The polar headgroup of the detergent governs the accessibility to water of tryptophan octyl ester in host micelles.

Many attempts have been made to rationalize the use of detergents for membrane protein studies [J. Biol. Chem. 264 (1989) 4907]. The barrier properties of the detergent headgroup may be one parameter critically involved in protein protection. In this paper, we analyzed these properties using a model system, by comparing the accessibility of tryptophan octyl ester (TOE) to water-soluble collisional quenchers (iodide and acrylamide) in three detergent micelles. The detergents used differed only in the chemical nature of their polar headgroups, zwitterionic for dodecylphosphocholine (DPC) and nonionic for octa(ethylene glycol) dodecyl monoether (C(12)E(8)) and dodecylmaltoside (DM). In all cases, in phosphate buffer at pH 7.5, the binding of 5 microM TOE was complete in the presence of a slight excess of detergent micelles over TOE molecules, resulting in a significant blue shift and greater intensity of TOE fluorescence emission. The resulting quantum yield of bound TOE was between 0.08 (in DPC) and 0.12 (in DM) with an emission maximum (lambda(max)) of approximately 335 nm whatever the detergent micelle. Time-resolved fluorescence intensity decays of TOE at lambda(max) were heterogeneous in all micelles (3-4 lifetime populations), with mean lifetimes of 1.7 ns in DPC, and 2 ns in both C(12)E(8) and DM. TOE fluorescence quenching by iodide, in detergent micelles, yielded linear Stern-Volmer plots characteristic of a dynamic quenching process. The accessibility of TOE to this ion was the greatest with C(12)E(8), followed by DPC and finally DM (Stern-Volmer quenching constants K(sv) of 2 to 5.5 M(-1)). In contrast, the accessibility of TOE to acrylamide was greatest with DPC, followed by C(12)E(8) and finally DM (K(sv)=2.7-7.1 M(-1)). TOE also presents less rotational mobility in DM than in the other two detergents, as shown from anisotropy decay measurements. These results, together with previous TOE quenching measurements with brominated detergents [Biophys. J. 77 (1999) 3071] provide reference data for analyzing Trp characteristics in peptide (and more indirectly protein)-detergent complexes. The main finding of this study was that TOE was less accessible (to soluble quenchers) in DM than in DPC and C(12)E(8), the cohesion of DM headgroup region being suggested to play a role in the ability of this detergent to protect function and stability of solubilized membrane proteins.

Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins.

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.

Tryptophan octyl ester in detergent micelles of dodecylmaltoside: fluorescence properties and quenching by brominated detergent analogs.

The fluorescence properties of tryptophan octyl ester (TOE), a hydrophobic model of Trp in proteins, were investigated in various mixed micelles of dodecylmaltoside (DM) and 7,8-dibromododecyl beta-maltoside (BrDM) or 10,11-dibromoundecanoyl beta-maltoside (BrUM). This study focuses on the mechanism via which these brominated detergents quench the fluorescence of TOE in a micellar system. The experiments were performed at a pH at which TOE is uncharged and almost completely bound to detergent micelles. TOE binding was monitored by its enhanced fluorescence in pure DM micelles or its quenched fluorescence in pure BrUM or BrDM micelles. In DM/BrUM and DM/BrDM mixed micelles, the fluorescence intensity of TOE decreased, as a nonlinear function of the molar fraction of brominated detergent, to almost zero in pure brominated detergent. The indole moiety of TOE is therefore highly accessible to the bromine atoms located on the detergent alkyl chain because quenching by bromines occurs by direct contact with the fluorophore. TOE is simultaneously poorly accessible to iodide (I(-)), a water-soluble collisional quencher. TOE time-resolved fluorescence intensity decay is heterogeneous in pure DM micelles, with four lifetimes (from 0.2 to 4.4 ns) at the maximum emission wavelength. Such heterogeneity may arise from dipolar relaxation processes in a motionally restricted medium, as suggested by the time-dependent (nanoseconds) red shift (11 nm) of the TOE emission spectrum, and from the existence of various TOE conformations. Time-resolved quenching experiments for TOE in mixed micelles showed that the excited-state lifetime values decreased only slightly with increases in the proportion of BrDM or BrUM. In contrast, the relative amplitude of the component with the longest lifetime decreased significantly relative to that of the short-lived species. This is consistent with a mainly static mechanism for the quenching of TOE by brominated detergents. Molecular modeling of TOE (in vacuum and in water) suggested that the indole ring was stabilized by folding back upon the octyl chain, forming a hairpin conformation. Within micelles, the presence of such folded conformations, making it possible for the entire molecule to be located in the hydrophobic part of the micelle, is consistent with the results of fluorescence quenching experiments. TOE rotational correlation time values, in the nanosecond range, were consistent with a hindered rotation of the indole moiety and a rotation of the complete TOE molecule in the pure DM or mixed detergent micelles. These results, obtained with a simple micellar model system, provide a basis for the interpretation of fluorescence quenching by brominated detergents in more complex systems such as protein- or peptide-detergent complexes.

