A study of the interactions of an immunoglobulin light chain with artificial and B-lymphocyte membranes.

The binding of an immunoglobulin lambda light chain (IgLC) to synthetic and biological membranes was monitored in real-time using a recently developed, time-resolved fluorescence technique. Lambda IgLC purified from the urine of patients with multiple myeloma, were used in studies of protein-membrane interactions. The association of the lambda IgLC dimer with B-lymphocytes was shown to be stabilised predominantly by non-polar interactions. Furthermore, it was found that following binding to synthetic phospholipid membranes, a reorientation of the light chain occurred which resulted in a change in the distribution of charged residues at the lipid-water interface. The rate constants associated with the binding event were calculated, and appear to comprise both temperature insensitive and sensitive components. The calculated activation energies of the binding and reorientation events were found to be 13.53 KJmol(-1) and 87.89 KJmol(-1), respectively. The large activation energy associated with the reorientation phase suggests the movement of large protein domains, possibly involving a whole immunoglobulin domain. The binding and reorganisation of the IgLC upon the phospholipid membrane may confer novel biological functions to the bound protein and potentially contribute to such phenomenon as myeloma-associated immuno-suppression.

New insights on the cytochrome c oxidase proton pump.

Cytochrome c oxidase vesicles were used to show that, under appropriate experimental conditions: (1) no net deprotonation of the vesicular membrane or of the incorporated enzyme occurs during the oxidation of ferrocytochrome c; (2) the pH equilibration kinetics of a respiration-induced pH gradient across the bilayer are a simple function of the ohmic proton-conductance properties of the membrane; (3) a fairly constant stoichiometry (0.8-0.7) of the numbers of protons pumped per molecule of ferrocytochrome c oxidized, i.e. the H+/e- ratio, over a wide range of dioxygen molecules reduced (1-12) is observed.

Proton translocation by a native and subunit III-depleted cytochrome c oxidase reconstituted into phospholipid vesicles. Use of fluorescein-phosphatidylethanolamine as an intravesicular pH indicator.

The existence of a proton pump associated with bovine cytochrome c oxidase (EC 1.9.3.1) has over the last few years been a matter of considerable dispute. In an attempt to resolve some of the problems with the measuring system we have synthesized fluorescein-phosphatidylethanolamine which when reconstituted with cytochrome c oxidase into phospholipid vesicles provided a reliable indicator of the intravesicular pH. It was observed that cytochrome c oxidase catalyzed the abstraction of almost 2 protons from the intravesicular medium/molecule of ferrocytochrome c oxidized. In parallel experiments whereby the extravesicular pH was measured with an electrode it was found that the enzyme appeared to be responsible for the appearance of almost 1.0 proton/molecule of ferrocytochrome c oxidized. Taken together these data unequivocally demonstrate that cytochrome c oxidase behaves as a proton pump. Furthermore, the other proton which was abstracted is believed to be used for the process of the reduction of oxygen. Similar experiments were performed with a cytochrome c oxidase preparation which was devoid of subunit III. Under these circumstances the enzyme appeared to be unable to translocate protons across the vesicular membrane but was competent to abstract protons from the intravesicular medium for the reduction of oxygen.

On the logic of the application of double-inhibitor titrations for the elucidation of the mechanisms of energy coupling.

In the last couple of years the chemiosmotic hypothesis has been severely criticised, such that many research groups now consider it to be a less than exact description of biological energy transduction. The most potent experimental support for this view is based upon the technique known as the double-inhibitor titration [e.g., (1982) Biochem. J. 206, 351-357]. The results of such experiments have been considered by many to exclude 'unequivocally' the chemiosmotic coupling model. It will be shown that such unequivocal statements are not possible. An argument is put forward which shows how the chemiosmotic model may explain these titrations without any further elaborations of the original hypothesis.

The use of fluorescein-dipalmitoylphosphatidylethanolamine for measuring pH-changes in the internal compartment of phospholipid vesicles.

The synthesis and characterisation of fluorescein-phosphatidylethanolamine (FPE) is described. The effects of dielectric constant, ionic strength and ambient pH upon the optical absorbance properties of FPE are presented. It is shown that under appropriate conditions, FPE rapidly and quantitatively reports the pH of the aqueous bulk phases when incorporated into phospholipid vesicles. It is also shown that, when the external medium is highly buffered, FPE is capable of specificity reporting only the pH of the intravesicular compartment. The application of FPE for studies of intravesicular pH changes of reconstituted membranous protein systems is discussed.

