pH dependence of phosphate transport across the red blood cell membrane after modification by dansyl chloride

Dansylation of the red blood cell membrane inhibits monovalent anion transport as measured by means of 36C1 and enhances divalent anion transport as measured by means of 35SO4 (Legrum, Fasold and Passow (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1573-1590 and Lepke and Passow (1982) J. Physiol. (London) 328, 27-48). In the present work the effect of dansylation on phosphate equilibrium exchange was studied over the pH range where the ratio between monovalent and divalent phosphate anions varies. At high pH, phosphate equilibrium exchange was enhanced; at low pH, exchange was inhibited. The pH maximum of phosphate equilibrium exchange, seen at pH 6.3 in untreated ghosts is now replaced by a plateau. The inverse effects of dansylation on the rates of exchange at high and low pH suggest that both monovalent and divalent phosphate anions are accepted as substrates by the anion transport protein. A tentative attempt to obtain a quantitative estimate of the ratio of monovalent and divalent phosphate transport indicates that in the untreated red cell membrane over the pH range 7.2-8.5 the transport of HPO42- is negligible compared to the transport of H2PO4-.     

The pH dependence of the inhibition of ascorbate oxidase by anions

Double-reciprocal plots of azide inhibition, with respect to ascorbate, of ascorbate oxidase indicate mixed-type inhibition at pH values above 6. This is in contrast to the simple competitive inhibition previously observed at pH 5.6. Linear replots of the slopes and intercepts of the double-reciprocal plots yield two inhibition constants. Both constants are pH dependent. Similar inhibition patterns are obtained with fluoride and thiocyanate. These results suggest the presence of two inhibitor binding sites, one of which is competitive with respect to ascorbate and the other uncompetitive.     

The temperature dependence of the facilitated transport ofd(+)-glucose across the human red cell membrane

Summary The rate of exit ofd(+)-glucose from human red cells was measured as a function of the extracellular glucose concentration over the temperature range 12 to 47°C. The results were analyzed at each temperature, according to the kinetic model of Widdas and of Rosenberg and Wilbrandt, in terms of the apparent maximum exit rate (V _max) and the apparent dissociation constant (K _m ) of the carrier-glucose complex. When the values ofV _max andK _m were obtained by the same graphical method as that used by Sen and Widdas, the results were very similar to theirs insofar as the effect of temperature is concerned. In particular, the apparent standard enthalpy of dissociation (H _m ) of the carrier-glucose complex does not vary with temperature, whereas the apparent activation energy (E _max) for the translocation of the carrier increases strongly with decreasing temperature. It is shown that the explanation of these findings given by Dawson and Widdas is internally inconsistent. Furthermore, the graphical method as used by these authors is unreliable at higher temperatures, whereK _m is large and consequently underestimatesK _m . An improved modification of the method, suggested by Bolis, Luly and Wilbrandt, overcomes this difficulty and leads to more reliable values forV _max andK _m . These new results show thatE _max decreases, and H _m increases, as the temperature is raised. This behavior is shown to be consistent with the modified kinetic model for sugar transport proposed by Wilbrandt, in which the translocation rate of the loaded carrier is assumed to be different from that of the empty carrier. The changes inE _max and H _m with temperature are the result of the difference in true activation energies for the translocation of the loaded and empty carrier.     

Influences of membrane curvature in lipid hexagonal phases studied by deuterium NMR spectroscopy

The presence of reversed hexagonal phase, HII, favoring lipids in membranes has been proposed to be significant in various biological processes. Therefore an understanding of the HII phase and the transition from the lamellar to hexagonal phase is of importance. We have applied deuterium NMR spectroscopy to study the bilayer and reversed hexagonal phases of 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamin e. The difference in packing between the HII and L alpha phases leads to smaller segmental order parameters in the former case. Since the order profiles are sensitive to the geometry of the aggregates, they can be used to extract structural information about the phases. We present a new means of calculating the radius of curvature, R1, for the HII phase from 2H NMR data. This method gives a value of R1 = 18.1 A, which is in agreement with current understanding of the structure of the HII phase and with x-ray diffraction data.     

