Cyclic-AMP receptors in the human spermatozoa membrane.

The binding properties of cyclic-AMP to human spermatozoa were studied. Incubation of whole human spermatozoa and of head and tail fractions with ¹⁴C-cyclic AMP induced the binding of 7.8^± 0.86 pmoles of the nucleotide per 5 × 10⁷ sperm cells showing an intrinsic association constant of 15 × 10⁻⁸ M. No significant inhibition of cyclic-AMP binding was caused by the addition of one hundred fold excess of AMP. However, when the excess AMP reached a thousand fold a slight but significant reduction (10%) was observed. These values were not modified by using sperm homogenates instead of intact sperm cells, which suggested that cyclic-AMP binding is to surface membranes and not to protein released from broken or damaged sperm. Binding was found to be unrelated with pH, between 6 to 8, but depended on the temperature of the incubation medium, showing a maximum at 20°C. The blocking of membrane sulfhydryl groups significantly inhibited (48%) cyclic-AMP binding to sperm cells.     

Participation of a membrane system in the potassium uptake rhythm in a duckweed, Lemna gibba G3

The period of the free-running potassium uptake rhythm in along-day duckweed, Lemna gibba G3, was lengthened by Li⁺ andshortened by ethanol. This suggests that a membrane system participatesin the circadian feed-back system. High temperature treatment(39°C) increased the motion of the rhythm when applied duringperiods of high potassium uptake (CT 18–06), and decreasedit when applied during low potassium uptake (CT 06–18),which indicates a circadian change in the properties of themembrane. However, modification of the potassium uptake level caused bytemperature change, application of valinomycin, or a changein the ambient potassium concentration caused no modificationin the phase of the rhythm. Thus, the oscillator involved probablyis not located in the cell membrane or the ectoplasm. ¹National Institute for Basic Biology, Myodaiji, Okazaki 444,Japan. ²Aichi-Gakuin University, Chikusa, Nagoya 464, Japan. (Received October 24, 1979; )     

Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane.

We analyzed the properties of a G protein-coupled receptor localized in cholesterol-poor vs. cholesterol-rich microdomains of the plasma membrane. For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK293 cells. To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor. Only 1% of the oxytocin receptors were present in cholesterol-rich detergent-insoluble domains. In contrast, employing a detergent-free fractionation scheme that preserves the functional activity of the receptor, we detected 10-15% of the receptors in cholesterol-rich low-density membranes and therein the high-affinity state receptors were twofold enriched. In cholesterol-poor vs. cholesterol-rich domains, high-affinity oxytocin receptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high-affinity oxytocin receptors localized in cholesterol-rich low-density membranes showed a markedly enhanced (t (1/2) approximately threefold) stability at 37 degrees C as compared with the oxytocin receptors localized in the cholesterol-poor high-density membranes. Addition of cholesterol to the high-density membranes fully protected the oxytocin receptors against loss of function. The importance of cholesterol to stabilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin receptors solubilized with Chapso. In conclusion, the data of this report suggest that functional properties of heptahelical receptor proteins could differ in dependence of their localization in different membrane microdomains.     

Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane.

The rapid amplification of beta-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein alpha- and betagamma-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of beta-adrenergic receptors with caveolin under resting conditions. beta1- and beta2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. beta1- and beta2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gsalpha- and betagamma-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of beta-adrenergic receptors with caveolin, indicating a nonrandom distribution of beta-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, beta-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize beta-adrenergic receptors.     

Specificity of siderophore receptors in membrane vesicles of Bacillus megaterium.