Spectroscopic studies of the interaction of Ca2+-ATPase-peptides with dodecyl maltoside and its brominated analog.

The transmembrane sector of sarcoplasmic reticulum Ca2+-ATPase comprises ten putative transmembrane spans (M1-M10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca2+ binding: peptide M6 (amino acid residues 785-810), peptide M7-L (amino acid residues 808-847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818-847) which contains a shorter version of loop 6-7 than M7-L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the alpha-helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7-L peptide, the L6-7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca2+-ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl beta-D-maltoside or quenching using the recently introduced brominated analog of n-dodecyl beta-D-maltoside: 7,8-dibromododecyl beta-maltoside [de Foresta, B., Legros, N., Plusquellec, D., le Maire, M. & Champeil, P. (1996) Eur J. Biochem. 241, 343-354]. Our results indicate that M7-L and M7-S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size-exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl beta-D-maltoside/7,8-dibromododecyl beta-maltoside, using tryptophan octyl ester and solubilized Ca2+-ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca2+-ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca2+-ATPase and the rather short alpha-helix span of M6 may lead to exposure of a significant part of the C-terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca2+ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site-directed mutagenesis, that this transmembrane segment should be essential in Ca2+ binding.

Ligand-induced movement of helix X in the lactose permease from Escherichia coli: a fluorescence quenching study.

Five single-Trp mutants were constructed by replacing Val315, Leu318, Val326, Leu329, or Val331 with Trp in transmembrane helix X of a functional lactose permease mutant devoid of Trp residues (Trp-less permease). Taking into account expression levels, each single-Trp permease except for Val331-->Trp exhibits significant activity. The intrinsic fluorescence emission of each single-Trp mutant does not change significantly after addition of beta-d-galactopyranosyl 1-thio-beta-d-galactopyranoside (TDG), indicating that ligand induces little change in the microenvironment of the Trp residues. However, fluorescence quenching studies with the brominated detergent 7,8-dibromododecyl beta,d-maltoside (BrDM) demonstrate that a Trp residue in place of Val315, Val326, or Val331 becomes less accessible to BrDM in the presence of TDG, while a Trp residue in place of Leu318 or Leu329 becomes more accessible. Acrylamide quenching studies with Leu318-->Trp and Val331-->Trp permeases or 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS)-labeled Thr320-->Cys and Glu325-->Cys permeases indicate that positions 318 and 325 also become more accessible to a hydrophobic environment in the presence of TDG, while positions 320 and 331 become less accessible. The findings are consistent with a recently proposed mechanism for energy coupling in lactose permease [Kaback, H. R. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5539-5543] in which substrate binding causes a conformational change resulting in movement of Glu325 to a nonpolar environment with a dramatic increase in pKa.

Dissociation of Ca2+ from sarcoplasmic reticulum Ca2+-ATPase and changes in fluorescence of optically selected Trp residues. Effects of KCl and NaCl and implications for substeps in Ca2+ dissociation.

