Lipid-protein interactions at the erythrocyte membrane. Different influence of glucose and sorbose on membrane lipid transition.

When observed over a temperature range, erythrocyte membrane lipids undergo a transition at 18-20 degrees C (Zimmer, G. and Schirmer, H. (1974) biochim. Biophys. Acta 345, 314-320). This observation has prompted an investigation of the effects that substrate binding has on the transition of the red cell membrane. Glucose and sorbose were compared, since transport kinetics of these sugars still pose unresolved questions. In membranes, preloaded with glucose, the break at the transition temperature was intensified, while it was abolished or reversed in membranes preloaded with sorbose. These results were corroborated using different solubilization procedures (sonication, sodium dodecyl sulfate treatment) of the membranes, and also different techniques (viscosimetry, 90 degrees light scattering, 1-anilino-naphthalene-8-sulfonate fluorescence). In extracted membrane lipids, viscosimetry indicated a break at transition temperature after preloading with either glucose or sorbose. Disc electrophoresis revealed a different binding pattern of the two sugars. It is suggested, that the amplification of the discontinuity in red cell membranes by glucose and the abolition or reversal of the break by sorbose are mediated by membrane protein- and/or membrane lipid-protein interaction.     

Muscular atrophy: activation of mitochondrial ATPase

Mitochondria isolated from atrophic rat gastrocnemius muscles are deficient in respiratory control. In this report, it is demonstrated that the ATPase activity of mitochondria isolated from atrophic muscles is higher than the activity of control preparations. Activation of ATPase may be a factor in the loss of respiratory control in mitochondria from disused muscles.     

A conformational study of N-phenyl-1-naphthylamine and 1-anilino-8-naphthalene sulfonate by the empirical method

Conformational analysis of N-phenyl-1-naphthylamine and 1-anilinonaphthalene-8-sulfonate (ANS) was carried out using the empirical method. Properties such as conformational energies and dipole moments were considered. Furthermore, the effect of solvent medium was examined through the effective dielectic constant. The N-phenyl-1-naphthylamine molecule showed two energy minima which were independent of dielectic constant. The ANS molecule also showed two energy minima but the minima changed positions when the dielectic constant increased from 1.0 (vacuum) to 80.0 (highly polar medium). Hydrogen bonding appeared to play an important role in stabilizing these conformations. The minimum energy conformations may have relevance to the binding of ANS to lipid bilayers and bimembranes. The dipole moment, in contrast to the energy minimum, was found to depend on orientation of the sulfonate group rather than of the benzene ring with respect to the naphthalene ring. Thus binding and fluorescence enhancement of ANS may be attributed to the orientation of the sulfonate group, which to a large extent may determine the magnitude of the dipole moment and the degree of electrostatic interactions between the probe and binding domains. Various dimensions like intra-atomic distances, volume and area of the ANS molecule were calculated.     

Effect of hesperetin on tyrosinase: inhibition kinetics integrated computational simulation study

Tyrosinase inhibitors have potential applications in medicine, cosmetics and agriculture to prevent hyperpigmentation or browning effects. Some of the flavonoids mostly found in herbal plants and fruits are revealed as tyrosinase inhibitors. We studied the inhibitory effects of one such flavonoid, hesperetin, on mushroom tyrosinase using inhibition kinetics and computational simulation. Hesperetin reversibly inhibited tyrosinase in a competitive manner with K_i = 4.03 ± 0.26 mM. Measurements of ANS-binding fluorescence showed that hesperetin induced the hydrophobic disruption of tyrosinase. For further insight, we used the docking algorithms to simulate binding between tyrosinase and hesperetin. Simulation was successful (binding energies for Dock6.3: −34.41 kcal/mol and for AutoDock4.2: −5.67 kcal/mol) and showed that a copper ion coordinating with 3 histidine residues (HIS61, HIS85, and HIS259) within the active site pocket was chelated via hesperetin binding. Our study provides insight into the inhibition of tyrosinase in response to flavonoids. A combination of inhibition kinetics and computational prediction may facilitate the identification of potential natural tyrosinase inhibitors such as flavonoids and the prediction of their inhibitory mechanisms.     

