Adaptation of biological membranes to temperature. The effect of temperature acclimation of goldfish upon the viscosity of synaptosomal membranes

The fluidity of synaptosomal membrane preparations isolated from goldfish acclimated to 5, 15 and 25°C and from rat has been estimated using the fluorescence polarisation technique with 1,6-diphenyl-1,3,5-hexatriene as probe. Membranes of cold-acclimated goldfish were more fluid than those of warm-acclimated goldfish when measured at an intermediate temperature, indicating a temperature-dependent regulation of this parameter. Similarly, membranes of warm-acclimated goldfish were more fluid than those prepared from rat brain. Liposomes prepared from the purified phospholipids of goldfish and rat synaptosomal preparations showed differences similar to those of the native membranes. Increased membrane fluidity of cold-acclimated goldfish was correlated with a decrease in the proportion of saturated fatty acids of the major phospholipid classes and an increased unsaturation index in choline phosphoglycerides. Rat membranes showed a substantial reduction in unsaturation index and an increase in the proportion of saturated fatty acids compared to the membranes of 25°C-acclimated goldfish. The cholesterol content of synaptosomal membranes of goldfish was unaffected by acclimation treatment.The role of homeoviscous adaptation in the compensation of the rates of membrane processes during thermal acclimation, and upon the resistance adaptation of poikilotherms to extreme temperatures is discussed.     

A differential polarized phase fluorometric study of the effects of high hydrostatic pressure upon the fluidity of cellular membranes

The effects of high hydrostatic pressure (up to 2 kbar) upon the fluidity and order of the synaptic and myelin membrane fractions of goldfish brain have been studied by using steady-state and differential polarized phase fluorometry. Probe motion provided a measure of membrane order (r infinity) and probe rotational rate (R). Membrane order became progressively greater as pressure was increased up to approximately 2 kbar. This effect was similar over the temperature range 5.6-34.3 degrees C. An increase in pressure of 1 kbar had an effect on membrane order that was equivalent to a 13-19 degrees C reduction in temperature. Membrane order was essentially identical during pressurization and depressurization. At 5.6 degrees C, pressurization caused a large increase in R, and similar, though less dramatic, anomalies occurred at higher temperatures. It is suggested that this is due to the segregation of probe molecules in highly ordered membranes, which leads either to excitation transfer between 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules or to changes in the rotational motion of DPH from "sticking" to "slipping".     

Homeoviscous theory under pressure: II. The molecular order of membranes from deep-sea fish

The molecular order of brain and liver membranes isolated from deep sea and continental shelf fish species have been estimated and compared using the fluorescence polarization technique in order to determine whether life in a high pressure habitat is associated with an adjustment of membrane order. Fish were trawled at depths between 200 m and 4000 m, liver and brain membranes were fractionated, and fluorescence polarization was measured at 4°C and ambient pressure. Polarization of the brain myelin fraction provided a statistically significant regression with depth of capture ($\text{P<0.001}$) with a slope of ?0.004 km^?1. This change in polarization with depth was sufficient to offset approximately half of the pressure-induced increase in polarization and thus represents the first structural evidence of homeoviscous adaptation to pressure. Polarization of the brain synaptic and liver mitochondrial fraction was not significantly related to depth. This may be due, at least in part, to a high individual variability of polarization compared to laboratory-acclimated freshwater fish.     

Aminoacyl-tRNA-synthetase activity of subcellular fractions of cerebral cortex]

The total activity of aminoacyl-tRNA-synthetases of myelin, synaptic membranes, heavy and light synaptosomes, mitochondria and soluble fractions of rat cerebral cortex was studied. It was found that the highest activity of the enzymes is localized in the fractions of synaptic membranes and heavy and light synaptosomes and is practically absent in the myelin fraction. The specific activity of the total aminoacyl-tRNA-synthetase fraction in the soluble fraction is 2 times as low as compared to the synaptic membranes and light and heavy synaptosomes.     