Sequential dissociation of the two Ca2+ ions bound to non-phosphorylated sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe. The kinetics of Ca2+ dissociation were deduced from the observed changes in quin2 fluorescence in the visible region (with lambdaex = 313 nm), while fluorescence detection in the UV region (with lambdaex = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying this dissociation under the same ionic conditions. In the absence of KCl or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence were monoexponential, with rate constants very close to those of the changes in ATPase tryptophan fluorescence, which also appeared monophasic. In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovalent ions but distinctly slower than the changes in tryptophan fluorescence, which were accelerated to a larger extent. In addition, the apparent kinetics of the Trp fluorescence changes depended on the excitation wavelength. Using an excitation wavelength of 296 nm, the Trp fluorescence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitation wavelength of 290 nm, the rapid drop in tryptophan fluorescence mainly monitors the dissociation of the first of the two Ca2+ ions to be released from Ca2+-ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived ATPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analysis) is faster than the true rate of dissociation of the first Ca2+ ion and in a subsequent slower rise related to dissociation of the second Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of these cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fluorescence level and significantly affects the cooperativity of Ca2+ binding at equilibrium.

Brominated detergents as tools to study protein-detergent interactions.

In order to study protein-detergent short-range interactions, we analyzed the quenching by brominated detergents of reticulum sarcoplasmic (SR) Ca(2+)-ATPase intrinsic fluorescence. For this purpose, 7,8-dibromododecyl beta-maltoside and 2-O-(10,11-dibromoundecanoyl)sucrose, brominated analogs of two non-ionic detergents, the frequently used dodecylmaltoside and the newly synthesized 2-O-lauroylsucrose respectively, were prepared. Rayleigh scattering measurements showed that the brominated detergents efficiently and rapidly solubilized SR vesicles like their non-brominated analogs although at slightly higher concentrations. Similarly, each analog had a slightly higher critical micellar concentration than its parent detergent. The partition coefficient K (expressed as the ratio of the molar fraction of detergent in the SR lipid phase to that in the aqueous phase, at pH 7.5 and 20 degrees C) was similar for brominated and non-brominated dodecyl maltoside (3.5-4 x 10(5)) and slightly lower for dibromoundecanoylsucrose (approximately 10(5)) than for lauroylsucrose (approximately 2 x 10(5)). At detergent concentrations too low to solubilize the membrane, the brominated detergents rapidly inserted (within seconds) into SR vesicles. In this concentration range, Ca(2+)-ATPase fluorescence quenching steadily increased with detergent concentration. When the membrane was saturated with detergent, the residual fluorescence was about half of its initial value, indicating significant protein-detergent, contacts, possibly due to a slightly higher affinity of Ca(2+)-ATPase for these detergents than for phospholipids. For higher detergent concentrations, solubilizing the membrane, the fluorescence continued to decrease with detergent concentration, with no evidence for a dramatic change in the average hydrophobic environment of the protein during the transition from bilayers to a soluble state. For still higher detergent concentrations, above that necessary for membrane solubilization, the fluorescence was further quenched to a residual relative value of about 20%, corresponding to further delipidation of the protein surface, in agreement with previous results [de Foresta, B., le Maire, M., Orlowski, S., Champeil, P., Lund, S., Møller, J.V., Michelangeli, F. & Lee, A.G. (1989) Biochemistry 28, 2558-2567]. Fluorescence quenching for solubilized Ca(2+)-ATPase was quickly reversed upon addition of excess non-brominated detergent. The effects of the four detergents on the Ca(2+)-ATPase hydrolysis of p-nitrophenyl phosphate were similar and correlated with the protein-detergent contacts evidenced above. In conclusion, both these brominated detergents appear to be promising tools to study protein-detergent interactions at the hydrophobic surface of a membrane protein, either in a membrane or in solubilized complexes.

Analysis of effects of corticotropin, forskolin and fluoride on activity of adenylate cyclase of bovine adrenal cortex.

