Photobleaching recovery studies of membrane events accompanying lectin stimulation of rabbit lymphocytes.

Fluorescence photobleaching recovery methods reveal marked changes in lateral mobilities of rabbit lymphocyte membrane components during the course of stimulation with succinyl concanavalin A (S Con A). The diffusion constant of S Con A receptors on T lymphocytes falls from 1.6×10^?10 cm²/sec to 6.5×10^?11 cm²/sec within 4 hr after stimulation, remains constant for 14 hr, and returns to its former value. The mobility of B cell receptors similarly falls from 1.4×10^?10 cm²/sec to 5.5×10^?11 cm²/sec but regains its unstimulated value much more slowly. In contrast, a fluorescent phospholipid analog shows constant mobilities of 1.9×10^?8 cm²/sec and 1.5×10^?8 cm²/sec in T and B cells, respectively, throughout the experiment.     

Headgroup behaviour of an uncharged complex glycolipid

A globoside spin labelled on the terminal sugar residue has been synthesized, and employed in model membranes to study headgroup behaviour of complex uncharged glycolipids. The labelled headgroup demonstrated a high degree of motional freedom limited to the aqueous region of the interface between lipid bilayer and surrounding medium. This observation was unaltered by the presence of a dense, tightly-bound surface layer of peripheral proteins or polysaccharide—which might be expected to reproduce conditions present at a cell surface. Headgroup dynamics were only very modestly correlated with the physical state (i.e., fluidity) of the membrane itself. In spite of the absence of charged sugar residues in globoside, the aspects of its headgroup behaviour monitored here we found to be similar to those of oligosaccharide chains on gangliosides and several sialic acid-rich glycoproteins.     

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity become extreme upon fertilization

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-(N-hexadecanoyl)-aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching indicating the existence of lipidic microdomains. In the unfertilized egg the mobile fraction of plasma membrane lipids (～50%) has a fivefold smaller lateral diffusion coefficient (D = 1.5 × 10^?8 cm²/sec) in the animal than in the vegetal plasma membrane (D = 7.6 × 10^?8 cm²/sec). This demonstrates the presence of an animal/vegetal polarity within the Xenopus egg plasma membrane. Upon fertilization this polarity is strongly (>100×) enhanced leading to the formation of two distinct macrodomains within the plasma membrane. At the animal side of the egg lipids are completely immobilized on the time scale of FPR measurements (D ? 10^?10 cm²/sec), whereas at the vegetal side D is only slightly reduced (D = 4.4 × 10^?8 cm²/sec). The immobilization of animal plasma membrane lipids, which could play a role in the polyspermy block, probably arises by the fusion of cortical granules which are more numerous here. The transition between the animal and the vegetal domain is sharp and coincides with the boundary between the presumptive ecto- and endoderm. The role of regional differences in the plasma membrane is discussed in relation to cell diversification in early development.     

The secretory response in dissociated acini from the rat submandibular gland

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to β-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of ¹⁴C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 × 10^?5 to 5 × 10^?7 M, and to 110% of controls at 5 × 10^?8 M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 × 10^?5 M and to 115% at 5 × 10^?6 M but had no effect at 5 × 10^?7 or 5 × 10^?8 M. The secretory response was blocked by the respective β-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.     

Headgroup behaviour of an uncharged complex glycolipid

A globoside spin labelled on the terminal sugar residue has been synthesized, and employed in model membranes to study headgroup behaviour of complex uncharged glycolipids. The labelled headgroup demonstrated a high degree of motional freedom limited to the aqueous region of the interface between lipid bilayer and surrounding medium. This observation was unaltered by the presence of a dense, tightly-bound surface layer of peripheral proteins or polysaccharide--which might be expected to reproduce conditions present at a cell surface. Headgroup dynamics were only very modestly correlated with the physical state (i.e., fluidity) of the membrane itself. In spite of the absence of charged sugar residues in globoside, the aspects of its headgroup behaviour monitored here we found to be similar to those of oligosaccharide chains on gangliosides and several sialic acid-rich glycoproteins.     

