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Bile salts are surfactants that partition into phospholipid bilayers. When liposomes or membranes are exposed to mixed solutions of bile salts, the more hydrophobic bile salt species associate preferentially with the lipid bilayer. As a consequence, in the aqueous phase, the free monomeric concentration of bile salt declines and the more hydrophilic species become relatively enriched. Above a critical saturating concentration of lecithin-associated bile salt, a phase transition occurs with loss of membrane integrity and formation of mixed micelles. In this paper we present a quantitative model which, for mixed solutions of bile salt taurine conjugates, predicts the distribution of bile salt monomers between large unilamellar vesicles composed of lecithin and cholesterol and the aqueous phase. The model is based on association isotherms for individual bile salts, determined by an ultrafiltration method with empirical curve fitting, and is critically dependent upon the observation that association coefficients of each bile salt are a function of the total bound bile salt/lecithin mole ratio. Given the concentrations of individual bile salts, lecithin and cholesterol, the model permits calculation of the membrane-bound bile salt/lecithin ratio and the concentration of each bile salt remaining free as soluble monomer in the aqueous phase, as well as the overall hydrophilic-hydrophobic balance (hydrophobicity index) of the bile salts remaining free in aqueous solution. Distribution data determined empirically for a variety of mixtures of bile salt taurine conjugates and large unilamellar vesicles of varying cholesterol:lecithin ratio agree closely with predictions. This model may be of value in predicting the physical, biological and toxic properties of mixed bile salt solutions.
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PMID:Distribution of mixtures of bile salt taurine conjugates between lecithin-cholesterol vesicles and aqueous media: an empirical model. 921 49

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, kon = 10(11) M-1 s-1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, kon is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (koff) is approximately 10 s-1 (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca2+-calmodulin (Ca2+.CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 micrM Ca2+.CaM. Our results suggest that Ca2+.CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4, 5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).
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PMID:Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin. 934 Nov 59

The zona pellucida is the extracellular coat that surrounds the mammalian oocyte. It forms a spherical shell of remarkably uniform thickness (5-10 microns in eutherian mammals). The mouse is currently the largest source of data on the zona pellucida and this review is built largely on these data. The zona pellucida is composed of three proteins in both mice and humans: ZP1, ZP2 and ZP3. These proteins are glycosylated and, in mice, have mature relative molecular masses of 200,000, 120,000 and 83,000, respectively. ZP1 is a dimer of two apparently identical subunits. All three mouse proteins have been sequenced and possess transmembrane domains at their C-terminal ends coupled with furin cleavage sites immediately upstream. Sequence data have been used to provide an accurate assessment of the mole ratios of the three proteins. The ratio of ZP2:ZP3 is close to 1:1, whereas ZP1 is approximately 9% of the combined mole amounts of ZP2 and ZP3. Ultrastructural evidence suggests that the mouse zona pellucida is composed of filaments constructed by head-to-tail association of globular proteins. The coordinate synthesis of the three zona pellucida proteins coupled with the near 1:1 stoichiometry of ZP2 and ZP3 is consistent with a model in which ZP2-ZP3 heterodimers are the basic repeating units of the filament, with cross-linking of filaments by dimeric ZP1. This model is also consistent with data from ZP2 and ZP3 gene knockout and antisense experiments. However, the structure remains unproven. The small amount of ZP1 relative to ZP2 and ZP3 may have important implications for the distribution of ZP1 cross-links, since the number of cross-linking sites potentially exceeds the number of ZP1 dimer molecules by a considerable margin. The evidence that ZP1, ZP2 and ZP3 are all synthesized via a membrane-bound step is discussed and two models are proposed for the assembly of the zona pellucida. The cortical reaction and its effect on the zona pellucida are examined in detail. It is shown that the amount of material released by cortical granules could be of the order of 30% by mass of ZP1, and that if this material was distributed predominantly on the inner face of the zona pellucida, its local concentration could approach that of ZP1. A model in which the zona block to polyspermy is caused by direct titration of zona pellucida binding sites is suggested as an alternative to the explanation that relies on enzyme cleavage of ZP2 to ZP2f. Finally, some of the major experimental and structural issues that remain to be addressed are identified.
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PMID:Three-dimensional structure of the zona pellucida. 941 78

