Model of interaction between a cardiotoxin and dimyristoylphosphatidic acid bilayers determined by solid-state 31P NMR spectroscopy.

The interaction of cardiotoxin IIa, a small basic protein extracted from Naja mossambica mossambica venom, with dimyristoylphosphatidic acid (DMPA) membranes has been investigated by solid-state 31P nuclear magnetic resonance spectroscopy. Both the spectral lineshapes and transverse relaxation time values have been measured as a function of temperature for different lipid-to-protein molar ratios. The results indicate that the interaction of cardiotoxin with DMPA gives rise to the complete disappearance of the bilayer structure at a lipid-to-protein molar ratio of 5:1. However, a coexistence of the lamellar and isotropic phases is observed at higher lipid contents. In addition, the number of phospholipids interacting with cardiotoxin increases from about 5 at room temperature to approximately 15 at temperatures above the phase transition of the pure lipid. The isotropic structure appears to be a hydrophobic complex similar to an inverted micellar phase that can be extracted by a hydrophobic solvent. At a lipid-to-protein molar ratio of 40:1, the isotropic structure disappears at high temperature to give rise to a second anisotropic phase, which is most likely associated with the incorporation of the hydrophobic complex inside the bilayer.     

Lipid-protein interactions. A comparative study of the binding of cardiotoxins and neurotoxins to phospholipid vesicles

It has recently been shown that cardiotoxin II from Naja mossambica mossambica specifically interacts with negatively charged phospholipids (Dufourcq, J. and Faucon, J.F. (1978) Biochemistry 17, 1170–1176). In order to investigate whether or not short neurotoxins give rise to similar interactions, four techniques have been used, namely intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene, turbidity measurements and release of 6-carboxyfluorescein trapped inside single shelled vesicles.Neurotoxin III from Naja mossambica mossambica and neurotoxin I from the venom of the scorpion Androctonus australis Hector, specifically interact with negatively charged phospholipids leading to changes in tryptophan fluorescence and to a decrease of the fluidity of the bilayer. Cardiotoxin II from the same snake venom gives similar results. On the other hand, it seems that either a very weak or no interaction at all occurs in the case of neurotoxin I from the same Naja venom.There are important differences in the behaviour of cardiotoxin and neurotoxins: (i) neurotoxins lead to only weak release of 6-carboxyfluorescein from lipid vesicles, whereas cardiotoxin II induces fast and quantitative escape of the dye and then a general breakdown of the vesicular structure; (ii) binding of neurotoxins can be easily reversed by 100–200 mM NaCl or less than 1 mM Ca²⁺ and so it is essentially electrostatic, whereas binding of cardiotoxin II seems to involve some hydrophobic contribution.The short neurotoxins and cardiotoxins from snake venom having a great homology in sequence, their differences on binding properties are discussed in terms of changes in a particular area of the sequence.     

Characterization by infrared spectroscopy of the interaction of a cardiotoxin with phosphatidic acid and with binary mixtures of phosphatidic acid and phosphatidylcholine.

