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Mitochondrial aspartate aminotransferase was selectively labeled with various maleimide-linked nitroxide spin labels at the conformationally sensitive Cys166. The mobility of the spin group was found to increase with increasing length of the spacer between the nitroxide and maleimide moiety. The label with the ethylcarbamoyl group, a spacer of intermediate length, responded sensitively to conformational changes of aspartate aminotransferase. The modification with this label decreased the enzymic activity to 30% of its initial value and increased the affinity for various substrates and inhibitors 5-10-fold. Identical ESR spectra were obtained for the pyridoxal and pyridoxamine form of the enzyme. These spectra are complex, consisting of an isotropic and at least two anisotropic components. The spectral complexity is attributed to different modes of interaction of the spin label with its local protein environment giving rise to different motional states. The same changes in the ESR spectra have been observed upon formation of the adsorption complex of the pyridoxal form with a competitive inhibitor and on formation of covalent intermediates of the transamination reaction. Essentially, the isotropic component is converted to a new anisotropic one as the local environment changes due to a conformational adaptation of aspartate aminotransferase. The ESR data are consistent with an equilibrium between two conformational states of the enzyme but inconsistent with individual protein conformations of the various intermediates of the transamination reaction. The two contormational states may be assigned to the open and closed conformations as defined by X-ray crystallography. In the adsorption complex of the pyridoxal enzyme, and in the covalent intermediates, the two-state equilibrium appears to be shifted towards the closed conformation in which the spin label is more rigidly bound, as also suggested by molecular dynamic simulations of the label modelled into aspartate aminotransferase. In contrast the formation of adsorption complexes between the pyridoxamine form and aspartate or maleate was not accompanied by the same shift of the conformational equilibrium. [ABSTRACT FROM AUTHOR]

The binding of Ca2+ to monolayers and bilayers of phosphatidylserine has been investigated as a function of pH, ionic strength (NaCl concentration) and Ca2+ concentration using surface and colloid chemical techniques. The molar ratio of lipid to bound calcium decreases to 2 as the Ca2+ concentration is increased to about 0.1 mM. At [Ca2+] > 0.1 mM a 1:1 complex is formed. The apparent binding constant Ka ranges from about 106-104 l/mol depending on the Ca2+ concentration. After allowing for electrostatic effects and neighbour group interactions, the intrinsic binding constant Ki of the phosphorylserine polar group at pH 7 (I = 0.0I M), where it carries a net negative charge of one, is ≈ 104 l/mol; consistent values for Ki were obtained using several independent approaches. Ka for Ca2+ binding decreases with increasing NaCl concentration because the monovalent cations compete with Ca2+ for the same binding site. Na + and K+ are equally effective in displacing 45Ca2+ adsorbed to monolayers of phosphatidylserine, both with respect to the kinetics and the equilibrium of the displacement. Ka for the reaction between phosphatidylserine and monovalent cations is about 103-fold smaller than that of Ca2+. An investigation of the binding of Mn2+ to phosphatidylserine by both surface chemical and nuclear magnetic resonance methods shows that this cation has a similar binding constant to that of Ca2+. The Ca2+-binding capabilities of monolayers containing only carboxyl groups (i.e. arachidic acid) and phosphodiester groups (i.e. dicetyl phosphate) have also been determined ; the apparent pK for the—COOH group in monolayers is [This symbol cannot be presented in ASCII format] 9 and that for the phosphodiester group is < 4. Since these groups do not retain the same pK values when they are in close proximity in the phosphorylserine group, the relative contributions of the two groups to the binding of Ca2+ to phosphatidylserine is not obvious. [ABSTRACT FROM AUTHOR]

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations > 0.1 mM. ¹H and 31P high-resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid. The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1-10. Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+-lecithin interactions. An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA ≈ 30 l/mol was calculated at lanthanide concentrations > 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding. The complete assignment of the ¹H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cationlecithin interaction. From spin-lattice relaxation-time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site. The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site. The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions. Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of stearic hindrance due to the extended conformation of the lecithin polar group. [ABSTRACT FROM AUTHOR]

