Incorporation of bovine thyroid peroxidase in liposomes

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropiate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (T_c) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37°C, the temperature at which thyroid peroxidase works ‘in vivo’. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.     

Rotational relaxation of 1,6-diphenylhexatriene in membrane lipids of cells acclimated to high and low growth temperatures.

Measurement of the time-resolved fluorescence depolarization of 1,6-diphenylhexatriene (DPH) in artificial bilayers of microsomal membrane lipids from Tetrahymena gives detailed information concerning the molecular motion of this probe and fluid properties of the membrane lipids which are obscured with steady-state methods. The rotational motion of DPH in these lipids from cells acclimated to 15 and 39.5 degrees C growth temperatures was anisotropic, which agrees with recent time-resolved studies of this probe in synthetic phospholipid systems. Evaluation of DPH polarization data obtained from these lipid fractions at their respective growth temperatures showed differences in physical properties which suggest that "viscosity", per se, of the microsomal lipids is not a strictly regulated as it is in prokaryotic systems. Rotational relaxation of DPH in 39.5 degrees C microsomal lipids measured at 15 degrees C is more complex than that of either lipid fraction measured at its actual growth temperature, suggesting that the probe has partitioned into two dissimilar environments within the bilayer. Similar effects are observed in the microsomes of 39.5 degrees C cells by freeze-fracture electron microscopy following rapid cooling to 15 degrees C. Under these conditions, two distinct regions are observed on the fracture faces, suggesting a correlation between lipid phase changes and alterations in membrane structure.     

Structural changes caused by thyrotropin in thyroid cells and in liposomes containing reconstituted thyrotropin receptor

Thyrotropin causes a rapid and significant increase in the fluorescence polarization of DPH when this hydrophobic probe is incorporated into a strain of functioning rat thyroid cells (FRTL5). This increase is ligand-specific and is not related to cAMP production. The phenomenon seems to reflect the interaction of thyrotropin with the glycoprotein component of its membrane receptor, as suggested by experiments in which thyrotropin causes increases in DPH fluorescence polarization in liposomes embedded with this receptor component but not with gangliosides. A strain of nonfunctioning rat thyroid cells (FRT), exhibiting no reactivity with monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, requires two orders of magnitude higher concentrations of thyrotropin to exhibit a comparable phenomenon.     

Cardiolipin Regulates the Activity of the Reconstituted Mitochondrial Calcium Uniporter by Modifying the Structure of the Liposome Bilayer

Reconstitution of mitochondrial calcium transport activity requires the incorporation of membrane proteins into a lipidic ambient. Calcium uptake has been measured previously using Cytochrome oxidase vesicles. The enrichment of these vesicles with cardiolipin, an acidic phospholipid that is found only in the inner mitochondrial membrane of eukaryotic cells, strongly inhibits calcium transport, in remarkable contrast with the activation effect that cardiolipin exerts upon other mitochondrial transporters and enzymes. The relation of the inactivation of calcium transport to the physical state of the bilayer was studied by following the polarization changes of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by flow cytometry in the cardiolipin-enriched liposomes with incorporated mitochondrial solubilized proteins. Non-bilayer molecular arrangements in the cardiolipin-supplemented liposomes, detected by flow cytometry, may produce the fluidity changes observed by fluorescence polarization of DPH. Fluidity changes correlate with the abolition of calcium uptake, but have no effect on the establishment of a membrane potential in the vesicles required for calcium transport activity. Changes in the membrane structure and uniporter function are observed in the combined presence of cardiolipin and calcium leading to a modified lipid configuration.     

Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.     

Hepatic plasma membranes from genetically obese (ob/ob) mice: studies on fluorescence polarization, phospholipid composition and 5'-nucleotidase activity

Hepatic plasma membrane lipids of lean (+/?) and obese (ob/ob) mice have been investigated using 1,6-diphenylhexatriene (DPH). Arrhenius plots of DPH fluorescence polarization in membranes showed the breakpoint in obese mice was reduced from 21 to 15 degrees C, whereas the breakpoint of 5'-nucleotidase activity was raised from 23 to 32 degrees C. Arrhenius break temperatures of DPH polarization and 5'-nucleotidase activity responded differently to housing mice at 34 degrees C and triiodothyronine (T3) treatment. Studies of DPH polarization in liposomes and phospholipid fatty acid composition suggested that differences in sphingomyelin acyl composition determine Arrhenius characteristics of hepatic 5'-nucleotidase in lean and obese mice.     

