Cardiac muscarinic receptors. Cooperativity as the basis for multiple states of affinity.

Cooperativity has been investigated as the mechanistic basis for effects observed with cardiac muscarinic receptors in washed membranes from Syrian hamsters. Specifically, N-[3H]methylscopolamine labeled only 66-75% of the sites labeled by [3H]quinuclidinylbenzilate at apparently saturating concentrations of each radioligand. Also, receptors labeled by N-[3H]methylscopolamine revealed three states of affinity for agonists, both in native membranes and following irreversible blockade of about 80% of the sites by propylbenzilylcholine mustard; in both preparations, guanylylimidodiphosphate (GMP-PNP) effected an apparent interconversion of sites from higher to lower affinity for agonists and from lower to higher affinity for the antagonist. Excellent and mechanistically consistent descriptions of the data were obtained in terms of a model comprising cooperative and noncooperative forms of the receptor; the former was described by a variant of the Adair equation, and the latter was included to account for low-affinity sites that survived treatment with the mustard. If differences in apparent capacity derive from negative cooperativity in the binding of N-[3H]methylscopolamine, the cooperative form of the receptor was at least trivalent in native membranes; otherwise, constraints imposed by the effects of GMP-PNP at the concentrations of radioligand used in the assays dictate that the cooperative form of the receptor was at least tetravalent. In contrast, a divalent receptor is sufficient with the data from alkylated membranes, in accord with the reduced likelihood of interactions between functional sites within an oligomeric array. A model is presented wherein the receptor interconverts spontaneously between two or more states differing in their cooperative properties. The effects of GMP-PNP can be rationalized as a shift in the equilibrium between the different states.

Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization.

Ligand-dependent quenching of tryptophan fluorescence in human erythrocyte hexose transport protein.

D-Glucose transport by the 492-residue human erythrocyte hexose transport protein may involve ligand-mediated conformational/positional changes. To examine this possibility, hydrophilic quencher molecules [potassium iodide and acrylamide (ACR)] were used to monitor the quenching of the total protein intrinsic fluorescence exhibited by the six protein tryptophan (Trp) residues in the presence and absence of substrate D-glucose, and in the presence of the inhibitors maltose and cytochalasin B. Protein fluorescence was found to be quenched under various conditions, ca. 14-24% by KI and ca. 25-33% by ACR, indicating that the bulk of the Trp residue population occurs in normally inaccessible hydrophobic regions of the erythrocyte membrane. However, in the presence of D-glucose, quenching by KI and ACR decreased an average of -3.4% and -4.4%, respectively; Stern-Volmer plots displayed decreased slopes in the presence of D-glucose, confirming the relatively reduced quenching. In contrast, quenching efficiency increased in the presence of maltose (+5.9%, +3.3%), while addition of cytochalasin B had no effect on fluorescence quenching. The overall results are interpreted in terms of ligand-activated movement of an initially aqueous-located protein segment containing a Trp residue into, or toward, the cellular membrane. Relocation of this segment, in effect, opens the D-glucose channel; maltose and cytochalasin B would thus inhibit transport by mechanisms which block this positional change. Conformational and hydropathy analyses suggested that the region surrounding Trp-388 is an optimal "dynamic segment" which, in response to ligand activation, could undergo the experimentally deduced aqueous/membrane domain transfer.

D-glucose binding increases secondary structure of human erythrocyte monosaccharide transport protein.

Purified hexose transport protein ("band 4.5") from human erythrocytes, reconstituted in vesicles of its endogenous lipids, displays minima in its circular dichroism (CD) spectrum at 222 and 207 nm, a pattern diagnostic for alpha-helical content of proteins. Upon addition of D-glucose, a saturable increment of +10-12% in negative ellipticity at 222 nm is observed stereospecifically and reproducibly. Addition of L-glucose had no effect on the CD spectrum of the transport protein. Addition of cytochalasin B (CB), a reversible inhibitor of hexose transport, had no effect itself on transporter CD spectra, but restored the spectrum at 222 nm to its original value when added in the presence of D-glucose. The observed D-glucose-induced increase in ordered secondary structure is proposed to result from incorporation into the membrane of a segment of the transport protein originally at a membrane-water interface.

Effect of hypobaric oxygen and oleic acid on respiration of Staphylococcus aureus.

The effect of hypobaric oxygen, with oleic acid in the nutrient substrate, on respiration and slime production by a pulmonary isolate of Staphylococcus aureus was investigated. Under hypobaric, but not normal oxygen pressure, the addition of oleic acid to the nutrient broth caused the bacteria to drastically diminish their demand for oxygen and initiate the secretion of extrapolymeric substances (slime). The decrease in oxygen demand was found to result from impairment of the capacity to reduce and oxidize the coenzyme NAD. Prior to the initiation of slime production, the rate of oxidation exceeded the rate of reduction of the coenzyme, whereas with slime production the rate of reduction was greatest. This could result in elevation of the cellular NADH, which could stimulate gluconeogenesis and thereby increase the synthesis of the carbohydrate component of the slime. The results suggest that staphylococcal infections, such as those of the pulmonary tract in cystic fibrosis and essential fatty acid deficiency, may occur in response to a peculiar chemical environment.

Binding of human normal and multiple sclerosis-derived myelin basic protein to phospholipid vesicles: effects on membrane head group and bilayer regions.