A mathematical model relating the activity of adenylate cyclase (AC) with concentrations of stimulators, equilibrium dissociation constants, specific activity and efficacies of AC depending on the states of its binding sites has been developed and used for analysis of the data on activation of AC of bovine adrenal cortex plasma membranes presented in (De Foresta et al. (1987) FEBS Lett. 216, 107-112). Equilibrium dissociation constants. chi h and chi l, corresponding to high- and low-affinity forskolin-binding sites were estimated to be 0.37 and 17 microM: these constants characterize forskolin's potency more adequately than does ED50, the concentration eliciting half-asymptotic activity of AC. Corticotropin does not affect the affinity of AC for forskolin whereas fluoride increases this affinity, thus augmenting forskolin's potency. Hormone receptor of adenylate cyclase of bovine adrenal cortex has been suggested to have two or more binding sites for corticotropin. Some unidentified factor(s) may be responsible for the differences found in adenylate cyclase activity in different experiments carried out under similar conditions. The model applied for the analysis may be thought to be the best means for the moment to relate dose-response dependencies with what is known or can be hypothesized about the mechanisms underlying activation of adenylate cyclase.

Cancellation of the cooperativity of Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase by the non-ionic detergent dodecylmaltoside.

The perturbation of the kinetics of the sarcoplasmic reticulum (SR) membranous Ca(2+)-ATPase cycle by the non-ionic detergent dodecylmaltoside (DM) has been shown to exhibit specific features which were not observed with the related detergents octa(ethylene glycol) monododecylether and Triton X-100 [de Foresta, B., Henao, F. & Champeil, P. (1992) Eur. J. Biochem. 209, 1023-1034]. This previous study has been completed here by a detailed analysis of the perturbation by DM of the interaction of Ca2+ with membranous ATPase, both in its unphosphorylated and phosphorylated form. Equilibrium binding measurements, performed at pH 7.5 and 20 degrees C, showed that only one 45Ca2+ was bound with high affinity to the ATPase in the presence of maximally perturbing concentrations of DM, as compared to two 45Ca2+ in the absence of detergent. This binding was also assessed by a small decrease in the tryptophan fluorescence intensity. Binding of a second Ca2+ occurred only with a much lower affinity. In the presence of DM, the pCa dependence of the phosphorylation by [gamma-32P]ATP of the ATPase shifted towards 50-fold higher Ca2+ concentrations than in its absence. Furthermore, DM completely inhibited the cooperativity of this dependence. This shift strongly suggests that the phosphorylation of DM-perturbed ATPase requires the binding of this second, low-affinity Ca2+. In order to assess this, samples of ATPase were intramolecularly cross-linked with glutaraldehyde. This treatment stabilized the phosphorylated intermediated with occluded Ca2+ [Ross, D. C., Davidson, G.A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. Both in the absence and presence of DM, the cross-linked enzyme occluded close to two Ca2+/phosphorylated molecule. Finally, the pCa dependences of the ATPase hydrolytic activity, measured with two different high-energy substrates, ATP or p-nitrophenylphosphate (PNpP), were also found to shift towards higher Ca2+ concentrations in the presence of DM, which was again consistent with a normal coupling ratio, i.e. two bound Ca2+/substrate hydrolyzed. As compared to other detergents, the maltoside head group of DM might favor a stronger interaction with membranous ATPase, resulting in its high perturbing effect on Ca2+ binding. The loss of cooperativity of Ca2+ binding evidenced here makes DM a useful tool in the analysis of the sequence of events occurring during Ca2+ binding.

Kinetic characterization of the perturbation by dodecylmaltoside of sarcoplasmic reticulum Ca(2+)-ATPase.