Acetyl-l-carnitine influences the fluidity of brain microsomes and of liposomes made of rat brain microsomal lipid extracts

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) was measured in different preparations (bovine spinal cord phosphatidylserine liposomes, rat brain microsomes, liposomes made with rat brain microsomal lipid having different phospholipid:cholesterol ratios) at temperatures ranging from 10° to 55°C. Phosphatidylserine liposomes exhibited an exponential relationship of rversus temperature, whereas the relationship shown by microsomes and liposomes prepared with microsomal lipid extracts was a linear one. The removal of protein and high phospholipid:cholesterol ratios decreased the slope of the lines (fluidity increased), although the intercept was unaffected. This means that differences were better appreciated at high temperatures and were well evident at 37°C. Acetyl-l-carnitine decreased r in rat brain microsomes and in liposomes made with microsomal lipids with different phospholipid:cholesterol ratios. The fluidifying effect of acetyl-l-carnitine was mild but statistically significant and could explain, at least in part, the data reported in the literature of acetyl-l-carnitine acting on some parameters affected by ageing. Besides, acetyl-l-carnitine seemed to oppose the changes of viscosity due to lipid peroxidation, which has been reported to increase in ageing and dementia.l-carnitine shares the properties of its acetyl ester, but only in part.Abbreviations DPH diphenylhexatriene - HEPES 4-(2-hydroxyethyl-l-piperazineethansulfonic) acid - r fluorescence anisotropy - SHB sucrose-HEPES-buffer (0.32 M sucrose, 2 mM HEPES, pH 7.0)     

Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids

Cytochrome P-450, purified from liver microsomes of phenobarbital-treated rabbits, was incorporated into dimyristoylphosphatidylcholine liposomes. The binding of benzphetamine to the liposome-bound cytochrome P-450 was examined by measuring the benzphetamine-induced spectral change at various temperatures. The van't Hoff plot of the apparent spectral dissociation constant showed a distinct break at the temperature of phase transition of the synthetic lipid. On the other hand, no such break was observed for benzphetamine binding to microsomal bound cytochrome P-450. These results suggest that the substrate binding site of cytochrome P-450 is embedded in the apolar interior of phospholipid bilayer membranes.     

Lipid thermotropic transitions in Triatoma infestans lipophorin

The structure and lipid thermotropic transitions of highly purified lipophorin of Triatoma infestans were examined by several techniques: steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), cis-parinaric acid (cis-PnA) and trans-parinaric acid (trans-PnA), light scattering fluorescence energy transfer between the lipophorin tryptophan residues and the bound chromophores, DPH, trans-parinaric acid cis-parinaric acid, gel electrophoresis, and gel filtration. Fluorescence polarization of PnAs and DPH revealed a reversible lipid thermotropic transition in intact lipophorin at about 20 degrees C and 18 degrees C, respectively. In lipophorin, lipid dispersion fluorescence polarization of DPH detected a lipid transition approximately at 20 degrees C, while trans-PnA showed a gel phase formation at a temperature below 30 degrees C. Similar experiments in which trans-PnA was incorporated into diacylglycerols and phospholipids extracted from the lipophorin revealed gel phase formation below 30 degrees C and 24 degrees C, respectively. Light scattering measurements showed that lipophorin particles aggregate irreversibly at 45 degrees C, increasing the molecular weight, as determined by gel filtration on Sephacryl S-300, from 740,000 to values larger than 1,500,000. The particle aggregation did not change the physical properties of the lipophorin studied by fluorescence polarization, indicating that the aggregation is apparently a non-denaturing process. Energy transfer between the lipophorin tryptophans and the bound chromophores cis-PnA, trans-PnA, and DPA revealed a different location of the fluorescent probes within the lipophorin. Temperature-dependence on the energy transfer efficiency for all probes confirmed a change in the ordering of the lipophorin lipids at 24 degrees C.     