The detailed interaction of human myelin basic protein (MBP) with charged lipids may be critical in organizing the myelin sheath into its biologically functional structure. Carbon-13 and phosphorus-31 nuclear magnetic resonance spectroscopy has been used to study this interaction by examining spectral consequences of additions of MBP to membrane preparations of the negatively charged lipid phosphatidylglycerol (PG). Lipid head group 13C and 31P linewidths were found to narrow upon addition of protein, while concomitant broadening was noted for bilayer carbon resonances. At intermediate MBP/PG ratios, two components in slow exchange on the NMR time scale (bulk PG and a protein-induced PG domain) were observed for the 13C resonance of the head group carbon atom adjacent to phosphate. These results, and other spectral evidence, suggested that head groups in free PG vesicles are motionally restricted by intermolecular interactions which are disrupted by competition with MBP Lys and Arg positively charged side chains. Titration of PG with the homopolypeptide poly-L-lysine produced comparable effects on PG 13C head group spectra, indicating that electrostatic attractions constitute the primary basis of the observed interactions. Vicinal and/or geminal 13C-31P coupling constants measured from the spectra of PG head group carbons were found to be essentially invariant for free PG in dimethyl sulfoxide solution, free PG vesicles, PG vesicles + MBP, and PG vesicles + poly-L-lysine. Comparison of the spectral effects induced in PG head group resonances by normal vs multiple sclerosis-derived MBP (MS-MBP) indicated that the MS-MBP is relatively less effective in converting PG to the protein-induced domain, a result which was attributed to increased protein self-aggregation arising from the reduced net positive character of the MS protein samples.

In vitro response of Staphylococcus aureus from cystic fibrosis patients to combinations of linoleic and oleic acids added to nutrient medium.

The effect of supplementing nutrient substrate with various combinations of concentrations of oleic and linoleic acids on the growth of 11 strains of Staphylococcus aureus was assessed. Whereas increasing the concentration of linoleic acid by itself greatly diminished the growth of all 11 strains, concomitant increases in oleic acid greatly diminished the inhibitory effect of linoleic acid. With oleic acid in the nutrient substrate, most of the strains were induced to produce slime which surrounded the cells. Since the slime incorporated oleic but not linoleic acid, such slime production isolated the cells from direct contact with the growth inhibitor, linoleic acid.

Reversible cryogenic alteration of the ultraviolet absorption spectra of the olefinic bonds in lipid membranes.

In earlier studies it was found that the association of basic polypeptides with lipid vesicles drastically altered ultraviolet absorption by the olefinic bonds of the lipid. Such an effect suggested that the polypeptide was increasing the polarity of the chromophore environment by either direct interaction with the acyl chains or by inducing their hydration. It is reported here that freeze-thaw cycling, which was expected to allow hydration of the olefinic-bond region of the membranes, caused the same spectral alteration as vesicle interaction with basic polypeptides. When these vesicles were subsequently placed under conditions that would be expected to accelerate the escape of water entrapped within the membranes (i.e., by placing them under vacuum or adding sucrose to establish a high osmotic gradient to their exterior), the absorption spectrum was rapidly restored to that for olefinic bonds in a nonpolar environment. Since placing the polylysine- dioleoylphosphatidylcholine (DOPC) vesicle interaction product under the same conditions restored the spectral intensity, at 190 nm, to between 80 and 85% of that for the lipid in a nonpolar environment, it seems that a major effect of polylysine on DOPC membranes may be though induction of hydration of their interior.

Vesicle surface charge and polylysine modification of ultraviolet absorption by the olefinic bonds in charged lipid.

The results of electrical conductivity and ultraviolet absorption studies on bilayer vesicles, composed of various mixtures of saturated dipalmitoylphosphatidylcholine (DPPC)and unsaturated bovine brain phoaphatidylserine (PS), in the presence and absence of polylysine indicate the following. (i) The two kinds of lipid maintain a strong transbilayer segregation over a wide range of proportions. The charged lipid (PS) preferentially locates in the inner, at PS proportions of 33% or less, and in the outer layer of the membranes when its proportion is increased to 50%. (ii) At PS proportions of 33% of less, where DPPC is the major component of the outer layer, the ability of polylysine to modify ultraviolet absorption by the olefinic bonds in the PS depends on the fluidity of the phosphatidylcholine. At higher PS proportions, where PS preferentially locates in the outer layer, modification of the ultraviolet absorption spectrum of the lipid by polylysine occurs only when the surface charge of the vesicles is diminished by the addition of bivalent metal ions. (iii) The polylysine-induced change in the ultraviolet absorption spectrum of the olefinic bonds in the vesicles was found to be very similar to that induced by dispersing unsaturated fatty acids in water rather than dissolving them in a nonpolar solvent. This suggests that polylysine modification of the vesicles spectrum may be the result of deep hydrophobic penetration by the polypeptide causing hydration and(or) parallel alignment of the dipole moments of the absorbing chromophores.

The effect of polypeptide-lipid interactions on the ultraviolet spectrum of the olefinic bonds of the lipid.

The effect of associating acidic and basic polypeptides with dilute suspensions of vesicles composed of various unsaturated phospholipids was assessed with regard to optical density and ultraviolet absorption. Associating basic polypeptides with phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol vesicles, or acidic polypeptide with phosphatidylcholine vesicles, caused an increase in the optical density of the preparations, with no measurable effect on the intensity of the ultraviolet spectrum of the olefinic bonds of the lipid. Associating basic polypeptides with phosphatidylcholine vesicles, in addition to causing similar increases in optical density, resulted in a large decrease in the intensity of ultraviolet absorption by the olefinic bonds. This implies that the interaction between the basic polypeptides and phosphatidylcholine vesicles results in major alterations in the microenvironment of the olefinic bonds, which would require intimate association of the polypeptide with the ninth carbon region of the acyl chains. These observations support the conclusion, drawn from our earlier studies, that the association of basic polypeptides and liquid crystalline phase phosphatidylcholine vesicles is peculiar in that it involves a major hydrophobic component.