Low- and high-affinity concanavalin A binding to thymocyte plasma membrane vesicles

Binding of concanavalin A to isolated thymocyte membrane vesicles occurs through (a) numerous (～6 × 10⁶/cell equivalent) low-affinity sites (K_a = 1.3 × 10⁵ M^?1) and (b) fewer (～0.4 × 10⁶/cell equipment) specific receptors (K_a = 6.8 × 10⁶ M^?1) defined as 55,000 D glycoprotein and its multimers. Specific binding is positively-cooperative, with a Hill coefficient of～1.8. Low concentrations of glutaraldehyde selectively crosslink the 55,000 D glycoprotein with replacement of positively-cooperative sites by high-affinity sites. It is proposed that concanavalin A-binding induces multimerization of the 55,000 D glycoprotein.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

Abnormality of 2-deoxyglucose uptake kinetics in fibroblasts at low concentrations

2-deoxyglucose uptake rates at low sugar concentrations (less than 500 μM) appeared to be lower than those predicted by the Michaelis-Menten model which correctly described higher concentrations. This phenomenon which we will call concentration-dependent transport lag, was also observed for L-glucose uptake which suggest that this phenomenon is carrier-independent. A model involving the perimembrane space is developed which, for L-glucose, gives k₁ = 0.931 ± 0.072 × 10^?6 l. mg protein^?1. minute^?1, k₂ = 2.97 ± 0.19 × 10^?7 l. mg protein^?1. minute^?1 and So = 88,8 ± 4,3 μM; where k₁ is the diffusion constant in the cell membrane, k₂ is the diffusion constant in the perimembrane space and S_o the sugar concentration required in the external medium in order to provide an équivalent sugar concentration in the transport carrier area.     

Effect of Melafen on functional efficiency of mitochondrial membranes from sugar beet taproots

Mitochondria were isolated from sugar beet (Beta vulgaris L) taproots and incubated in the presence of low concentrations of Melafen (2 × 10^?9 and 4 × 10^?12 M). This treatment of mitochondrial membranes induced an appreciable decrease in microviscosity of superficial lipids in the lipid bilayer and a parallel increase in microviscosity of the deeply immersed lipid regions adjacent to membrane proteins. Melafen had no effect on fluorescence of lipid peroxidation products in membranes of freshly prepared mitochondria but declined this fluorescence to control values in artificially aged mitochondria. Melafen raised the maximum rates for oxidation of NAD-dependent substrates, elevated the efficiency of oxidative phosphorylation, and activated electron transport in the terminal (cytochrome oxidase) step of mitochondrial respiratory chain, which implies the activation of energy metabolism within the cell. The acceleration of electron transport through the terminal step of mitochondrial respiratory chain was apparently accompanied by retardation of lipid peroxidation, which prevented impairment of mitochondrial membranes under stress conditions. A proposal is put forward that some properties of Melafen are favorable for adaptogenesis because its effects on mitochondrial energy metabolism depended on the functional state of mitochondria.     

Headgroup oligosaccharide dynamics of a transmembrane glycoprotein

Glycophorin, a major integral membrane glycoprotein of the human erythrocyte, has been spin labelled on oligosaccharide chains. Electron paramagnetic resonance studies of this glycoprotein in systems of controlled complexity have provided a degree of insight into its headgroup behaviour. (i) When glycophorin is free in solution its oligosaccharide chains exhibit uniformly high freedom of motion. This motional freedom is not attributable to the presence of N-acetyl-neuraminic acid residues. (ii) No evidence has been found of a finite tendency for headgroup sugars to associate with hydrophobic regions of phospholipid or glycoprotein. (iii) Headgroup oligosaccharide dynamics are essentially independent of the state of and interactions of the polypeptide hydrophobic portion (that portion which traverses the membrane). (iv) Nonspecific interaction with proteins and polysaccharides can readily reduce oligosaccharide chain mobility by some 25%, but does not alter their basic behaviour. (v) Binding of wheat germ agglutinin, dramatically immobilizes (terminal) N-acetylneuraminic acid residues. (vi) The above observations hold over the temperature range 0-40 degrees C. (vii) Headgroup carbohydrate mobility is at a minimum in the region of headgroup neutrality (pH 2.6-3.5) and is pH invariant over several pH units in the physiological range.     