Association of axonally transported heparan sulfate with isolated synaptic plasma membrane

Studies on isolated synaptic plasma membranes (SPM) have detected little if any heparan sulfate or other glycosaminoglycans (GAGs), while more recent studies employing proteoglycan antibodies have localized heparan sulfate proteoglycan in presynaptic plasma membrane of intact tissue. To further address the issue of proteoglycans in synaptic plasma membrane of intact tissue. To further address the issue of proteoglycans in synaptic plasma membrane, we have investigated the possible presence of axonally transported GAGs in SPM isolated from the goldfish optic tectum. SPMs isolated from tecta following rapid axonal transport of³⁵SO₄ labeled molecules down the optic nerve, showed specific radioactivity approximately two-fold higher than the starting homogenate. Treatment of the transport labeled SPM with the enzyme heparitinase liberated 21% of the radioactivity, indicating the presence of a significant fraction of trnasported label in heparan sulfate. In a separate series of experiments a GAG fraction was isolated from transport labeled SPM and was found to consist of heparan sulfate containing 28% of transported radioactivity. Chondroitin (4 or 6) sulfate, which undergoes axonal transport in the goldfish optic system, was not found associated with SPM. Taken together the results support immunological evidence for the presence of heparan sulfate proteoglycans in presynaptic plasma membrane.To whom to address reprint request..     

Uptake and distribution in rat brain of organic and inorganic selenium

Polyunsaturated fatty acids (PUFAs) occur in relatively high amounts in phospholipids of the synapses. PUFAs may thus determine the fluidity of the synaptosomal membrane and, hereby, they may regulate the neuronal transmission. It was therefore tempting to suggest a system in the brain, that inhibits autooxidation of PUFAs. In order to trace such a protection system, Wistar rats were equally loaded with 4500 kBq of 75-Se either as selenite or as L-Se-methionine. By means of gradient ultracentrifugation, particulate fractions of the brains were isolated, and the radioactivity as well as the glutathione-transferase and -peroxidase activities were estimated. The distribution of the two selenium components among the particulate fractions was different. Thus, selenite gave higher radioactivity in myelin, then followed by the light synaptosomal and the vesicular fraction. L-Se-methionine was more equally incorporated in all particulate fractions, although highest activity was found in the mitochondrial fraction. Myelin and synaptic vesicles were devoid of transferase activity. On the other hand, the synaptosomal fraction showed highest specific transferase activity. The glutathione peroxidase activity was highest in the myelin fraction, followed by the vesicular and the synaptosomal fractions. The data obtained thus support the idea that the PUFAs of the synaptic compartment are protected against peroxidation, at least in part, by the selenium containing glutathione peroxidase.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions: Effects of local anesthetics,cholesterol and protein

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 °C). Incorporation of cholesterol (30–50%) increased the microviscosity of lipid phases by 200–500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b₅ did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since the latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracaine and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of the anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at the 25 °C varied as follows:polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erytherocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol : phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important fuctional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

SUBCELLULAR DISTRIBUTION AND SOME PROPERTIES OF ACETYL-COENZYME A HYDROLASE IN THE BRAIN

—The detailed subcellular distribution and some properties of acetyl-CoA hydrolase were studied in the rat brain. The brain homogenate (S₁) hydrolysed acetyl-CoA at a rate of approx 2·3 nmol/min/mg of protein at 37°C. The total activity of acetyl-CoA hydrolase was distributed in the following order: soluble > mitochondrial > microsomal, synaptosomal > myelin fraction. The order of the specific activity of the enzyme was: soluble, microsomal > mitochondrial > synaptosomal > myelin fraction. The synaptic vesicle fraction (D) had relatively high specific activity among the intraterminal particulate fractions, having two or three times higher specific activity than that of the synaptic cytoplasmic membrane fraction (F or G). Attempts to de-occlude acetyl-CoA hydrolase in the particulate fraction showed that only the enzyme activity in the myelin fraction was increased markedly by the treatment with ether or Triton X-100. Lineweaver-Burk plots gave straight lines for each subcellular fraction and apparent K_m values for acetyl-CoA were between 0·1 and 0·2 mM. Neither diisopropyl fluorophosphate nor physostigmine at the concentration of 0·1 mm inhibited the enzyme activity.     

A steady state and differential polarised phase fluorimetric study of the liver microsomal and mitochondrial membranes of thermally acclimated green sunfish (Lepomis cyanellus)

The liver mitochondrial and microsomal membranes of green sunfish and rat were examined by steady state polarisation and differential polarised phase fluorimetry to determine the effects of seasonal adaptation of membrane dynamic structure to temperature. Steady state polarisation studies indicated that the liver mitochondria of green sunfish acclimated to different temperatures showed a greater partial compensation of membrane fluidity for the altered acclimation temperature than did liver microsomal membranes. The fatty acid composition of both membrane preparations generally became more unsaturated at lower acclimation temperatures, though the differences between 5°C and 25°C acclimated fish were more pronounced in the mitochondrial fraction than in the microsomal fraction.Differential polarised phase fluorimetric studies indicated that the rotations of diphenylhexatriene in mitochondrial and microsomal membranes were highly hindered, though the hindrance offered by membranes of 25°C acclimated green sunfish was far greater than that offered by the membranes of 5°C acclimated fish, thus supporting the concept of homeoviscous adaptation. The absolute rotational rate was not consistently affected by acclimation treatment.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