The effect of cardiotoxin IIa from Naja mossambica mossambica, a small basic protein extracted from snake venom, on dimyristoylphosphatidic acid (DMPA) and on equimolar mixtures of DMPA and dimyristoylphosphatidylcholine (DMPC) has been studied by Fourier transform infrared spectroscopy. The interaction of cardiotoxin with DMPA dispersions decreases both the cooperativity of the phase transition of the lipid and the molecular order of the lipid acyl chains in the gel phase. This effect increases with the proportion of the toxin in the complexes and leads to the total abolition of the phase transition of DMPA at a lipid-to-protein molar ratio of 5. Small-angle X-ray results demonstrate that the structure of the lipid-protein complexes is poorly ordered and gives rise to broad diffusion peaks rather than to well-resolved diffraction patterns. Infrared spectra of oriented cardiotoxin-DMPA films show that the protein is not homogeneously oriented with respect to the bilayer surface. The destabilization of the gel-phase structure of DMPA by cardiotoxin also results in a deeper water penetration in the interfacial region of the lipid since more carbonyl ester groups appear to be hydrogen bonded in the presence of the toxin. The infrared results on the phosphate group vibrations also indicate clearly that the basic residues of cardiotoxin interact strongly with the phosphate group of DMPA that becomes partly ionized at a pH as low as 6.5. The results obtained on the interaction of cardiotoxin with an equimolar mixture of DMPA and DMPC clearly demonstrate the ability of this toxin to induce lateral phase separation in this mixture with one phase containing DMPA-rich domains perturbed by cardiotoxin while the second phase is composed of regions enriched in DMPC. Comparison of the results of the current study with those obtained on other basic proteins and polypeptides suggests that charge-induced phase separation occurs only when the charge density on certain regions of the protein structure is high enough to lead to efficient electrostatic interactions with anionic phospholipids. This condition occurs only when the conformation of the protein or polypeptide is well-ordered at the lipid interface.     

中华眼镜蛇毒心脏毒素对人肝癌细胞株细胞膜的影响

目的和方法：运用５７０ 型粘附式细胞仪光漂白后荧光再分布法测定单个细胞膜脂流动性的动态变化和流式细胞仪测定细胞群细胞膜电位,以观察中华眼镜蛇毒心脏毒素对人肝癌细胞株细胞膜的影响。结果：心脏毒素使肝癌细胞膜脂流动性下降,并且使肝癌细胞膜电位下降。结论：心脏毒素对人肝癌细胞株Ｈ７４０２细胞膜有损伤作用     

Adenosine triphosphatase activity of mycoplasma membranes

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.     

Isolation and partial characterization of basolateral membranes from rat proximal colonic epithelial cells

The isolation of basolateral membranes from rat proximal colonic epithelial cells is described. Cells were harvested using a technique combining chelation of divalent cations with mechanical dissociation. After homogenization, differential centrifugation yielded a 'crude' membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 10-14-fold over homogenate in ouabain-sensitive sodium-potassium dependent adenosine triphosphatase and ouabain-sensitive potassium-dependent phosphatase specific activities. SDS-polyacrylamide gel electrophoresis of this membrane revealed at least 18 protein bands with molecular weights of 14600-200000. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, free cholesterol and fatty acids were the major lipid components of this membrane. The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. Membranes and their liposomes were studied, using the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH), by steady-state fluorescence polarization. The fluorescence anisotropy was greater in the intact membranes compared to their liposomes, indicating greater fluidity in the liposomes. Compositional studies suggested that the high fluidity of this membrane was due to its low ratios of protein/lipid (w/w), cholesterol/phospholipid (mol/mol), and sphingomyelin/phosphatidylcholine (mol/mol).     

Protein-Associated Lipid of Bacillus stearothermophilus

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.     

Penetration of a cardiotoxin into cardiolipin model membranes and its implications on lipid organization

The interaction of cardiotoxin II of Naja mossambica mossambica with cardiolipin model membranes was investigated by binding, fluorescence, resonance energy transfer, fluorescence quenching, 31P NMR, freeze-fracture, and small-angle X-ray experiments. An initially electrostatic binding appeared to be accompanied by a deep penetration, most likely into the acyl chain region of the phospholipids, indicating a hydrophobic contribution to the strong interaction (KD congruent to 5 X 10(-8) M). This binding results in a fusion of unilamellar vesicles as indicated by a fluorescence-based fusion assay, freeze-fracture, and X-ray diffraction. In these fused structures freeze-fracture electron microscopy reveals the appearance of particles, which is accompanied by the induction of an isotropic component in 31P NMR. The well-defined particles are interpreted as inverted micelles, and the localization of the cardiotoxin molecule in these structures is discussed.     