Membrane vesicles of Bacillus megaterium strains SK11 and Ard1 bound the ferrischizokinen and ferriferrioxamine B siderhores (iron transport cofactors). An approximately equimolar uptake of both labels of [3H, 59Fe]ferrischizokinen indicated binding of the intact chelate. Binding reached equilibrium in 2 to 5 min, was temperature independent, and was unaltered by the addition of several energy sources. A 91% dissociation of bound [Fe]ferrischizokinen was achieved in 60 s by the addition of excess ferrischizokinen. Ferriaerobactin, a siderophore which is structurally related to ferrischizokinen, caused no detectable release of bound [59Fe]ferrischizokinen. Of several other ferrigydroxamates tested, only ferriferrichrome A achieved the release (11%) of [Fe]ferrischizokinen. Rapid dissociation (92%) of bound [59Fe]ferriferrioxamine B by the addition of ferriferrioxamine B was observed, and a 67% release of [59Fe]ferriferrioxamine B was caused by ferriA2265, its structural relative. Ferrischizokinen, ferriferrichrome A, and ferrirhodotorulic acid produced a 6, 25, and 29% dissociation, respectively, of [59Fe]ferriferrioxamine B; ferriaerobactin caused no dissociation. [59Fe]ferriaerobactin was bound by the membranes, but its dissociation was not effected by unlabeled ferriaerobactin, suggesting no specific receptors for this chelate. The respective binding affinity constants and maximal binding capacities of membrane vesicles of strain SK11 were 2 x 10(7) M-1 and 280 pmol per mg of protein for ferrischizokinen and 7 x 10(7) M-1 and 37 pmol per mg of protein for ferriferrioxamine B. These values in strain Ard1 were, respectively, 1.4 x 10(7) M-1 and 186 pmol per mg of protein for ferrischizokinen and 11 x 10(7) M-1 and 23 pmol per mg of protein for ferriferrioxamine B. Separate, specific binding sites (receptors) for ferrischizokinen and ferriferrioxamine B exist on the vesicles. The ferrischizokinen receptors have a lower affinity but a higher binding capacity (eightfold) than that shown by the ferriferrioxamine B receptor. These receptors may be components of independent transport systems.     

Phage receptors of Escherichia coli. Basic components of the outer membrane of E. coli as phage receptors

Major outer membrane components which determine the structure and the barrier function of membrane Gram-negative bacteria are receptors for many bacteriophages. LPS--the major component of the outer membrane of Enterobacteria can be used by some phages with wide host range specificity. The other component of the outer membrane frequently include phage receptor component is OmpA protein. OmpA protein different areas can be used as receptors for different phages T--even group. A large group of phage receptors compose porin proteins, which are discovered in 32 species of bacteria. The synthesis of major porin proteins, which a receptor for several phages, are regulated by sufficiently complex system of some genes. These genes are sensitive to the changes of environment.     

High-frequency oscillations and circadian rhythm of the membrane potential in Spinach leaves

The microelectrode technique was used to follow oscillations in membrane potential in mesophyll cells of spinach (Spinacia oleracea L.) during exposure do different photoperiodic conditions. Both high-frequency oscillations and circadian variations were observed. The circadian rhythm was imposed on the period of high-frequency oscillation during short days as well as in continuous light: The free-running period was 25.2 h. The average period of high-frequency oscillation increased from 7.64 min in the dark to 19.95 min in the light within several minutes after dark to light transition. This period length coincides with the established period length for oscillations in the redox potential in the chloroplast suspensions of spinach.Abbreviations CL continuous light - SD short day - MP membrane potential     

Paticipation of cannabinoid receptors in the regulation of cardiac rhythm and cardiac contractility

It has been found that i. v. administration of cannabinoid receptor (CB) agonists (HU-210, ACPA, anandamide, methanandamide) induced a decrease in the heart rate (HR) in anesthetized rats. Pretreatment with CB1 receptor antagonist SR141716A completely abolished a negative chronotropic effect of CB receptor agonist HU-210. The CB2 receptor antagonist SRI 44528 did not prevent a HU-210-induced decrease in the HR. Pretreatment with the ganglion blocker hexamethonium had no effect on the negative chronotropic action of HU-210. Addition of HU-210 (100 nM) to perfusion solution induced a decrease in the HR, left ventricular development pressure, rate of contractility and relaxation of isolated perfused rate heart without change in end diastolic pressure. These data suggest that cardiac CBI receptor activation induces a decrease in the HR both in vivo and in vitro. An occupancy of the same receptors mediates a negative inotropic effects of cannabinoids.