We investigated the functional aspects of the interaction between the sarcoplasmic reticulum (SR) membranous Ca(2+)-ATPase and the non-ionic detergent dodecylmaltoside, using detergent concentrations allowing perturbation of the membrane but not its solubilization. At pH 7.5, the effects of dodecylmaltoside on ATPase activity and delipidation had previously been shown to resemble, in some respects, those of octa(ethylene glycol) monododecylether (C12E8), an appropriate detergent for ATPase studies. Our aim here was to explore the specific effects of dodecylmaltoside on the different steps in the ATPase catalytic cycle, which may owe their specificity to the difference between the polar head groups of dodecylmaltoside and C12E8. This was done at 20 degrees C, both at pH 6 in the absence of KCl and at pH 7.5 in the presence of 100 mM KCl, two conditions under which the characteristics of unperturbed ATPase have already been well defined. Preliminary estimation of dodecylmaltoside partition between water and SR membranes at pH 6 yielded a partition coefficient K close to 4 x 10(5) (ratio of the molar fraction of dodecylmaltoside in the lipid to that in the aqueous phase at a low detergent concentration, assuming that most of this detergent was present in the lipid phase). At near saturation of SR membranes, bound dodecylmaltoside was roughly equimolar with the constituent phospholipids. Non-solubilizing concentrations of dodecylmaltoside inhibited SR ATPase activity by up to 65-70% at pH 7.5, but not at pH 6, unlike the results of similar experiments with C12E8. The rates of the four main steps in the ATPase catalytic cycle were measured by fast kinetic techniques; they were similarly modified at both pH. Dodecylmaltoside slowed down both the rate of calcium-saturated ATPase phosphorylation and the rate of ATPase isomerization after phosphorylation, two steps which were not targets of perturbation by C12E8. The slowing down of the isomerization step by dodecylmaltoside might well explain why it inhibited overall ATPase activity at pH 7.5. In contrast to C12E8, dodecylmaltoside did not affect the dephosphorylation step, which was the main target of inhibition by C12E8 and the main rate-limiting step at pH 6. However, like C12E8, dodecylmaltoside accelerated the calcium binding-induced transition of nonphosphorylated ATPase. Another striking feature of the perturbation induced by dodecylmaltoside was that it significantly altered the binding of 45Ca2+ to the ATPase and the corresponding conformational changes. At pCa 5-5.5, it almost halved calcium binding to the ATPase but ATPase phosphorylation was unimpaired.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic characterization of the normal and procaine-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump.

We investigated the effect of the local anesthetic procaine on the activity of the calcium pump protein of sarcoplasmic reticulum (SR) vesicles. Procaine slowed down the rate of calcium uptake by SR vesicles without enhancing the vesicles' passive permeability. This slowing of the unidirectional pumping rate was reflected by the inhibition of the maximal rate of the transport-coupled Ca(2+)-ATPase activity. The inhibition was dependent on Mg2+ concentration; at optimal (i.e. low) concentrations of magnesium, half-maximal inhibition occurred with procaine concentrations close to 15-20 mM. Inhibition of ATPase was not mediated by a change in the properties of the bulk lipid phase. Procaine moderately reduced the true affinity of ATPase for ATP, whereas equilibrium binding of calcium to ATPase in the absence of ATP was virtually not modified by procaine. In fast-kinetics studies, we explored the various intermediate steps in the ATPase catalytic cycle, in order to determine which of them were targets for inhibition by procaine. We found that procaine slowed down ATPase dephosphorylation, an effect which is at least partly responsible for the observed inhibition of overall ATPase activity. In contrast, procaine accelerated the calcium-induced transconformation of unphosphorylated ATPase in the absence of ATP, and altered neither the rate of the Ca(2+)-dependent phosphorylation of ATPase, nor the rate of the dissociation of Ca2+ from phosphorylated ATPase towards the SR lumen, a critical step, the rate of which was measured by a novel fast-filtration method. These results are discussed with respect to the possible site(s) of binding of this amphiphile on the ATPase, and in relation to the contribution of individual steps in the catalytic cycle to the rate limitation of unperturbed SR ATPase activity.

Different classes of tryptophan residues involved in the conformational changes characteristic of the sarcoplasmic reticulum Ca2(+)-ATPase cycle.