Carbonic anhydrase localization in the elasmobranch rectal gland

Carbonic anhydrase (CA) activity was histochemically localized in the elasmobranch rectal gland at the light and electron microscopic levels. Reaction product in the secretory tubules was localized coincident with that reported for sodium-potassium activated adenosine triphosphatase (Na-K-ATPase): along the highly amplified basolateral plasma membranes of the epithelial cells. Reaction product was also localized along the plasma membrane of adjacent central canal epithelial cells. The results suggest that CA plays a role in modulating the environment of the intercellular space which in the secretory tubule is believed to be the paracellular pathway for sodium. The results also draw attention to the possible role of the central canal epithelium in modification of the secreted fluid.     

Ontogeny of proximal colon basolateral membrane lipid composition and fluidity in the rabbit.

Basolateral membranes from rabbit proximal colon were prepared from isolated colonocytes throughout postnatal maturation, using a modification of published techniques. In suckling (14-20 day) and post-weaning/mature (35-49 day) animals, membranes were purified approx. 10-fold, based upon the enrichment of ouabain-sensitive, sodium-potassium dependent adenosine triphosphatase activity. Membrane lipid analyses demonstrated age-dependent increases in total cholesterol and the cholesterol/phospholipid molar ratio, as well as decreases in phosphatidylethanolamine content and the fatty acid unsaturation index. Fluidity of basolateral membranes and membrane liposomes, determined from fluorescence anisotropy measurements using the lipid probes 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid, demonstrated significant, ontogenic decreases in fluidity; and, additional studies showed that fluidity changes occurred early in the weaning period (by day 24 postnatally). Arrhenius plots of liposome anisotropies suggested a bilayer lipid thermotropic transition temperature of 22 degrees C in sucklings 26 degrees C in mature rabbits. These findings demonstrate that ontogeny of colonic basolateral membranes is associated with significant modulations in lipid composition and fluidity.     

Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes

Lipid dynamics and lipid-protein interactions were examined in basolateral membranes prepared from rat proximal and distal colonic epithelial cells. The results demonstrate that: (1) these membranes have a high lipid fluidity, as assessed by steady-state fluorescence polarization studies using seven fluorescent probes; (2) lipid compositional differences exist between these membranes but their fluidity is similar; (3) fluorescence polarizations studies, using diphenylhexatriene (DPH), detect a thermotropic transition at 22–23°C in each membrane; (4) several membrane protein activities, including adenylate cyclase and sodium-potassium dependent adenosine triphosphatase ($\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$) appear to be functionally dependent on the physical state of the proximal basolateral membrane's lipid.     

The hydroxylation of tyrosine by an enzyme from third-instar larvae of the blowfly Calliphora erythrocephala.

1. A procedure was developed for the preparation of plasma membranes from experimental granulation tissue of the rat without the addition of enzymes. The yield is better than 20% and the purification at least tenfold. 2. Values are given for the activities of 5'-nucleotidase, Na-+, k-+-activated Mg-2+dependent adenosine triphosphatase and leucine beta-naphthylamidase, for lipid composition, and for the gel-electrophoretic patterns of proteins and glycoporteins in the membrane preparations. 3. The plasma membranes from the mature granulation tissue contain proportionally more protein in the lipid phase, but the specific activities of 5'-nucleotidase and Na-+,K-+-activated Mg-2+-dependent adenosine triphosphatase are smaller than in the proliferating tissue. Certain differences were repeatedly observed in the gel-electrophoretic patterns of the developmental phases. 4. The plasma membranes from the granulation tissue were compared with those from rat peritoneal macrophages and from embryonic-chick tendon cells.     

Membrane interactions of amphiphilic polypeptides mastoparan, melittin, polymyxin B, and cardiotoxin. Differential inhibition of protein kinase C, Ca2+/calmodulin-dependent protein kinase II and synaptosomal membrane Na,K-ATPase, and Na+ pump and differentiation of HL60 cells.