Inhibition of thyrotropin binding to human thyroid plasma membranes by thyroid hormones and "reverse triiodothyronine".

Triiodothyronine, reverse triiodothyronine and thyroxine were found to inhibit ¹²⁵I labelled thyrotropin binding to human thyroid plasma membranes in vitro. Both the thyrotropin binding and the effect of the above iodoamino-acids on this binding were pH, temperature and time dependent, 50% inhibition of thyrotropin binding was observed at 2×10^?7M concentration of reverse triiodothyronine or thyroxine and at 1.1 × 10^?6M concentration of triiodothyronine. The kinetic studies of thyrotropin binding revealed that the maximal capacity of receptor sites for the pituitary hormone is unaffected by the presence of thyroid hormones. On the other hand the association and dissociation constants for thyrotropin binding changed when iodoaminoacids were present in the incubation medium /Ka 8.13 × 10⁷M^?1 vs 1.6 × 10⁸M^?1 and Kd 1.14 × 10^?8M vs 4.55 × 10^?9M respectively, depending on the pH/. The double reciprocal plots showed competitive mechanism of inhibition. The present study suggest that triiodothyronine, reverse triiodothyronine and thyroxine are able to modify the thyrotropin binding to membrane receptors.     

Steroid-protein interactions. Fluorescence quenching of progesterone-binding globulin and alpha1-acid glycoprotein upon binding of steroids.

The influence of progesterone and four other steroids on the intrinsic fluorescence of progesterone-binding globulin was investigated. The corresponding effect of progesterone on α₁-acid glycoprotein was also studied. The intrinsic fluorescence of the progesterone-binding globulin and of α₁-acid glycoprotein was quenched by about 60 and 17%, respectively, upon forming stoichiometric complexes with progesterone. Graphical analysis of fluorescence quenching titrations with progesterone gave affinity constants at 23 °C of 2 × 10⁹m^?1 for progesterone-binding globulin and 1 × 10⁶m^?1 for α₁-acid glycoprotein. With progesterone-binding globulin, affinity constants of 1 × 10⁹m^?1 were determined for desoxycorticosterone, 1 × 10⁸m^?1 for testosterone, and 2 × 10⁶m^?1 for cortisol. The fluorescence quenching of PBG by 5-pregnen-3β-ol-20-one, 5α-pregnanedione, and 5β-pregnanedione, steroids lacking the Δ⁴-3-keto grouping, was too small to be evaluated; however, binding of the pregnanediones to progesterone-binding globulins was demonstrated when the progesterone-progesterone-binding globulin complex was “unquenched” as a result of competitive displacement of progesterone by addition of the pregnanediones. The quenching phenomenon is assumed to be mainly due to radiationless transfer from protein to the near uv (n → π1) absorption band of steroids containing the Δ⁴-3-keto chromophore.     

A pulsed N.M.R study of D2O bound to 1,2 dipalmitoyl phosphatidylcholine

Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (²H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D₂O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from ?20°C to 65°C.The proton (¹H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10^?10 sec and ≈ 10^?6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D₂O molecule, hydrogen bonded to the negatively charged phosphate headgroup.     