Pressure stability of the complex formed between F1-ATPase and the inhibitor protein (IP) was studied in the membrane-bound and soluble, purified forms of beef-heart mitochondrial enzymes. A latent preparation of submitochondrial particles (SMP-MgATP) initially exhibits low hydrolytic activity. Dissociation of IP increases the activity about 10-fold. This increase occurs in parallel with an increase in sensitivity to pressure inactivation. The membrane-bound, latent IP-F1-ATPase complex is activated 2.5-fold when incubated at a pressure of 1.7 kbar, suggesting dissociation of IP. A fully active preparation of submitochondrial particles depleted of IP (AS-particles) is highly pressure labile when compared with the latent form. In the absence of IP, soluble purified F1-ATPase is also inactivated by pressure. In contrast, the soluble IP-F1-ATPase complex is very resistant to pressure, as evidenced by enzymatic and fluorescence studies. Based on the pressure-titration experiments, binding of IP stabilizes the F1-ATPase complex by 1.54 kcal per mole of complex. The substrate MgATP confers additional protection on both preparations only in the presence of IP. Glycerol appears to prevent dissociation of IP and therefore protects SMP-MgATP from pressure inactivation. Our results demonstrate that in addition to its regulatory role in catalysis, IP stabilizes the structure of the F1-ATPase complex. The pressure-induced dissociation of IP from F1-ATPase and its prevention by glycerol suggest that nonpolar in addition to electrostatic interactions are important for the binding of IP to the regulatory site.
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PMID:Pressure effects on the interaction between natural inhibitor protein and mitochondrial F1-ATPase. 944 19

Similarly to the recently described methanol dehydrogenase (MDH) from Methylocystis sp. GB 25 (Grosse et al. 1997) MDH from Methylosinus sp. WI 14 is able to catalyse the oxidation of methanol to formate directly. The enzyme was purified about 9-fold to electrophoretic homogeneity and is localised in the soluble fraction. The relative molecular mass of the native enzyme has been determined to be 140 kDa. It is composed of two identical subunits of relative molecular mass 70 kDa. The amino terminal sequence shows a strong similarity (a match of 80% over the first 20 amino acids) to the MDH from Methylocystis sp. GB 25. PQQ could be detected as the prosthetic group of MDH in the purified enzyme fraction by using the apoenzyme of a membrane-bound glucose dehydrogenase from Pseudomonas aeruginosa. A PQQ ratio of 1.3 per mole MDH was estimated. The purified enzyme has an optimum activity at pH 9.0 and at 57 degrees C. MDH from Methylosinus sp. WI 14 oxidises only primary alcohols up to octanol and several aldehydes. The estimated K(m)-values vary between 0.18 mM for the sorbic alcohol and 6.3 mM for butanol and show no dependence upon the chain length.
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PMID:Purification and properties of methanol dehydrogenase from Methylosinus sp. WI 14. 972 24

We have conducted a detailed study of the effect of membrane cholesterol content on the initial hydrolytic activity of Crotalus durissus terrificus venom phospholipase A2 (sPLA2) in large unilamellar vesicles of cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and cholesterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC) at 37 degrees C. The activity was monitored by using the acrylodan-labeled intestinal fatty acid binding protein and HPLC. In contrast to conventional approaches, we have used small cholesterol concentration increments ( approximately 0.3-1.0 mol %) over a wide concentration range (e.g., 13-54 mol % cholesterol). In both membrane systems examined, the initial hydrolytic activity of sPLA2 is found to change with cholesterol content in an alternating manner. The activity reaches a local minimum when the membrane cholesterol content is at or near the critical cholesterol mole fractions (e.g., 14.3, 15.4, 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol) predicted for cholesterol regularly distributed in either hexagonal or centered rectangular superlattices. According to the sterol regular distribution model [Chong, P. L.-G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10069-10073; Liu et al. (1997) Biophys. J. 72, 2243-2254], the extent of lipid superlattices is maximal at the critical cholesterol mole fractions, at which the membrane free volume is minimal. Thus, our present data can be taken to indicate that the initial hydrolytic activity of sPLA2 is governed by the extent of cholesterol superlattice. These data provide the first functional evidence for the formation of cholesterol superlattices in both saturated (e.g., DMPC) and unsaturated (e.g., POPC) liquid-crystalline phospholipid bilayers. The data also illustrate the functional importance of cholesterol superlattice and demonstrate a new type of regulation of sPLA2. Furthermore, upon binding to cholesterol/POPC large unilamellar vesicles, the intrinsic fluorescence intensity of sPLA2 shows an alternating variation with cholesterol content, exhibiting a minimum at the critical cholesterol mole fractions. This result suggests that either the number of sPLA2 bound to lipid vesicles or the conformation of membrane-bound sPLA2 or both vary with the extent of the cholesterol superlattice in the plane of the membrane.
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PMID:Evidence for a regulatory role of cholesterol superlattices in the hydrolytic activity of secretory phospholipase A2 in lipid membranes. 1019 97