Various classes of tryptophan residues in the Ca2(+)-ATPase of sarcoplasmic reticulum membranes have been distinguished on the basis of their sensitivities to certain fluorescence quenchers: the brominated phospholipid 1,2-bis(9,10-dibromostearoyl)-sn-glycero(3)phosphocholine, the calcium ionophore calcimycin (A23187) and its brominated analog (4-bromo-A23187), and the nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate. We show that tryptophans located at the protein-lipid interface are the main contributors to the well-known fluorescence intensity change occurring in parallel with the conformational rearrangement induced by addition of calcium to the ATPase or its removal; Trp-794 on the ATPase chain may be one of these tryptophans. We also show that tryptophans more deeply embedded in the transmembrane protein structure contribute to the fluorescence change observed upon phosphorylation from inorganic phosphate of the calcium-free ATPase. This phosphorylation step involves opposite changes in the fluorescence quantum yield of tryptophans located in the membrane and in the cytoplasmic regions of the ATPase. This result is in agreement with models in which phosphorylation from inorganic phosphate not only changes the ATPase conformation locally around the catalytic center, but also reorganizes the membrane portion of the ATPase by long-range action, allowing, for instance, the calcium sites to become accessible from the luminal medium.

How to evaluate the distribution of an "invisible" amphiphile between biological membranes and water.

To evaluate the distribution of an amphiphile or its binding to membranes whose properties are affected by such binding, it is only necessary to establish to what extent the dose-response to the amphiphile depends on the membrane concentration. The measured response only needs to reflect local events. This method of evaluation does not depend on the precise shape of the dose-response curve and is particularly useful for amphiphiles devoid of properties like fluorescence or radioactivity which would allow their direct assay. In this work, we establish the validity of this approach by comparing it with direct conventional determinations. Two parameters are especially suitable for such evaluation: the perturbation of an enzyme's activity, produced by many amphiphiles, and the fluorescence quenching of membrane-embedded proteins by chromophoric amphiphiles through long-range Förster transfer. We illustrate this approach in sarcoplasmic reticulum membranes containing Ca2(+)-ATPase as the main protein constituent. The equilibrium distribution of the antioxidant 4-nonylphenol was deduced from its inhibition of ATPase activity, whereas the equilibrium distribution of the calcium ionophore calcimycin (A23187) and of its brominated analog 4-bromo-A23187 were determined from their quenching of ATPase fluorescence. Apparent partition coefficients K* in the range of 10(5) (expressed as (moles of lipid/liter)-1) were obtained for these highly hydrophobic molecules.

Effects of ionizing radiations on proteins. Evidence of non-random fragmentations and a caution in the use of the method for determination of molecular mass.

We have reinvestigated the use of ionizing radiations to measure the molecular mass of water-soluble or membrane proteins. The test was performed by using the most straightforward aspect of the technique, which consists of SDS/PAGE analysis of the protein-fragmentation process. We found that exposure of purified standard proteins to increasing doses of ionizing radiation causes progressive fragmentation of the native protein into defined peptide patterns. The coloured band corresponding to the intact protein was measured on the SDS gel as a function of dose to determine the dose (D37.t) corresponding to 37% of the initial amount of unfragmented protein deposited on the gel. This led to a calibration curve between 1/D37.t and the known molecular mass of the standard proteins whose best fit gave Mr = 1.77 x 10(6)/D37.t at -78 degrees C, i.e. 35% higher than the generally accepted value at that temperature obtained from inactivation studies. However, we have to conclude that this method is useless to determine the state of aggregation of a protein, since, for all the oligomers tested, the best fit was obtained by using the protomeric molecular mass, suggesting that there is no energy transfer between promoters. Furthermore, SDS greatly increases the fragmentation rate of proteins, which suggests additional calibration problems for membrane proteins in detergent or in the lipid bilayer. But the main drawback of the technique arises from our observation that some proteins behaved anomalously, leading to very large errors in the apparent target size as compared with true molecular mass (up to 100%). It is thus unreliable to apply the radiation method for absolute molecular-mass determination. We then focused on the novel finding that discrete fragmentation of proteins occurs at preferential sites, and this was studied in more detail with aspartate transcarbamylase. N-Terminal sequencing of several radiolysis fragments of the catalytic chain of the enzyme revealed that breaks along the polypeptide chains are localized close to the C-terminal end. Examination of the three-dimensional structure of aspartate transcarbamylase suggests that radiolysis sites (fragile bonds) might be localized in connecting loops.