Further characterization of gradient-fractionated sub-mitochondrial membrane fragments from beef heart mitochondria

We have recently reported that with a linear sucrose density gradient centrifugation two distinct types of membrane fragments, designated as X- and Y-fragments are obtained (Huang, C. H., Keyhani, E. and Lee, C. P. (1973) Biochim. Biophys. Acta 305, 455–473). Further characterization of these two membrane fragments is reported. (1) Potassium chloride at the concentration of 0.15 M extracts 7% and 30% of cytochrome c from the X- and Y-fragments, respectively. (2) When cytochrome c was added to the mitochondrial suspension prior to sonication, the cytochrome c content was increased by 6–8-fold in both X- and Y-fragments. Subsequently KCl extraction resulted in loss of cytochrome c by 1/4 in the X- and by 2/3 in the Y-fragments. (3) With partially inhibitory concentrations of KCN, cytochrome c in either the X- or the KCl extracted X-fragments showed uncoupler-sensitive, biphasic reduction kinetics upon the addition of NADH to the oligomycin-supplemented system. Under identical conditions rapid first order reduction kinetics were seen for cytochrome c in Y-fragments supplemented with either oligomycin or oligomycin + carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (4) When cytochrome c was added to the mitochondrial suspension after sonication, a significant amount of cytochrome c was bound to both X- and Y-fragments, but was readily removed with a high ionic strength medium. (5) Lubrol had little effect on the ATPase activity of the X- and the Y-fragments, suggesting a lack of membrane-buried ATPase. (6) Partial depletion of ATPase in X-fragments did not induce an increase in reactivity towards externally added cytochrome c. (7) Both the X- and the Y-fragments showed an energy-linked fluorescence enhancement of 8-anilinonaphthalene-1-sulfonate and an energy-linked fluorescence decrease of quinacrine. (8) In the presence of K⁺ nigericin alone or in combination with valinomycin exhibited a stimulating effect on the rate of NADH oxidase of the oligomycin-supplemented X- and Y-fragments.     

Oxidative phosphorylation in Azotobacter vinelandii. Energy-linked pH changes and fluorescence changes of atebrin and 1-anilinonaphthalene-8-sulphonate

1. Phosphorylating particles from Azotobacter vinelandii show a rapid, respiration-induced reversible increase in pH of the suspending medium; this is not found with non-phosphorylating particles.2. The observed pH response requires the presence of low concentrations of Mg²⁺ or of higher concentrations of Na⁺ or K⁺.3. Between 40 and 10 °C the rates of proton influx and efflux have similar temperature coefficients; below 10 °C the effect of temperature is greater on proton efflux.4. The kinetics of the energy-linked enhancement of fluorescence 1-anilinonaphthalene-8-sulphonate are slower than that of the quenching of the fluorescence of atebrin.     

Neurite formation and membrane changes of mouse neuroblastoma cells induced by valinomycin

A clonal cell line of mouse neuroblastoma cells was found to undergo morphological differentiation in the presence of a K⁺ ionophore, valinomycin, in the assay medium. This effect was blocked by increasing the concentration of KCl of the medium, suggesting that the changes in resting membrane potential and ion fluxes may be involved in the mechanism of the formation of neurites. No enhancement of the neurite formation was observed in salines containing high concentrations of KCl in the absence of valinomycin. Depolarizing agents including veratridine, gramicidin and ouabain did not stimulate the outgrowth of neurites. Neither electrophoretic mobility of the cells nor molecular anisotropy of fluorescence probes in the membranes was modified by the treatment of valinomycin. Instead, it modified the slow binding phase in kinetics of the interaction of 1-anilinonaphthalene-8-sulfonate (ANS) with the cells, which is related to the penetration process of the probe into membranes. Valinomycin also enhanced the fluorescence intensity of ANS by increasing the binding sites in neuroblastoma cells.     

A study on the membrane potential and pH gradient in chromatophores and intact cells of photosynthetic bacteria

Generation of membrane potential (Δψ) and transmembrane pH difference (ΔpH) was studied in PP_i-energized chromatophores of Rhodospirillum rubrum by means of measurements of carotenoid and bacteriochlorophyll absorption changes, atebrin and 8-anilinonaphthalene-1-sulphonate fluorescence responses, and phenyldicarbaundecaborane transport.The data obtained are consistent with the suggestion that carotenoid, bacteriochlorophyll and phenyldicarbaundecaborane responses are indicators of Δψ, while an atebrin response is an indicator of ΔpH. The fluorescence of 8-anilinonaphthalene-1-sulphonate is affected both by Δψ and ΔpH.     

Different binding sites for glucose and sorbose at the erythrocyte membrane,studied by gel filtration and infrared spectroscopy

Summary Human red blood cell membranes were solubilized with sodium dodecylsulfate and incubated with various concentrations of¹⁴C-glucose and¹⁴C-sorbose. After gel filtration on Sephadex G-100, which separated lipoproteins of differing lipid content, it was observed that the radioactivity of the bound glucose coincided with the protein peak. Radioactivity of bound sorbose was found mainly before and after the protein peak. This distribution of bound sugars was confirmed by double labeling experiments in which³H-glucose and¹⁴C-sorbose were applied simultaneously. Infrared spectroscopy revealed differences between the membranes loaded with sorbose and glucose. Particularly, the band in the C–O–C and P=O region at 1,225 cm^–1 was intensified in the sorbose-loaded membranes. Compared to serum albumin, the erythrocyte membranes were found to bind 4 times as much¹⁴C-glucose per mg of protein. It is concluded from the results obtained by gel filtration that glucose and sorbose preferentially bind at different sites of the erythrocyte membrane. The results obtained by infrared spectroscopy correspond with this conclusion.     