Differences in the response of several cell types to inhibition of surface receptor mobility by local concanavalina binding

Concanavalin A (conA) modulates the lateral mobility of cell surface receptors differently on different cell types. This was demonstrated by using fluorescence photobleaching recovery (FPR) to measure the inhibition of the lateral mobility of conA receptors by localized binding of conA on lymphocytes, fibroblasts, and macrophages. On mouse spleen lymphocytes, binding of conA platelets above a threshold coverage (about 12% of the upper cell-surface area) reduced the diffusion coefficient of mobile TMR-SconA-receptor complexes from 3.0×10^?10 cm²/sec to 0.6× 10^?10 cm²/sec (a 5-fold decrease), and the fraction of mobile receptors was concomitantly reduced from 0.4 to 0.11. Below the threshold occupancy, no effect on either parameter was detected. On 3T3 cells, a qualitatively similar threshold phenomenon was observed: coverage of over 9% of the upper cell surface by conA platelets induced a 3-fold reduction in the diffusion coefficient of TMR-SconA-receptor complexes from 5×10^?10 cm²/sec to 1.7× 10^?10 cm²/sec. However, no effect on the mobile fraction (about 0.4) was observed. In contrast, neither the diffusion coefficient nor the mobile fraction of TMR-SconA-receptor complexes on mouse peritoneal macrophages (both resident and thioglycolate-stimulated) or on the mouse macrophage cell line P388D₁ were affected by the binding of conA platelets in amounts covering over 50% of the upper cell surface (approx. 4.6× 10^?10 cm²/sec and 0.5 for the diffusion coefficient and mobile fraction, respectively). These differences are correlated to the different cytoskeletal functions of the various cell types studied, and are discussed regarding the mechanism of the conA-induced modulation.     

Membrane receptors for Vicia graminea anti-N lectin and its binding to native and neuraminidase-treated human erythrocytes

Membrane receptors for Vicia graminea (Vg) lectin on human red cells were analyzed using deoxycholate lysates obtained from ¹²⁵I-erythrocyte membranes incubated with a purified lectin immobilized on Sepharose 4B. The glycoproteins (GP) specifically bound to the gel were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Using native erythrocytes the results obtained demonstrate that N red cells have exposed Vg receptors located on GPα (synonym glycophorin A) and GPδ (synonym glycophorin B) whereas on M erythrocytes the Vg receptors are restricted to GPδ. The presence of Vg receptors was also found on the hybrid glycoprotein (made of the N-ter of GPδ and C-ter of GPα) carried by St(a+) erythrocytes. A similar amount of radioactivity was bound to Vg-Sepharose incubated with neuraminidase-treated N or M membranes. The material eluted was tentatively identified as asialo GPα and asialo GPδ, suggesting that numerous receptors have been uncovered mainly on asialo GPα species from M erythrocytes. No glycoprotein component could be identified from the material eluted from Vg Sepharose incubated with native or neuraminidase-treated membrane from a Tn(+) individual. Scatchard plot analysis obtained from binding experiments at equilibrium with M, N, and St(a+) cells revealed the existence of at least two classes of receptors both on native and neuraminidase-treated erythrocytes. Desialylation of the M, N, and St(a+) erythrocytes resulted in an increase in the number of low- and high-affinity binding sites but had no significant effect on the association constants. However, high-affinity binding constants were about six times higher with N (7.07 × 10⁷ and 6.61 × 10⁷m^?1 for native and neuraminidase-treated N cells, respectively) as compared to M erythrocytes (1.13 × 10⁷ and 1.17 × 10⁷m^?1 for native and neuraminidase-treated M cells, respectively) whereas the low-affinity binding constants were similar for all types of cells (in the range of 0.1 to 0.3 × 10⁷m^?1). The number of Vg binding sites increases from 0.085 × 10⁵ to 0.8 × 10⁵ (high affinity) and from 2.10 × 10⁵ to 6.25 × 10⁵ (low affinity) per native and neuraminidase-treated N cell, respectively. On native and neuraminidase-treated M cells the number of Vg receptors increases from 0.011 × 10⁵ to 0.51 × 10⁵ (high affinity) and 0.13 × 10⁵ (low affinity), respectively. The large increase in the number of Vg receptors on neuraminidase-treated M cells is correlated with a large increase in agglutinability. Under similar treatment St(a+) cells behave like N erythrocytes whereas only 0.16 × 10⁵ Vg receptors of low affinity could be detected on neuraminidase-treated Tn erythrocytes. The results demonstrate that sialic acid is not required for binding and favor the view that the binding site of V. graminea lectin accommodates with two types of erythrocyte membrane receptors, one including both a contribution of polypeptide and oligosaccharide chains and a second which involves a simple interaction with sugar sequence Galβ1–3GalNAc available only when sialic acids are removed. The latter disaccharide is recognized by the Arachis hypogea lectin which therefore inhibits further binding of the V. graminea to neuraminidase-treated erythrocytes.     