Interactions of certain naturally occurring, amphiphilic polypeptides with membranes were investigated. Mastoparan (wasp venom toxin), melittin (bee venom toxin), cardiotoxin (cobra venom toxin), and polymyxin B (antibacterial antibiotic) inhibited protein kinase C stimulated by phosphatidylserine bilayer or arachidonate monomer and blocked binding of [3H] phorbol 12,13-dibutyrate to protein kinase C in the presence of phosphatidylserine bilayer, with IC50 values (concentrations causing 50% inhibition) of 1-8 microM. Mastoparan and polymyxin B were much less inhibitory (IC50, 10-20 microM), whereas melittin and cardiotoxin were similarly inhibitory (IC50, 1-4 microM), when protein kinase C was activated instead by synaptosomal membrane. Kinetic analysis indicate that mastoparan inhibited protein kinase C, assayed using phosphatidylserine or synaptosomal membrane as the phospholipid cofactor, competitively with the phospholipid cofactor, in a mixed manner with CaCl2 or diacylglycerol, noncompetitively with histone, and uncompetitively with ATP, with apparent Ki values of 1.6-18.7 microM. Inhibition of Na,K-ATPase in the membrane by these polypeptides had relative potencies different from those for their inhibition of protein kinase C activated by the same membrane preparation; mastoparan and melittin inhibited the two activities with comparable potencies, but polymyxin B and cardiotoxin were far less effective in inhibiting Na,K-ATPase. The same relative inhibitory potencies of the polypeptides (melittin greater than mastoparan greater than polymyxin B) for inhibition of Na,K-ATPase were also noted for their inhibition of Ca2+/calmodulin-dependent protein kinase II, 86Rb uptake (Na+ pump) by HL60 cells and the phorbol ester-induced differentiation of the leukemia cells. These findings were consistent with discrete interactions of the polypeptides with functionally distinct sites on the membrane, leading to differential inhibition of biological activities associated with the membrane. Actions of certain polypeptides appeared to be more specific compared to those of lipid second messengers such as lyso-phosphatidylcholine and sphingosine, and the antineoplastic ether lipid analogs such as 1-O-octadecyl-2-methyl-rac-glycero-3-ophosphocholine.     

Membrane adenosine triphosphatase in synchronous cultures of Rhodobacter sphaeroides

Studies of intracytoplasmic membrane biogenesis utilizing synchronized cultures of Rhodobacter sphaeroides have revealed that most intracytoplasmic membrane proteins accumulate continuously throughout the cell cycle while new phospholipid appears discontinuously within the intracytoplasmic membrane. The resulting changes in the structure of the membrane lipids was proposed to influence the activities of enzymes associated with the intracytoplasmic membranes (Wraight, C.A., Leuking, D.R., Fraley, R.T. and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). We have extended the study of intracytoplasmic membrane biogenesis in R. sphaeroides to include the membrane adenosine triphosphatase. The membrane bound Mg2+-dependent, oligomycin-sensitive adenosine triphosphatase activity was measured throughout the cell cycle for steady-state synchronized cells of R. sphaeroides and found to accumulate discontinuously. Following treatment with an uncoupling reagent (2,4-dinitrophenol) the intracytoplasmic membrane associated adenosine triphosphatase activity was stimulated uniformly in membranes isolated at different stages of the cell cycle. The adenosine triphosphatase was also measured by quantitative immunoblots utilizing specific antibody to compare the enzyme activity and enzyme protein mass. Immunologic measurement of the adenosine triphosphatase in isolated membranes indicated a constant ratio of enzyme to chromatophore protein exists during the cell cycle in contrast to the discontinuous accumulation of adenosine triphosphatase activity. These results are discussed in light of the cell-cycle specific synthesis of the intracytoplasmic membrane.     

Cytochemical study of liposome and lipid vesicle phagocytosis

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94–99 mol% ‘fluid’ lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37°C or ‘solid’ lipid (dipalmitoylphosphatidylcholine at 37°C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1–2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the ‘fluidity’ of the target membrane, or the presence of phosphatidylserine in the target membrane.     