Nitrate,fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step.In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate.The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth.An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.     

Energization of mitochondrial inner membranes caused by l-malate

It was found that 0.06 μg antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c₁ completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.     

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature

The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant ($\text{K′}{}_{\mathrm{<mtext>s</mtext>}}$) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome $\text{P-450}$ is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome $\text{P-450}$ binding reaction and in the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$.     

Effect of poly(ethylene glycol) on phospholipid hydration and polarity of the external phase

The hydration properties of phosphatidylcholine (PC)/water dispersions on the addition of poly(ethylene glycol) were studied by means of ²H-NMR. The quadrupole splittings and their temperature dependences correspond to measurements of PC/water dispersions at low water content. It is concluded that the bound water is partly extracted by poly(ethylene glycol) but the binding properties of the water in the inner hydration shell of about five water molecules are not changed. The ability of some phospholipid/water dispersions to undergo phase transitions to nonlamellar structures upon dehydration is discussed. Dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine do not form nonlamellar structures on addition of purified poly(ethylene glycol), as was demonstrated by means of ³¹P-NMR. Poly(ethylene glycol) decreases the polarity of the aqueous phase and the partition of hydrophobic molecules between the membrane and the external phase is changed. This was demonstrated using the excimer fluorescence of pyrene in a ghost suspension. It is suggested that the changes in polarity and hydration on the addition of poly(ethylene glycol) can contribute to the alterations in the membrane surface observed under conditions of membrane contact and fusion.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. II. Protein-mediated transfer of monosaccharides and anions

In order to investigate the influence of membrane lipids on transport via the protein domain of the erythrocyte membrane, a number of facilitated diffusion processes was studied by tracer flux techniques in whole cells after cleavage of up to 65% of the phosphatidylcholine or the sphingomyelin by phospholipase A₂ from Naja naja or bee venom, or by sphingomyelinase, respectively.The mediated fluxes of l-arabinose, which is transported by the glucose carrier, and of l-lactate, which uses a specific monocarboxylate carrier, were markedly inhibited by cleavage of either phosphatidylcholine or sphingomyelin. These phospholipid dependencies are in line with earlier data on cholesterol dependencies (Deuticke, B. (1977) Rev. Physiol. Biochem. Pharmacol. 78, 1–97). They can only in part be explained by changes of membrane fluidity. More specific interactions of the degradation products with the carrier proteins seem also to play a role.Sulfate and oxalate transfer, which proceed via the inorganic anion-exchange system, are essentially unaffected by cleavage of phosphatidylcholine and less sensitive to sphingomyelin cleavage than the two other processes. This also agrees with earlier data on cholesterol independency of sulfate transfer. The inorganic anion-exchange protein thus seems to be less dependent on the surrounding lipids in its conformation and its mode of action than the two other carriers.     

Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent

The localization of proteins in myelin was studied by the use of a non-penetrating penetrating reagent. Tritiated 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with ³H-labelled, 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

Nanosecond dynamics of charged fluorescent probes at the polar interface of a membrane phospholipid bilayer

Molecular relaxation fluorescence methods were applied to analyze the nature and characteristic times of motions of amphiphilic molecules absorbed in the polar region of a phospholipid bilayer. The fluorescence probes 2-toluidinonaphthalene-6-sulfonate and 1-anilinonaphthalene-8-sulfonate in egg phosphatidylcholine vesicles were studied. The methods of edge excitation fluorescence red shifts, nanosecond time-resolved spectroscopy, fluorescence quenching by hydrophilic and hydrophobic quenchers and emission wavelength dependence of polarization were used. The structural (dipolar) relaxation is shown to be a very rapid (subnanosecond) process. The observed nanosecond phenomena are related to translational movement of the chromophore itself towards a more polar environment and its rotation. The polar surface area of the phospholipid membrane appears to be a highly mobile liquid-like system.     

Impact of intra-subunit domain-domain interactions on creatine kinase activity and stability

Creatine kinase (CK) is a key enzyme in vertebrate excitable tissues. In this research, five conserved residues located on the intra-subunit domain-domain interface were mutated to explore their role in the activity and structural stability of CK. The mutations of Val72 and Gly73 decreased both the activity and stability of CK. The mutations of Cys74 and Val75, which had no significant effect on CK activity and structure, gradually decreased the stability and reactivation of CK. Our results suggested that the mutations might modify the correct positioning of the loop contributing to domain-domain interactions, and result in decreased stability against denaturation.