The morphology and structure of lipoprotein complexes reconstituted from apoprotein from porcine high-density lipoprotein and dimyristoyl lecithin have been studied by a range of physical techniques. At pH 5.6 and ionic strength I ... 0.1. disc-shaped particles are observed with a radius of 4.0-4.5 nm and a thickness of 4.36 ± 0.07 nm. the latter value being in good agreement with that of the average lipid bilayer thickness. The values for the number. weight and Z-average particle weights are Mn = 227000 daltons < Mw = 325000 daltons < Mz = 410000 daltons, respectively. and indicate the polydispersity of these complexes. The number-average particle weight is in good agreement with that of 236000 daltons determined from sedimentation and diffusion experiments. The latter particle weight is also consistent with the particle volume. determined from the dimensions of the disc. and the experimental partial specific volume of the complex. V25 = 0.917 ml/g. At the point inhere all the apoprotein is complexed the average lipid/protein molar ratio is 100 ± 6. Particle size and weight analysis indicate that each lipoprotein disc of radius 4.0-4.5 nm consists of 200 lipid and 2 apoprotein molecules. If lecithin is present in excess of the complex described above, all the lipid may interact with apoprotein up to molar lipid 'protein ratios of 200: 1 depending on the sonication conditions used. The lipoprotein discs undergo an ionic-strength-dependent monomer-dimer equilibrium with low ionic strength leading to the side-by-side aggregation of discs. At an ionic strength I ≈ 0.01 the disc radius is about twice that observed at I ≥ 0.1. The results with dimyristoyl lecithin — apoprotein complexes arc compared with those obtained with dilauroyl and dipalmitoyl lecithin — apoprotein complexes. Lecithin and apoprotein interact to form a loose. disc-shaped complex in which the apoprotein is oriented at the lipid-water (hydrophobic-hydrophilic) interface such that all the polar groups of both lipid and protein are readily accessible to the aqueous medium. Most of the lipid is present as a bilayer, however the cooperative motion of the lipid is significantly reduced due to the interaction with apoprotein. The latter shows a significant increase in α helical content from 50% to about 60-65% when complexed with lecithin. The dispersing (solubilizing) effect which the apoprotein has on unsonicated multilayers of lecithin is explained in terms of its surface activity and conformational properties which magi allow for the regional separation (accumulation) of opposite charges. [ABSTRACT FROM AUTHOR]

The intestinal brush-border membrane contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompanied by a partial loss of vesicle contents. Proteolytic treatment of the brush-border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush-border membrane. Activation of intrinsic proteinases of the brush-border membrane liberates water-soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush-border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr < 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G-75 SF yielding three protein peaks of apparent Mr ≥ 70000, Mr = 22000 and Mr = 11500. All three protein fractions show similar phosphatidylcholine-exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr > 70000 has the highest fusogenic activity while the protein fraction of apparent Mr = 11500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable. [ABSTRACT FROM AUTHOR]

The detailed molecular structure of the complex formed by the apoprotein from porcine high density lipoprotein and dimyristoyl phosphatidylcholine (lecithin) has been investigated by a range of physical techniques. The complex, an oblate ellipsoid with major axis 11.0 nm and minor axis 5.5 nm (see the accompanying paper), is comprised of a section of lecithin bilayer with apoprotein at the surface. The main site of interaction between protein and lipid is in the lipid glycerophosphorytcholine group region; as with native high density lipoprotein the surface of the particle consists of a mosaic of lecithin polar groups and protein. The formation of this mosaic reduces the cooperativity of the lecithin chain motions and changes the curvature of the lipid-water interface, as compared to a bilayer. Otherwise, there are no major changes in lecithin motions indicating that no strong binding of lipid to protein occurs. The interaction involves the intercalation of amphipathic, 60 % α-helical, apoprotein molecules among the lecithin molecules so that the protein resides at the lipid-water interface. The apoprotein has a high affinity for the lipid-water interface but specific lipid-protein interactions are not involved. [ABSTRACT FROM AUTHOR]