Cation-dependent light-induced structural changes in visual receptor membranes

A stearamide spin probe was used to study the light-induced structural changes in Rod Outer Segment Membranes in the presence of sodium and calcium ions. The correlation time (τ_C) for the reorientation of the probe was calculated in the dark and light. In the presence of sodium ions, τ_C = 3.3 × 10^?9 sec in the dark, and 2.7 × 10^?9sec in the light while the opposite was noticed in the presence of calcium ions, τ_C = 2.9 × 10^?9 sec in the dark and 3.6 × 10^?9 sec in the light. The correlation times for reorientation of the probe were also calculated in aqueous glycerol solutions of varying viscosities at 20°C. Comparison of the values of τ_C (dark and light) suggests a change in local mobility in the ROS corresponding to a macroscopic viscosity difference of approximately 150 cp. The significance of calcium ion interaction with negatively charged groups and the formation of a Schiff base is emphasized.     

Purification and some properties of γ-conglycinin in soybean seeds

A 7S globulin (γ-conglycinin) which was one of four major antigenic components in soybean globulins was purified and found to be homogeneous on ultracentrifugation, disc electrophoresis, immunoelectrophoresis and disc electrofocusing by gel filtration, preparative-scale disc electrophoresis and two kinds of affinity chromatography. Subsequently, some physico-chemical properties of the protein were determined. The sedimentation coefficient, isoelectric point, MW and diffusion constant were 6·55S, pH 5·80, 104000 and 5·80 × 10^?7 cm²/sec, respectively. The protein was a glycoprotein which contained 5·49% total carbohydrate per protein. The protein did not aggregate and dissociate with a change of ionic strength from 0·1 to 0·5.     

Effect of chlordimeform on nerve-muscle system of the larva of the waxmoth, Galleria mellonella

The action of chlordimeform on the nerve-muscle preparation of the larvae of the waxmoth Galleria mellonella has been studied by means of microelectrodes. Exitatory junction potential evoked by nerve stimulation is reversibly suppressed by 2 × 10^?3 M chlordimeform, and spike-like component is abolished. The resting membrane potential of the muscle fibre and the action potential from the nerve terminal are not affected at 5 × 10^?3 M chlordimeform. The depolarizing membrane response caused by outward current and the effective membrane resistance are not appreciably affected. It appears that chlordimeform exerts its blocking action on the neuromuscular junction rather than the conductance mechanism of muscle fibre membrane.     

Kinetic Behavior of Microencapsulated β-Galactosidase

Escherichia coli β-D -galactosidase (E.C. 3.2.1.23) was immobilized in cellulose nitrate membrane microcapsules and the reaction kinetics with o-nitrophenyl-β-D -galactopyranoside (ONPG), lactose, and whole milk were studied using both continuous stirred tank and packed bedreactor configurations. The results of the experiments gave effectiveness factors of 0.3 for ONPG, 0.6 to 0.7 for lactose in solution, andclose to unity for lactose in milk. Using a coupled mass transfer and kinetic model, it was possible to estimate the permeability of the microcapsule membrane from the reactor data. Membrane permeabilities on the order of 5 × 10^?3 and 3 × 10^?4 cm/sec were estimated for ONPG and lactose, respectively. It was determined that the membrane was the limiting mass transfer resistance for the overallreaction. The analysis showed that within the microcapsule, the reaction is reaction rate limited for lactose and slightly diffusion limitedfor ONPG.     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.