Homeoviscous adaptation and its effect upon membrane-bound proteins

Synaptic membranes of goldfish showed compensatory adjustments in fluidity when the fish were acclimated to high or low temperature. This was associated with changes in the thermal stability of the synaptic (Na⁺+K⁺) ATPase at high inactivating temperatures. The importance of membrane fluidity to the structural stability of membrane-bound enzymes was supported by the labilising effects of the fluidising anaesthetic, n-hexanol, upon the (Na⁺+K⁺) ATPase. These results indicate that homeoviscous adaptation elicits adaptive changes in the (Na⁺+K⁺) ATPase.     

Investigation of phospholipids of the pulmonary extracellular lining by electron paramagnetic resonance. The effects of phosphatidylglycerol and unsaturated phosphatidylcholines on the fluidity of dipalmitoyl phosphatidylcholine.

The membranous structures of the pulmonary extracellular lining were removed from the lungs of rabbits by pulmonary lavage and isolated by differential centrifugation. This membranous fraction contained 93% of the total extracellular phospholipids present in lavage effluents and consisted of membranous vesicles, membrane fragments, tubular myelin and secreted lamellar bodies. The fraction was rich in phosphatidylcholine (79.4%) containing 85.2% palmitic acid in the 1-position and 57.4% palmitic acid in the 2-position. Phosphatidylglycerol was the next most abundant phospholipid, accounting for 9.4% of the total. E.p.r. spectra, obtained by using 5-doxylmethylstearate as a probe, showed that the extracellular phospholipids of the pulmonary lining were organized into structures which were much more fluid than erythrocyte-ghost membranes. The fluidity of phosphatidylcholine isolated from the membranous fraction was similar to that of the fraction itself, indicating that the minor phospholipids had very little influence on the fluidity of the major phospholipid. At physiological temperature, the fluidity of dipalmitoyl phosphatidylcholine was relatively low, but could be markedly increased by the presence of 1-palmitoyl-2-oleoyl phosphatidylcholine or phosphatidylglycerol (10%). Protein present in the extracellular phospholipid fraction did not affect the fluidity of the fraction. These studies indicate that the unsaturated phosphatidylcholines could play a major role in determining the fluidity of the important surface-tension-lowering phospholipids such as dipalmitoyl phosphatidylcholine.     

Immunohistochemical Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Myelin Basic Protein in the Chick Retina

A GTP-dependent regulatory component of adenylate cyclase was found in myelin from rat brain. The fraction solubilized from myelin contained a component that reconstituted guanine nucleotide-responsive adenylate cyclase activity when combined with the catalytic unit of adenylate cyclase prepared from rat brain. Purified myelin demonstrated little adenylate cyclase activity, even in the presence of F- or Mn2+. The reconstituted activity was dependent on the amount of the solubilized myelin fraction and required the presence of 5'-guanylylimidodiphosphate, a hydrolysis-resistant analog of GTP. The elution pattern of the component solubilized from myelin in gel filtration was very similar to that of a GTP-dependent regulatory component from synaptic plasma membranes. The content of the regulatory component-like activity in myelin was estimated to be 50-60% of that in synaptic plasma membranes. Cholera toxin ADP-ribosylated proteins having molecular weights of 48,000, 38,000, 23,000, 20,000, and 15,000 and other minor peptides in myelin, some of which were also present in synaptic plasma membranes. We conclude that myelin contained a GTP-dependent regulatory component of adenylate cyclase despite the apparent lack of adenylate cyclase activity in myelin.     

Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation

A method is described for the subcellular fractionation of brain to obtain a preparation highly enriched in synaptic plasma membranes. The enriched fraction is recovered from the interface of a two-step sucrose density gradient on which a hypotonically lysed crude mitochondrial fraction from brain has been separated by simultaneous sedimentation and flotation centrifugation. Enzyme marker activities associated with the neuronal plasma membrane are enriched in the synaptic plasma membrane-containing fraction while less than 10% of enzyme markers associated with the major probable contaminants, myelin and mitochondria, are found in the same fraction. Morphological examination of the enriched fraction suggests that about 80% of the profiles are recognisably synaptic in origin. Compared to previously described methods for obtaining synaptic plasma-enriched fractions of equivalent purity, the procedure reported here is simpler, shorter, and of greater capacity.     