Membrane-potential-dependent changes of the lipid microviscosity of mitochondria and phospholipid vesicles.

The effects of a transmembrane potential difference upon the lipid microviscosity of cytochrome oxidase vesicles (COVs) and rat liver mitochondria (RLM) were investigated. COVs and RLM were labelled with the fluorescent probe 1,6-diphenylhexa-1,3,5-triene (DPH). The fluorescence polarization of the probe was then measured when potentials of different magnitudes were induced across the membranes of these particles. It was shown that the absolute value of the microviscosity changes to quite a significant extent, owing to the imposition of large membrane potentials. On relaxation of the membrane potential the lipid microviscosity was also shown to return to the value before the induction of the potential. The largest change in lipid microviscosity was observed when coupled respiration was initiated. This occurred in both the COV system and the RLM system. The absolute value of the lipid microviscosity was shown to change by as much as 22% with the induction of membrane potentials, owing to respiration. To confirm the viscosity measurements made with DPH, lipid microviscosity was also measured with the spin-labelled fatty acid 5-doxyl stearate. Measurements of the order parameters indicated that, in agreement with the results of fluorescence experiments, viscosity changes occurred that were due to the induction of a membrane potential. The significance of these findings to the regulation of metabolism is briefly discussed, the main conclusion being that, although there is certainly a significant variation of lipid microviscosity with electric field, mechanistic interpretations will require further studies.

The current-voltage relationships of liposomes and mitochondria.

Current-voltage relationships were determined for various membrane systems. We show that phospholipid and mitochondrial membranes exhibit linear relations between H+ flux and pH gradients. These membranes, however, exhibited non-linear relationships when the applied voltage was a membrane potential. The current-voltage relationship approximated to an exponential function. This relationship was found to be linearized when the membranes were treated with an electrogenic proton ionophore. The incorporation of cytochrome c oxidase (EC 1.9.3.1) was found to have no effect on the current-voltage characteristics of the phospholipid membranes. When a membrane potential of more than 140 mV was imposed across vesicular and mitochondrial membranes, they exhibited reversible di-electric breakdown. This phenomenon was correlated with the requirement of a permeant ion for the experimental demonstration of proton translocation by so-called 'proton pumps'.

A quantitative characterisation of H+ translocation by cytochrome c oxidase vesicles.

A quantitative analysis of H+ extrusion by reconstituted cytochrome c oxidase vesicles is presented with particular regard to the decay kinetics of the extruded proton pulse and to the structural heterogeneity of the vesicle preparation. The decay of the extruded H+ pulse under conditions typical of those used for its measurement is much slower than expected from the passive proton permeability of the vesicle membranes. It is shown that this apparent anomaly results from insufficient transmembrane charge equilibration via valinomycin and K+ during oxidase turnover. This situation can be remedied by increasing the valinomycin concentration or by replacing this counterion system with 1 mM tetraphenylphosphonium. Under these latter conditions, the decay kinetics can be described as the sum of two exponential terms. To facilitate interpretation of the proton pump decay kinetics, a structural analysis of the oxidase vesicle preparation is presented. The bulk of the reconstituted vesicles (i.e., those representing approx. 80% of the total oxidase and lipid) are 30-62 nm in diameter. At least 70% of the reconstituted oxidase molecules are contained individually in separate vesicles, indicating that the enzyme monomer is competent in H+ translocation.

The relationship between the rate of respiration and the protonmotive force. The role of proton conductivity.

It is shown by titrating a suspension of rat liver mitochondria with either ADP or an uncoupler that a specific rate of respiration may not have a unique associated value of the protonmotive force. Alternatively, a specific protonmotive force may not be associated with a unique rate of respiration. It seems that the rate of respiration and the protonmotive force are more sensitive to the agents used for the titrations than to each other. Such observations are not easily explained by the chemiosmotic hypothesis. It is, however, possible provided that the proton conductivities, i.e. the rates of dissipation of the protonmotive force, are considered to be different for each of the agents used to titrate the rate of respiration at the same protonmotive force, or vice versa.