Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide γ-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10–40°C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol dramatically decreases partition (2100 falls to 100, at 10°C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.     

Partition of DDT in synthetic and native membranes

Partition of DDT (2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane) was determined in artificial and native membranes. Partition in egg phosphatidylcholine of about 260 000 is independent of temperature over the range from 10 to 40 degrees C, in which the lipid is in the liquid-crystalline state. Incorporation of 50 mol% cholesterol decreases DDT partition to about 120 000. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in DDT partitioning. Partition decreases symmetrically in the temperature ranges to both sides of the phase transition. The insecticide is preferentially accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 336 000, 180 000 and 88 000 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. Partition values in native membranes decrease sequentially as follows: sarcoplasmic reticulum, mitochondria, myelin, brain microsomes and erythrocytes. This sequence is similar to that observed in related liposomes of total extracted lipids, although the absolute partitions showed decreased values. Partition of DDT in native membranes exhibits a negative temperature coefficient not apparent in related lipid dispersions. The effect of intrinsic membrane cholesterol on partition of DDT was also investigated.     

Effect of short-chain primary alcohols on fluidity and activity of sarcoplasmic reticulum membranes

Intramolecular excimer formation with the fluorescent probe 1,3-di(1-pyrenyl)propane, differential scanning calorimetry, and X-ray diffraction were used to assess the effect of ethanol, 1-butanol, and 1-hexanol on the bilayer organization in model membranes, sarcoplasmic reticulum (SR) lipids and native SR membranes. These alcohols have fluidizing effects on membranes and lower the main transition temperature of dimyristoylphosphatidylcholine (DMPC), but only 1-hexanol alters the cooperativity of the phase transition and significantly increases the thickness of DMPC bilayers. The interaction of the three alcohols with the SR Ca2+ pump was also investigated. Hydrolysis of ATP and coupled Ca2+ uptake are differently sensitive to the three alcohols. Whereas ethanol and 1-butanol inhibited the Ca2+ uptake, 1-hexanol stimulated it. Nevertheless, the energetic efficiency of the pump (Ca2+/ATP) is not significantly affected by ethanol or 1-hexanol, but uncoupling was observed with 1-butanol at high concentrations. The different effects of alcohols on the activity of SR membranes rule out an unitary mechanism of action on the basis of fluidity changes induced in the lipid bilayer. Depending on the chain length, the alcohols interact with the SR membranes in different domains, perturbing differently the Ca2+-pump activity.     

Modulation of sarcoplasmic reticulum Ca2+-pump activity by membrane fluidity

Intramolecular excimerization of 1,3-di-1-pyrenylpropane [Py(3)Py] was used to assess the fluidity of sarcoplasmic reticulum membranes (SR); on the basis of the spectral data, the probe incorporates completely inside the membrane probably somewhere close to the polar head groups of phospholipid molecules, however not in the very hydrophobic core. The excimerization rate is very sensitive to lipid phase transitions, as revealed by thermal profiles of dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Cholesterol abolishes pretransitions and broadens the thermal profiles of the main transitions which vanish completely at 50 mol % sterol. Excimer formation in liposomes of SR total lipid extracts does not show any sharp transitions, as in the case of DMPC and DPPC. However, the plots display discontinuities at about 20 degrees C which are broadened by cholesterol and not observed at 50 mol % sterol. Also cholesterol has been incorporated in native SR membranes by an exchange technique allowing progressive enrichment without changing the phospholipid/protein molar ratio. As in liposomes, discontinuities of excimer formation at 20 degrees C are broadened by cholesterol enrichment. The full activity of uncoupled Ca2+-ATPase is only affected by cholesterol above a molar ratio to phospholipid of 0.4. However, a significant decrease in activity (about 20%) is only noticed at a ratio of 0.6 (the highest technically achieved); at this ratio, about 28 lipid molecules per Ca2+-ATPase are expected to be relatively free from cholesterol interaction. The vesicle structure is still intact at this high ratio, as judged from the absence of basal activity (not Ca2+ stimulated).(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of urease into liposomes.

1. Plasma membranes were isolated from ascites hepatoma AH-130 and rat livers with or without partial hepatectomy or bile duct ligation. Reciprocal manifestations of two marker enzymes for plasma membranes were observed in these membrane preparations; alkaline phosphatase activity was found much higher in the hepatoma membrane than in any preparations of the liver membranes, whereas 5'-nucleotidase activity was much lower in the former than in the latter. 2. Effects of lectins and anti-plasma membrane antiserum on these two marker enzymes were examined. The results showed that about 50% of apparent activity of 5'-nucleotidase found in the hepatoma membrane was exhibited by alkaline phosphatase. 3. Localizations of alkaline phosphatase and 5'-nucleotidase in polyacrylamide gels after electrophoresis were demonstrated using 5'-AMP and 5-Br, 4-Cl-indoxylphosphate as substrate. There was a difference in electrophoretic mobility between the alkaline phosphatase of the hepatoma and that of the liver.     