Modulation of red cell K transport by membrane lipids

Alterations in the state of the membrane lipids affect human red cell K⁺ transport. Depletion of membrane cholesterol by 29–34% significantly inhibited both total K⁺ influx and ouabain-sensitive K⁺ influx. Addition of the hydrophobic anesthetic, chlorpromazine, in concentration from 2 · 10⁻⁵ to 2 · 10⁻⁴ M increased both total K⁺ influx and ouabain-sensitive K⁺ influx. In each case the effect on both processes was almost identical which indicates a linkage between K⁺ “pump” and “leak”. Further, these results demonstrate that red cell K⁺ transport can be modulated by local conditions in the micro-environment of the transport system.     

Interactions between mastoparan B and the membrane studied by 1H NMR spectroscopy

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic alpha-helical structure from Lys2 to Leu14. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp9 are in close contact with methylene protons of SDS micelles. T1 relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp9 with Ala9, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp9 into the membrane, as well as the amphiphilic alpha-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

Actin dynamics studied by solid-state NMR spectroscopy

Solid-state nuclear magnetic resonance spectroscopy was used to study the motion of ²H and ¹⁹F probes attached to the skeletal muscle actin residues Cys-10, Lys-61 and Cys-374. The probe resonances were observed in dried and hydrated G-actin, F-actin and F-actin-myosin subfragment-1 complexes. Restricted motion was exhibited by ¹⁹F probes attached to Cys-10 and Cys-374 on actin. The dynamics of probes attached to dry cysteine powder or F-actin were very similar and the binding of myosin had little effect indicating that the local probe environment imposes the major influence on motion in the solid state. Correlation times determined for the solid state probes indicated that they were undergoing some rapid internal motion in both G-actin and F-actin such as domain twisting. The probe size influenced the motion in G-actin and appeared to sense monomer rotation but not in F-actin where segmental mobility and intramonomer co-ordination appeared to dominate.     

Protein folding studied by real-time NMR spectroscopy

Real-time NMR spectroscopy developed to a generally applicable method to follow protein folding reactions. It combines the access to high resolution data with kinetic experiments allowing very detailed insights into the development of the protein structure during different steps of folding. The present review concentrates mainly on the progress of real-time NMR during the last 5 years. Starting from simple 1D experiments, mainly changes of the chemical shifts and line widths of the resonances have been used to analyze the different states populated during the folding reactions. Today, we have a broad spectrum of 1D, 2D, and even 3D NMR methods focusing on different characteristics of the folding polypeptide chains. More than 20 proteins have been investigated so far by these time-resolved experiments and the main results and conclusions are discussed in this report. Real-time NMR provides comprehensive contributions for joining experiment and theory within the 'new view' of protein folding.     

Millisecond protein folding studied by NMR spectroscopy

Proteins are involved in virtually every biological process and in order to function, it is necessary for these polypeptide chains to fold into the unique, native conformation. This folding process can take place rapidly. NMR line shape analyses and transverse relaxation measurements allow protein folding studies on a microsecond-to-millisecond time scale. Together with an overview of current achievements within this field, we present millisecond protein folding studies by NMR of the cold shock protein CspB from Bacillus subtilis.     

Regulation of intracellular pH in rat uterine smooth muscle, studied by P NMR spectroscopy

Intracellular pH (pH_i) affects smooth muscle function, yet little is known concerning its regulation. I have therefore investigated pH regulation in rat uterus, using ³¹P-NMR spectroscopy. A change in extracellular pH(pH_e) of 1 pH unit (7.4 to 6.4) elicited a 0.29 change in pH_i; smaller changes in pH_o were accompanied by proportionately smaller changes in pH_i. The pH changes were reversible. There was no fall of uterine ATP or phosphocreatine during the pH changes.     

Regulation of intracellular pH in rat uterine smooth muscle,studied by 31P NMR spectroscopy

Intracellular pH (pH_i) affects smooth muscle function, yet little is known concerning its regulation. I have therefore investigated pH regulation in rat uterus, using ³¹P-NMR spectroscopy. A change in extracellular pH(pH_e) of 1 pH unit (7.4 to 6.4) elicited a 0.29 change in pH_i; smaller changes in pH_o were accompanied by proportionately smaller changes in pH_i. The pH changes were reversible. There was no fall of uterine ATP or phosphocreatine during the pH changes.     