Detergent structure and associated lipid as determinants in the stabilization of solubilized Ca2+-ATPase from sarcoplasmic reticulum.

The properties of detergents required to substitute the lipid environment of sarcoplasmic reticulum Ca2+-ATPase with retention of good functional properties were determined by the use of a large number of diverse detergents and delipidated enzyme. Detergents having an intermediate chain length (approximately equal to C12) and a polyoxyethylene glycol or carbohydrate polar group were optimal for Ca2+-ATPase function and stabilization, while detergents with short alkyl chain (C8) or bulky head groups and many zwitterionic detergents led to rapid inactivation. Under optimal conditions (including solubilization in the E1 state), stability of delipidated Ca2+-ATPase approximated that obtained by solubilization of Ca2+-ATPase with a layer of bound lipid. Some detergents (in particular long chain members of the Tween family) were characterized by an inadequate interaction with delipidated Ca2+-ATPase, resulting in biphasic inactivation. According to analytical ultracentrifugation and high performance liquid chromatography experiments, the rapid and slow components of biphasic inactivation were due to the formation of monomeric and oligomeric Ca2+-ATPase, respectively. It is concluded that both hydrophobic and polar interactions are important for the detergent effect and that solubilizing detergents of intermediate and short chain length may be bound as a monolayer, differently than the membrane lipid. Long chain detergents cause protein aggregation and, despite their resemblance to natural lipids, are inferior in their activity-retaining properties. The previous use of such detergents to prepare oligomeric Ca2+-ATPase with long term retention of activity (cf. Møller, J. V., Anderson, J. P., and le Maire, M. (1988) Methods Enzymol. 157, 261-270) is shown to depend on the presence of residual lipid in these preparations.

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction.

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex.

Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylate cyclase of bovine adrenal cortex plasma membranes. Divergence between corticotropin and fluoride combined effects with forskolin.

The diterpene forskolin maximally stimulated bovine adrenal cortex adenylate cyclase activity 9-fold with a concentration producing half-maximum effect (ED50) of about 4 microM. The effects of forskolin and the fully active corticotropin fragment ACTH (I 24) were additive over nearly the whole range of concentration of both effectors, indicating separate and independent mechanisms of action. By contrast, 10 mM NaF blocked forskolin action in the nanomolar range of the diterpene concentration, while it allowed a partial stimulation by forskolin in the micromolar range. NaF thus reveals a heterogeneity of forskolin action in the adrenal cortex plasma membranes. Moreover, our data suggest that ACTH and NaF activation effects, both mediated by the stimulatory regulatory protein Gs, proceed through different mechanisms.

Cardiolipin vesicles can accommodate cholesterol up to 0.80 mole fraction, i.e. one molecule per cardiolipin fatty acid chain.

Cardiolipin-cholesterol interactions were studied by time-resolved fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene as probe. The residual anisotropy parameter, r infinity (reflecting the fatty acid chain packing), was measured in the liquid crystalline phase as a function of cholesterol addition. Two main results are reported: (i) a slight increase of the order parameter, S, computed from the r infinity value as S = (r infinity/r0)1/2, in the physiological concentration range of cholesterol; (ii) a sharp enhancement of S from a cholesterol mole fraction (X chl) of 0.20 and up to X chl of 0.80. This is in contrast to unsaturated lecithin systems for which a continuous increase of the order parameter was monitored, culminating at X chl = 0.50, the well-known maximum level of incorporation of cholesterol into lecithin model membranes.