Alkalinization within sarcoplasmic reticulum during the uptake of calcium ions.

The pH indicator, bromothymol blue, was incorporated into sarcoplasmic reticulum vesicles which bind more than 90% of the total added dye. The sequestered dye does not respond to changes in external pH upon addition of acid to the medium, since the decrease of absorbance at 616 nm is very slow. The absorbance of sequestered dye at 616 nm increases suddenly after triggering the transport of Ca²⁺ by ATP at a rate much higher than that of Ca²⁺ uptake, and declines when Ca²⁺ has been accumulated. When the uptake of Ca²⁺ is followed in the presence of oxalate, the absorbance of the indicator declines after the first phase of Ca²⁺ uptake. The results suggest that a transient alkalinization occurs rapidly inside the vesicles and reflects the formation of a transmembrane proton gradient responsible for sustaining the Ca²⁺ transport.     

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

The translational diffusion of bovine rhodopsin, the Ca2+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein greater than or equal to 3000/1) in liquid-crystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100 000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X 10(-8) cm2/s. In consideration of the sizes of the membrane-bound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbrück, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113]. Lipid diffusion was also examined in th same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X 10(-8) cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Tr?uble, H., & Sackmann. E. (1972) J. Am. Chem. Soc. 94, 4499-4510] may have to be invoked to explain the lipid diffusion behavior.     

Excretion of total phosphorus,dissolved reactive phosphorus,ammonia, and urea by Lake MichiganMysis relicta

Rates of nutrient release byMysis relicta in Lake Michigan were measured on five nights at a 45-m station near Milwaukee, Wisconsin, U.S.A., in the summer of 1977. Nocturnal vertical migrations of the mysids were monitored with both echosounder tracings and vertical net tows. Estimates of the total areal dry mass of the mysids ranged from 600 to 1 820 mg m–2. Rates of release of dissolved reactive phosphorus, total phosphorus, ammonia, and urea were measured in dark incubations on shipboard. Excretion experiments were initiated immediately after mysids were collected from each of several vertical net hauls. The depths of maximum mysid densities corresponded approximately with a deep phytoplankton peak located in the vicinity of the thermocline. Semiquantitative demands for N and P by phytoplankton within this peak were obtained from¹⁴C estimates of primary production from a previous study, assuming a constant C:N:P ratio for the algae. These algal nutrient demands were compared to potential N and P release by the mysids to obtain a first approximation of the relative rates of nutrient supply and demand for the field phytoplankton populations. Our analysis indicates that mysids may directly supply about 1–10% of the daily N and P demands of the phytoplankton in the deep peak. However, indirect interactions betweenMysis relicta and other organisms, such as small zooplankton and fishes, could be major factors in nutrient recycling within the metalimnion and subthermocline region of Lake Michigan.Contribution No. 238, Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Wisconsin.Contribution No. 238, Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Wisconsin.     

Oxalate transfer across the membranes of sarcoplasmic reticulum during the uptake of Ca++

The kinetic profile of Ca++ uptake in the presence of oxalate is biphasic. An initial phase independent on oxalate is followed by an oxalate-dependent phase delayed in time. The ionophore X-537A only abolishes the net Ca++ uptake if added before the onset of the oxalate phase. However, during this phase, X-537A suddenly releases an amount of Ca++ similar in quantity to that released in the initial phase. The delay of the oxalate-dependent phase is a function of pH. At pH of about 5.5, the oxalate phase and simultaneous calcium oxalate precipitation would theoretically start at the beginning, with no delay. Ejection of protons during Ca++ uptake is strongly depressed by oxalate, but not by other organic anions which do not trap Ca++. It is suggested that oxalate is transferred to the inside of the vesicles as a monoprotonated species at expense of protons ejected by the Ca++-pump during the uptake of Ca++.     

Exploring the carbohydrate‐binding ability of Canavalia bonariensis lectin in inflammation models

Lectins are a group of proteins of non‐immune origin recognized for their ability to bind reversibly to carbohydrates. Researchers have been intrigued by oligosaccharides and glycoconjugates for their involvement as mediators of complex cellular events and then many biotechnological applications of lectins are based on glycocode decoding and their activities. Here, we report a structural and biological study of a ConA‐like mannose/glucose‐specific lectin from Canavalia bonariensis seeds, CaBo. More specifically, we evaluate the binding of CaBo with α‐methyl‐D‐mannoside (MMA) and mannose‐1,3‐α‐D‐mannose (M13) and the resultant in vivo effects on a rat model of acute inflammation. A virtual screening was also carried out to cover a larger number of possible bindings of CaBo. In silico analysis demonstrated the stability of CaBo interaction with mannose‐type ligands, and the lectin was able to induce acute inflammation in rats with the participation of the carbohydrate recognition domain (CRD) and histamine release. These results confirm the ability of CaBo to interact with hybrid and high‐mannose N‐glycans, supporting the hypothesis that CaBo's biological activity occurs primarily through its interaction with cell surface glycosylated receptors.