Conformations of neurotensin in solution and in membrane environments studied by 2-D NMR spectroscopy

Two-dimensional HOHAHA and ROESY nuclear magnetic resonance techniques are used to obtain complete proton resonance assignments and to perform a conformational investigation of the neuropeptide neurotensin (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) in aqueous solution, methanol, and membrane-mimetic [deuterated sodium dodecylsulfate (SDS)] environments. Results suggest the absence of discernible elements of secondary structure in water and methanol. ROESY spectra confirm that Lys-Pro and Arg-Pro peptide bonds are all-trans, but that a significant population of cis Arg-Pro bonds arises in aqueous solution, which increases in the environment of SDS micelles. The conformational ensemble of the peptide is observed to narrow as it becomes bound through its cationic mid-region to SDS micelles, with the accompanying advent of local extended structure. The overall results indicate the inherent conformational flexibility of neurotensin, and emphasize the environmental dependence of conformation in peptides of medium length.     

Hsp90 structure and function studied by NMR spectroscopy

The molecular chaperone Hsp90 plays a crucial role in folding and maturation of regulatory proteins. Key aspects of Hsp90's molecular mechanism and its adenosine-5'-triphosphate (ATP)-controlled active cycle remain elusive. In particular the role of conformational changes during the ATPase cycle and the molecular basis of the interactions with substrate proteins are poorly understood. The dynamic nature of the Hsp90 machine designates nuclear magnetic resonance (NMR) spectroscopy as an attractive method to unravel both the chaperoning mechanism and interaction with partner proteins. NMR is particularly suitable to provide a dynamic picture of protein-protein interactions at atomic resolution. Hsp90 is rather a challenging protein for NMR studies, due to its high molecular weight and its structural flexibility. The recent technologic advances allowed overcoming many of the traditional obstacles. Here, we describe the different approaches that allowed the investigation of Hsp90 using state-of-the-art NMR methods and the results that were obtained. NMR spectroscopy contributed to understanding Hsp90's interaction with the co-chaperones p23, Aha1 and Cdc37. A particular exciting prospect of NMR, however, is the analysis of Hsp90 interaction with substrate proteins. Here, the ability of this method to contribute to the structural characterization of not fully folded proteins becomes crucial. Especially the interaction of Hsp90 with one of its natural clients, the tumour suppressor p53, has been intensively studied by NMR spectroscopy. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Effects of superoxide anions on red cell deformability and membrane proteins.

The effect of superoxide anions (O2-) on red blood cells (RBC) deformability and membrane proteins was investigated using hypoxanthine-xanthine oxidase system. Exposure of RBC to O2- caused a marked decrease in RBC deformability with a concomitant increase in cell volume and shape changes. The RBC exposed to O2- also displayed pronounced degradation of membrane proteins such as band 3 protein and spectrin; new bands of low molecular weight products appeared as the original membrane proteins tended to diminish, without the appearance of high molecular weight products. Since the membrane proteins are involved in processes regulating membrane properties such as permeability and viscoelasticity, the decreased deformability induced by O2- may be attributable to changes in membrane proteins. Interestingly, resealed ghosts exposed to O2- did not show any significant change in membrane proteins, which suggests the existence of further generation of O2- and subsequent production of other active oxygen species mediated by O2(-)-initiated autoxidation of hemoglobin in intact RBC. Furthermore, electrophoretic analysis suggested that active oxygens increased the endogenous proteolytic susceptibility of RBC. In conclusion, a close linkage was suggested between RBC deformability and the membrane proteins.     

Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization.

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.     

Mouse polyomavirus mediated effects on the infected cell membrane studied by dielectric spectroscopy

The effect of polyomavirus on the infected cell has been investigated by dieclectric spectroscopy. This technique has a great potential in the study of the ion transport properties of the cell membrane. The results presented in this communication suggest a correlation between progression of the viral infection and dielectric features of the infected, cell plasma membrane.