Effects of ketamine anesthesia on rat-brain membranes: fluidity changes and kinetics of acetylcholinesterase

This investigation shows that the effects of general anesthetics previously observed in vitro on membrane fluidity and on enzymic activities and occurring at concentrations calculated to be clinically relevant can be reproduced in vivo in anesthetized animals. Anesthesia with 2-chlorophenyl-2-methylaminocyclohexanone (ketamine) induces a more fluid state of rat-brain synaptic and mitochondrial membranes, as shown by the rotational correlation times of the spin labels 16-doxylstearate and 5-doxylstearate. Changes in acetylcholinesterase activity, with a decrease in Vmax and no change in the Km for acetylcholine, closely follow the fluidity increase.     

On the sensitivity of intact cells to perturbation by ethanol

A comparison was made of ethanol's effects on the order of plasma membranes in intact cells and some isolated membrane preparations. Order was assessed by steady-state fluorescence polarization techniques using the non-permeant probe, TMA-DPH. The data show that two cultured cells, rat neonatal astroglial and N2A neuroblastoma, were sensitive to significant ethanol-induced disordering within the anesthetically relevant range (100 - 200 mM). Human erythrocytes, cultured fibroblasts and homogenized astroglial cells required higher ethanol concentrations (greater than 250 mM) to produce a similar effect. Intact erythrocytes were approximately twice as sensitive as erythrocyte ghost membranes to ethanol-induced perturbation. The neonatal glial and N2A cells were approximately five times more sensitive than synaptic membranes to ethanol effects. DMPC and DMPC + cholesterol liposomes and myelin membranes were insensitive to ethanol's effects. The incorporation of 10 mole % ganglioside GM1 sensitized the liposomes to ethanol-induced perturbation.     

Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Further characterization of mitochondrial contact sites: effect of short-chain alcohols on membrane fluidity and activity

Two mitochondrial contact site-enriched fractions were further characterized by freeze-fracture electron microscopy. Examination of the replicas revealed that these two fractions are in the form of vesicles with membrane sheets attached to the vesicles. The physical state of these fractions has been investigated by means of steady-state fluorescence polarization to assess the effects of alcohols on their fluidity properties and activity. Comparison between intact membranes and their extracted lipids shows that butanol and hexanol induce a differential increase of the overall membrane fluidity in the two contact site-enriched fractions. This alteration in the membrane dynamics leads to a complex modulation of cytochrome c oxidase activity in both fractions. These results bring further evidence for the existence of morphologically and functionally distinct domains in mitochondrial membranes.     

Effects of hydrostatic pressure on the location of PRODAN in lipid bilayers and cellular membranes

The effects of hydrostatic pressure on the location of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), an environmentally sensitive fluorescent probe, in phosphatidylcholine lipid bilayers and in goldfish brain synaptic membranes have been studied by fluorescence spectroscopy over the pressure range of 0.001-2 kbar. The emission spectrum of PRODAN in all the membrane systems examined exhibits two local maxima: one centers at around 435 nm and the other at about 510 nm. The intensity ratio of these two peaks, F435/F510, increases as pressure increases; in the particular case of dimyristoyl-L-alpha-phosphatidylcholine multilamellar vesicles [DMPC(MLV)], a dramatic change in F435/F510 appears at the lipid phase transition pressure. As pressure varies, an isoemissive point is seen in both egg yolk phosphatidylcholines and goldfish brain synaptic membranes; however, no isoemissive point is observed in DMPC(MLV). The presence of an isoemissive point is attributed to a pressure-induced relocation of PRODAN from the "polar" disposition (the 510-nm peak) to the "less polar" disposition (the 435-nm peak). The absence of an isoemissive point in the case of DMPC(MLV) is probably due to the lack of void space in the lipid matrix, as a result of tight lipid packing. Apparently, the probe relocation takes place in unsaturated systems, and PRODAN favors a more hydrophobic environment under pressure. However, on the basis of the emission spectra, PRODAN seems to remain more or less at the interfacial region over the pressure range examined. In goldfish brain synaptic membranes, the PRODAN polarization increases with pressure, giving dT/dP values of 15-20 degrees C kbar-1 for both dispositions.(ABSTRACT TRUNCATED AT 250 WORDS)