Temperature-dependent spectral changes of a hemin-lipophilic imidazole complex incorporated into liposomes prepared from dipalmitoyl-, dimyristoyl- and egg yolk-phosphatidylcholine

The optical spectra of the hemin-lipophilic imidazole complex incorporated into liposomes prepared from three types of phosphatidylcholine were studied at various temperatures. The Soret peak of the system with dipalmitoyl- and dimyristoyl-phosphatidylcholine liposomes showed a marked bathochromic shift at the phase transition temperature, but in egg yolk phosphatidylcholine liposomes no spectral change was observed in the temperature range of 10° to 50°.     

Chloroplast thylakoid membrane fluidity and its sensitivity to temperature

In order to investigate membrane fluidity, the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), has been incorporated into intact isolated thylakoids and separated granal and stromal lamellae obtained from the chloroplasts of Pisum sativum. The steady-state polarization of DPH fluorescence was measured as a function of temperature and indicated that at physiological values the thylakoid membrane is a relatively fluid system with the stromal lamellae being less viscous than the lamellae of the grana. According to the DPH technique, neither region of the membrane, however, showed a sharp phase transition of its bulk lipids from the liquid-crystalline to the gel state for the temperature range -20° to 50° C. Comparison of intact thylakoids isolated from plants grown at cold (4°/7°C) and warm (14°/17° C) temperatures indicate that there is an adaptation mechanism operating which seems to maintain an optimal membrane viscosity necessary for growth. Using a modified Perrin equation the optimal average viscosity for the thylakoid membrane of the chill-resistant variety used in the study (Feltham First) is estimated to be about 1.8 poise.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - Hepes N-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

A novel hypothyroid dwarfism due to the missense mutation Arg479Cys of the thyroid peroxidase gene in the mouse

Recently, we found a novel dwarf mutation in an ICR closed colony. This mutation was governed by a single autosomal recessive gene. In novel dwarf mice, plasma levels of the thyroid hormones, T3 and T4, were reduced; however, TSH was elevated. Their thyroid glands showed a diffuse goiter exhibiting colloid deficiency and abnormal follicle epithelium. The dwarfism was improved by adding thyroid hormone in the diet. Gene mapping revealed that the dwarf mutation was closely linked to the thyroid peroxidase (Tpo) gene on chromosome 12. Sequencing of the Tpo gene of the dwarf mice demonstrated a C to T substitution at position 1508 causing an amino acid change from arginine (Arg) to cysteine (Cys) at codon 479 (Arg479Cys). Western blotting revealed that TPO protein of the dwarf mice was detected in a microsomal fraction of thyroid tissue, but peroxidase activity was not detected. These findings suggested that the dwarf mutation caused a primary congenital hypothyroidism by TPO deficiency, resulting in a defect of thyroid hormone synthesis.     

The effect of alpha-lactalbumin on the thermotropic phase behaviour of phosphatidylcholine bilayers, studied by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy

The effects of bovine alpha-lactalbumin on the thermotropic properties of dimyristoylphosphatidylcholine liposomes are studied by Raman spectroscopy, fluorescence polarization and differential scanning calorimetry. The Raman spectrum reveals the drastic effects of the protein on the phospholipid structure. The transition temperature shifts downwards and the inter- and intrachain order in the lipid matrix progressively diminish with increasing protein concentration. Up to a lipid to protein molar ratio R = 25, the bilayer structure however is maintained. From fluorescence polarization data we conclude that the protein restricts the mobility of the DPH probe. In view of the Raman results, the lower probe mobility obviously cannot be associated with a more rigid lipid matrix. Nevertheless the transition temperatures of the alpha-lactalbumin-phospholipid complex increases. DSC measurements give no decisive way out for this discrepancy. These results confirm that different types of lipid order are involved in lipid-protein interactions. Compared to the free protein, the alpha-helicity of the protein has increased in the complex.     

