionine SR-sulfoximine (MSOX) and nucleotide may result from the phosphorylation of MSOX. The n2 Mn(II) site is not affected by adenylylation in all the enzyme-metal complexes studied, which suggests that the regulatory effects of adenylylation may only act at the n1 Mn(II) sites.
In ligand binding studies, it is often difficult to apply kinetic analyses because of an uncertainty in experimental data obtained at high ligand concentrations. Under such circumstances, Kd value (an index of the affinity) and the binding site concentration may be estimated more accurately from the binding of a fixed concentration of labelled ligand observed in the presence of various concentrations of the non-labelled ligand, if the fraction of both labelled and non-labelled ligand bound is small. When there is no cooperative effect of the ligand binding, the Kd value may be calculated by subtracting the concentration of the labelled drug from the concentration of the non-labelled drug to cause a 50% reduction of the saturable binding of the labelled drug. From above values, the binding site concentration may be calculated. The proposed method is capable of examining the cooperativity of the ligand binding, the labelled drug concentration and the specific radioactivity of the labelled drug and does not require large amounts of the labelled drug.
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1. Plasma membranes isolated from rat livers and ascites hepatoma cells (AH-130, AH-7974) were assayed for specific Ca2+ binding sites using 45Ca2+ and a Millipore filtration technique. The presence of higher (Kd = 1.4--1.5 . 10(-5) M) and lower (Kd = 0.9--1.0 . 10(-4) M) affinity sites in both liver and hepatoma membranes was observed. The hepatoma plasma membranes however, showed 1.4--2.1-fold as many Ca2+ binding sites (higher and lower affinity sites) as the liver plasma membranes on the basis of protein. 2. Concanavalin A stimulated the specific Ca2+ binding to liver and hepatoma plasma membranes, showing a maximal stimulation (3--5-fold) at 100 microgram/ml. Succinyl concanavalin A was less effective, whereas wheat germ agglutinin and ricinus lectin were ineffective. 3. Concanavalin A stimulated the Ca2+ uptake by AH-7974 cells. The concanavalin A-mediated stimulation of Ca2+ uptake showed lectin-concentrations and Ca2+-concentration dependencies similar to those in the concanavalin A-mediated stimulation of Ca2+ binding.
The presence of insulin receptors in the heart muscle was investigated by measuring the binding of 125I-insulin to specific subcellular fractions of the rat and mouse myocardium. 125I-insulin bound to the plasma membrane fraction with a high degree of specificity and affinity. Insulin analogues competed with 125I-insulin in direct proportion to their biologic potency in vitro. Unlabeled insulin within the range of its concentrations in vivo inhibited 15 to 60 per cent of the 125I-insulin binding. The specific binding sites were finite in number and represented about 90 per cent of the total binding. The insulin-binding capacity of the plasma membrane fraction was twelve- to fifteenfold higher than that of the mitochondrial fraction. As in the liver, the binding was time- and temperature-dependent with a slower but higher binding achieved at a lower temperature. The binding sites appeared to be heterogeneous with respect to affinity. At 5 degrees C., the "higher-affinity" site had a K of about 2 times 10(9) M-1. No more than 10 per cent of the 125I-insulin was degraded by the heart plasma membranes after one hour at 30 degrees C. or twenty-two hours at 5 degrees C. Studies in the obese hyperglycemic (ob/ob) mouse revealed that the insulin binding is impaired in the heart muscle of this animal. Over a wide range of insulin concentrations, the plasma membrane fraction of ob/ob mice bound only 25 to 40 per cent as much insulin as did membranes of the thin littermates, suggesting that, as in the liver, the fat tissue, and the thymic lymphocyte, the number of insulin-binding sites is decreased in the heart of the ob/ob mouse. This defect selectively affected the plasma membrane fraction and could not be explained by differences in membrane purification or insulin-degrading activity. Heart and liver membranes of forty-hour fasted ob/ob mice bound two to three times as much insulin as did membranes of ob/ob mice fed ad libitum. These studies demonstrate and characterize the binding of insulin to heart muscle membranes; they extend to the heart muscle the insulin receptor defect also found in liver membranes and cells, in fat cell membranes, and in thymic lymphocytes of the ob/ob mouse.
Highly purified subsynaptic membrane fragments prepared from Torpedo marmorata electric organ (specific activity, greater than 4 mumol of Naja nigricollis alpha-[3H]toxin per mg of protein) exhibit, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, two major protein bands of apparent molecular weight 40,000 and 43,000, respectively. Dissolution of these membranes by the nondenaturing detergents Triton X-100 and Berol 043 followed by standard fractionation yielded (i) the 9S acetylcholine-receptor protein which still binds the alpha-[3H]toxin and after further purification yielded, in the presence of sodium dodecyl sulfate, the 40,000-dalton component, covalently labeled by the affinity reagent 4-(N-maleimido)phenyl[3H]trimethylammonium; only serine was found as the NH2-terminal amino acid of this protein; and (ii) a high molecular weight aggregate named 43,000 protein which was resolved in denaturing gels almost exclusively as the 43,000-dalton band, In the absence of detergents, the 43,000 protein binds compounds known to interact with the acetylcholine ionophore: a fluorescent local anesthetic quinacrine and histrionicotoxin (apparent dissociation constant, 7 +/- 1 X 10(-7) M). The regulation of quinacrine fluorescennce by carbamylcholine, observed in the intact membrane, no longer occurs with the isolated 43,000 component.
The binding of oestradiol to a nuclear fraction extracted from human breast carcinomatous tissue was demonstrated. The material, which was extracted with KCl, sedimented at 3--4S and bound oestradiol with high affinity (dissociation constant approximately 2 X 10(-10) mol/l). Oestriol, diethylstilboestrol and 5 alpha-dihydrotestosterone (100-fold excesses) competed with [3H]oestradiol for the binding sites (binding inhibited by 89 +/- 8 (S.D.), 92 +/- 6 and 57 +/- 8% respectively), whereas progesterone and cortisol (100-fold excesses) did not (binding suppressed by 5 +/- 5 and 2 +/- 3% respectively). Similar competition patterns were found for cytoplasmic material which bound oestradiol. The binding occurred at 4 degrees C and was therefore considered to be a measure of the amount of binding material unoccupied by endogenous oestrogen, Unoccupied binding sites for oestradiol in the nucleus and cytoplasm were measured in 35 samples of breast carcinomatous tissue using sucrose gradient centrifugation. In 17 out of 35 tumorus, unoccupied nuclear and cytoplasmic 8S and 4S binding sites could be detected. Three out of 35 tumours contained unoccupied nuclear binding sites and 4S cytoplasmic binding sites. Nuclear binding sites only were found in two out of 35 tumours. Unoccupied nuclear binding sites were not detected in 13 out of 35 tumours and ten of these tumours also did not contain unoccupied cytoplasmic binding sites.
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