Human erythrocyte calmodulin. Further chemical characterization and the site of its interaction with the membrane.

Human erythrocyte and bovine brain calmodulins were indistinguishable by tryptic peptide mapping, indicating that the primary sequence of the two proteins is either very similar or identical. Calcium binding determinations of human erythrocyte calmodulin, by equilibrium dialysis and fluorescence titration, were in close agreement with previous studies on other calmodulins. The calcium-activated adenosine triphosphatase which is stimulated by calmodulin was shown to be firmly associated with smooth erythrocyte plasma membranes devoid of spectrin and actin. Kinetic titration demonstrated that there are 4500 calmodulin binding sites per erythrocyte and that the turnover number of this calcium-activated adenosine triphosphatase is 3000 mumol of Pi . (mumol of site)-1 . min-1 which is similar to the turnover numbers of other transport adenosine triphosphatases. Furthermore, calmodulin stimulates calcium-activated adenosine triphosphatase by a simple enzyme-ligand association.     

The role of sulfatide lipid domains in the membrane pore-forming activity of cobra cardiotoxin

Cobra CTX A3, the major cardiotoxin (CTX) from Naja atra, is a cytotoxic, basic β-sheet polypeptide that is known to induce a transient membrane leakage of cardiomyocytes through a sulfatide-dependent CTX membrane pore formation and internalization mechanism. The molecular specificity of CTX A3-sulfatide interaction at atomic levels has also been shown by both nuclear magnetic resonance (NMR) and X-ray diffraction techniques to reveal a role of CTX-induced sulfatide conformational changes for CTX A3 binding and dimer formation. In this study, we investigate the role of sulfatide lipid domains in CTX pore formation by various biophysical methods, including fluorescence imaging and atomic force microscopy, and suggest an important role of liquid-disordered (ld) and solid-ordered (so) phase boundary in lipid domains to facilitate the process. Fluorescence spectroscopic studies on the kinetics of membrane leakage and CTX oligomerization further reveal that, although most CTXs can oligomerize on membranes, only a small fraction of CTXs oligomerizations form leakage pores. We therefore suggest that CTX binding at the boundary between the so and so/ld phase coexistence sulfatide lipid domains could form effective pores to significantly enhance the CTX-induced membrane leakage of sulfatide-containing phosphatidylcholine vesicles. The model is consistent with our earlier observations that CTX may penetrate and lyse the bilayers into small aggregates at a lipid/protein molar ratio of about 20 in the ripple P(β)' phase of phosphatidylcholine bilayers and suggest a novel mechanism for the synergistic action of cobra secretary phospholipase A2 and CTXs.     

Effect of phospholipase-D on rat kidney mitochondria

Incubation of purified rat kidney mitochondrial fraction with phospholipase-D resulted in the accumulation of phosphatidic acid in the membrane due to the degradation of membrane-bound phosphatidylcholine, -serine and-ethanolamine Simultaneously with the hydrolysis of the phospholipids, cholesterol and protein were released from the mitochondrial membrane into the medium, and binding of Ca²⁺ by mitochondrial membranes increased. Phospholipase Dtreated mitochondrial fraction exhibited increased swellingin vitro in the early stages of incubation (15 min) after which the mitochondria were ruptured. Membrane-bound adenosine triphosphatase was partially inactivated and the enzyme activity was not significantly restored by incubation with sonicated dispersions of phosphatidylcholine,-serine and cholesterol. These results indicate that removal of choline, serine and ethanolamine from membrane-bound phospholipids disrupt phospholipid-cholesterol and phospholipid-protein association and affect functions of the membrane. Communication no, 2468.     

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.     

Immunochemical Analysis of Triton X-100-Insoluble Residues from Micrococcus lysodeikticus Membranes

Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.