Temperature-Induced Changes in Lipid Fluidity Alter the Conformation of Proteins in Senescing Plant Membranes

Complementary biophysical techniques have provided evidencefor an effect of temperature-induced changes in lipid fluidityon the conformation of proteins in senescing plant membranes.Smooth microsomal membranes were isolated from bean cotyledons(Phaseolus vulgaris L. (cv Kinghorn)) at various stages of senescence.Lipid fluidity was measured by fluorescence polarization afterlabelling the membranes with diphenyl hexatriene (DPH). Alterationsin protein conformation were determined by labelling the membraneswith the paramagnetic sulfhydryl reagent, 3-maleimido proxyl(3-MP), and following changes in the ratio of weakly immobilizedto strongly immobilized (w/s) spin label. Plots of w/s as afunction of temperature featured characteristic break pointsfor each age of membrane. Corresponding break points at virtuallyidentical temperatures were also observed in plots of DPH polarizationversus temperature for membranes as well as for liposomes preparedfrom lipid extracts of membranes. In at least one instance thesebreak points in DPH polarization did not correspond to liquid-crystallineto gel phase transitions in the lipid. The fluidity of microsomalmembranes decreased with advancing senescence of the cotyledons,and there were also changes in the parameter w/s for membraneproteins with advancing age. The results indicate that subtlechanges in the molecular ordering of lipid bilayers alter therelative proportions of weakly and strongly immobilized sulfhydrylgroups in the membrane proteins, which can be interpreted asreflecting changes in protein conformation. ¹ Present address: Agronomy Department, Cornell University,Ithaca, N.Y. 14853, U.S.A. (Received January 19, 1987; Accepted April 14, 1987)     

Thermotropic phase transitions in the plasma membrane of ram spermatozoa

A steady-state fluorescence polarization technique, using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), showed that separately detectable transitions occurred in the regions of 17, 26 and 36 degrees C in isolated preparations of ram sperm plasma membrane. An independent technique based on the temperature-related behaviour of calcium- and magnesium-activated ATPase detected a single phase transition in the region of 24 degrees C. Modulation of ATPase by neighbouring lipid composition was inferred from findings that phospholipase A2 caused significant stimulation of the enzyme. Cholesterol-rich liposomes caused an upward shift of the phase-transition temperature from 24 degrees C to 30 degrees C, but the reasons for this are unclear. It is considered that these phase transitions may have profound effects on sperm survival and physiology, both during normal fertilization processes and in response to cryostorage.     

Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

Effect of the fungicides tributyltin acetate and tributyltin chloride on multilamellar liposomes: fluorescence studies.

The influence of tri-n-butyltin acetate (TBTA) and tri-n-butyltin chloride (TBTC) on the physico-chemical state of charged and neutral phospholipids was investigated using multilamellar liposomes. The thermal dependence of steady state fluorescence polarization of DPH and its charged derivative TMA-DPH was recorded. The two fungicides lowered DPPC phase transition temperature and broadened the temperature range of the transition in different ways. The effects were concentration-dependent. The results show that TBTC interacts more effectively with DPPC model membranes rather than TBTA. Moreover, TBTC broadens and shifts the main phase transition (Tm) more effectively in DPPC rather than in DMPC liposomes. Below Tm, TBTC decreases fluorescence polarization (P) in all phospholipids used. Above Tm P is almost constant in phospholipids with saturated acyl chains, except for DMPG. In fact, an increase of P is detectable in this lipid as in PLs with unsaturated acyl chains. It is suggested that the effects of TBT on liposomal membranes are dependent on the anion moiety and phospholipids characteristics.     

Changes Caused by Fruit Extracts in the Lipid Phase of Biological and Model Membranes

The aim of the study was to determine changes incurred by polyphenolic compounds from selected fruits in the lipid phase of the erythrocyte membrane, in liposomes formed of erythrocyte lipids and phosphatidylcholine liposomes. In particular, the effect of extracts from apple, chokeberry, and strawberry on the red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC liposomes was studied. In the erythrocyte population, the proportions of echinocytes increased due to incorporation of polyphenolic compounds. Fluorimetry with a laurdan probe indicated increased packing density in the hydrophilic phase of the membrane in presence of polyphenolic extracts, the highest effect being observed for the apple extract. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The polyphenolic extracts slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The studies have shown that the phenolic compounds contained in the extracts incorporate into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The compounds also penetrate the outer part of the external lipid layer of liposomes formed of natural and DPPC lipids, changing its packing order.     

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.