Type II beta phosphatidylinositol phosphate kinase is a representative phosphatidylinositol phosphate kinase that is active against membrane-bound substrates. The structure of the enzyme contains a flattened basic face that spans the crystallographic dimer interface and is adjacent to the active site. Analytical ultracentrifugation shows that phosphatidylinositol phosphate kinase is a dimer in solution. Modeling suggested that the flattened face binds to acidic phospholipids by electrostatic interactions. The enzyme binds to acidic vesicles containing phosphatidylserine, phosphatidic acid, or phosphoinositides mixed with phosphatidylcholine, but not to neutral phosphatidylcholine vesicles. Binding to acidic vesicles is abolished in the presence of 1.0 M NaCl, consistent with an essential electrostatic contribution to the free energy of binding. The +14 charge on the flattened face of the dimer was reduced to +2 in the triple mutant Lys72Glu/Lys76Glu/Lys78Glu. The mutation has no effect on dimerization, but reduces the apparent KA for 25% phosphatidylserine/75% phosphatidylcholine mixed vesicles by 16-fold. The reduction in the level of binding can be ascribed to a loss of electrostatic interactions based on the finite difference solution to the Poisson-Boltzmann equation. The mutant reduces catalytic activity toward phosphatidylinositol 5-phosphate by approximately 50-fold. The wild-type enzyme binds half-maximally to phosphatidylinositol 4,5-bisphosphate-containing vesicles at a mole fraction of 0.3% in a phosphatidylcholine background, as compared to a 22% mole fraction in phosphatidylserine. The binding to phosphatidylinositol 4,5-bisphosphate-containing membranes is less sensitive to salt and to the triple mutation than binding to phosphatidylserine-containing membranes, suggesting that at least part of phosphatidylinositol 4,5-bisphosphate's interaction with the enzyme is independent of the flattened face. It is concluded that the flattened face of type II beta phosphatidylinositol phosphate kinase binds to membranes through nonspecific interactions, and that this interaction is essential for efficient catalysis.
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PMID:The flattened face of type II beta phosphatidylinositol phosphate kinase binds acidic phospholipid membranes. 1056 96

The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.
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PMID:High-level expression of human liver monoamine oxidase B in Pichia pastoris. 1104 57

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.
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PMID:Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain. 1168 30

Apoptosis is characterised by a series of typical morphological features, such as nuclear and cellular convolution, chromatin condensation and the final disintegration of the cell into membrane-bound apoptotic bodies, which are phagocytosed, by neighbouring cells. Relocation of phosphatidylserine residues from the inner leaflet of the cellular membrane to being exposed on the cell surface is a necessary event for the phagocytic elimination of apoptotic cell debris. Using the MOLT-4 lymphoblastoid leukaemic cell line we investigated whether the formation of apoptotic bodies and loss of phosphatidylserine asymmetry were causally related. We have previously demonstrated that classical apoptotic morphology, including production of apoptotic bodies, was only possible in etoposide-treated MOLT-4 cells when administered in the presence of non-cytotoxic doses (200 microM) of aurin tricarboxylic acid (ATA). Electron microscopic analysis, followed by the quantitation of the ultrastructural morphological features of apoptotic MOLT-4 cells, demonstrated that the etoposide and ATA co-treatment, which caused the cellular fragmentation into apoptotic bodies, was closely associated with extensive chromatin condensation in individual cells. In this model however, the addition of ATA to frank cytotoxic doses of etoposide (50 microM), which we confirmed lead to formation of apoptotic bodies, caused no further increase in externalisation of phosphatidylserine moieties as determined by staining with fluorescence labelled annexin V. Consequently, in MOLT-4 cells undergoing etoposide-induced apoptosis, the mole-cular mechanisms leading to loss of phosphatidylserine asymmetry and the formation of apoptotic bodies are not causally related.
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PMID:Etoposide-induced apoptosis in lymphoblastoid leukaemic MOLT-4 cells: evidence that chromatin condensation, loss of phosphatidylserine asymmetry and apoptotic body formation can occur independently. 1177 6


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