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T Gulik-Krzywicki

Publications and source records attributed to T Gulik-Krzywicki.

At least 19 recordsLinked to original sources
PubMed1992

Calcium-induced acetylcholine release and intramembrane particle occurrence in proteoliposomes equipped with mediatophore.

Proteoliposomes obtained from the mediatophore, a purified Torpedo electric organ nerve terminals protein, and endogenous lipids were used for a study of calcium-induced release of acetylcholine and freeze-fracture electron microscopy. Large intramembrane particles were induced by the influx of calcium into proteoliposomes, as previously observed for synaptosomes or stimulated electric organ nerve terminals. The involvement of mediatophore in a calcium dependent acetylcholine translocation seems therefore to be related to the occurrence of a category of intramembrane particles in the course of the release process.

Acetylcholine↗
PubMed1992

Interactions between genes involved in exocytotic membrane fusion in paramecium.

Crosses between members of two independent collections of Paramecium tetraurelia mutants blocked in the final membrane fusion step of trichocyst release (nd mutants) allowed us to define 13 complementation groups comprising 23 alleles. The mutant nd9a was then used as a target in a mutagenesis experiment designed to screen both revertants and new mutants in order to identify interacting genes. This mutant was chosen because it is the best known of its class to date and seems to be altered in assembly of the material connecting the trichocyst membrane to the plasma membrane and in assembly of the "rosette," a complex array of intramembranous particles in the plasma membrane at the trichocyst insertion sites. No revertants were obtained but two new mutants deficient for rosette assembly were identified, nd16b and nd18, whose gene products appear to interact with that of nd9. Indeed, the double mutants grown at 18 degrees, a permissive temperature for each of the single mutants, are characterized by a deficiency in exocytosis and in rosette assembly, as are also double mutants combining other allelic forms of the same genes. Moreover, aberrant dominance relationships among alleles of nd9 and of nd16 indicate the existence of interactions between identical subunits, which most likely assemble into multimeric structures. The nd16 gene product was shown by microinjection experiments to be a cytosolic factor, as is the nd9 gene product. It is therefore tempting to propose that the nd16 gene product also belongs to the connecting material and is involved in rosette assembly, in cooperation with nd9 and nd18.

Animals↗
PubMed1991

Involvement of the protein-protein interactions in the thermodynamics of the electron-transfer process in the reaction centers from Rhodopseudomonas viridis.

Reaction centers from Rhodopseudomonas viridis were reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) liposomes. Freeze-fracture electron micrographs were performed on the samples frozen from temperatures above and below the phase transition temperatures of those lipids (Tc = 23 and 9.5 degrees C, in DMPC and DEPC, respectively). Above Tc, in the fluid conformation of the lipids, the reaction centers are randomly distributed in the vesicle membranes. Below Tc, aggregation of the proteins occurs. The Arrhenius plots of the rate constants of the charge recombination between P+ and QA- display a break at about 24 degrees C in DMPC vesicles and about 10 degrees C in DEPC vesicles (P represents the primary electron donor, a dimer of bacteriochlorophyll, and QA the primary quinone electron acceptor). This is in contrast to what was previously observed for the proteoliposomes of egg yolk phosphatidylcholine and for chromatophores [Baciou, L., Rivas, E., & Sebban, P. (1990) Biochemistry 29, 2966-2976], for which Arrhenius plots were linear. In DMPC and DEPC proteoliposomes, the activation parameters were very different on the two sides of Tc (delta H degrees for T less than Tc = 2.5 times delta H degrees for T greater than Tc), leading however, to the same delta G degrees values. Taking into account the structural and thermodynamic data, we suggest that, in vivo, protein-protein interactions play a role in the thermodynamic parameters associated with the energy stabilization process within the reaction centers.

Dimyristoylphosphatidylcholine↗
PubMed1991

The egg-shell of Drosophila melanogaster. VI, Structural analysis of the wax layer in laid eggs.

Utilizing freeze-fracturing conventional electron microscopy and scanning electron microscopy methods, a wax layer was identified, sealing the oocyte of Drosophila melanogaster. In mature egg-shells wax forms a hydrophobic layer surrounding the oocyte and lying between, and in very close contact with the vitelline membrane (interiorly) and the crystalline intermediate chorionic layer (exteriorly). In cross-fractured views it is less than 50 A thick whereas in longitudinal fracturing it reveals smooth fracture faces of a multilayered material in the form of hydrophobic areas or plaques (0.5-1 microns in diameter) which are partially overlapping and highly compressed between the vitelline membrane and the innermost chorionic layer. The evidence for this layer being a wax are the facts that a) it is not preserved in conventional fat-extracting electron microscopy methods, b) it directs laterally the fracture planes during freeze-fracturing and reveals smooth fracture faces. Analysis of the structural features of wax in mature egg-shell in various species of Drosophilidae have shown that the wax layer exhibits indistinguishable (among the species) hydrophobic plaques, which have the same size and thickness with Drosophila melanogaster. These data provide structural evidence explaining the physiological resistance of the insect eggs studied, against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. In addition, the data demonstrate how an extracellular substance can be organized to perform that function.

Animals↗
PubMed1990

Monomeric state and Ca2+ transport by sarcoplasmic reticulum Ca2(+)-ATPase, reconstituted with an excess of phospholipid.

The structural basis for Ca2+ transport was examined in vesicles reconstituted with an excess of phospholipid by a cholate dialysis procedure. Unincorporated protein and vesicles with a relatively high protein content were removed by sucrose density centrifugation (3-12%), leaving a fraction of lipid-rich vesicles (lipid to protein weight ratio 800-900:1) with a high coupling ratio (1.0) and transport capacity (25 mumol/mg protein, after Ca-phosphate loading). Freeze-fracture analysis showed that the reconstituted vesicles had a remarkably narrow size distribution (diameter 794 +/- 77 A (S.D.], suitable for stereological analysis. Intramembranous particles were dispersed and occurred with a low frequency in the fractured shells, also before sucrose fractionation. It was calculated that the number of intramembranous particles corresponded to the number of Ca2(+)-ATPase polypeptide/vesicle. A ratio of unity between particles and polypeptide chains was also obtained from the density of particle distribution on flat surfaces of fused vesicles, prepared by sucrose fractionation. The size of the particles formed a broad distribution, having a peak value around 60-67 A, both in the reconstituted preparation and sarcoplasmic reticulum vesicles. No evidence for protein-protein interactions was found in chemical cross-linking experiments. It is concluded that the intramembranous particles in the reconstituted preparations are referable to monomeric Ca2(+)-ATPase which is capable of transporting Ca2+ inside the vesicles. The implications of the observations for the associational state of Ca2(+)-ATPase at high protein concentration are considered in relation to previous ultrastructural investigations of membranous Ca2(+)-ATPase in native and two-dimensional-crystalline forms.

Animals↗
PubMed1987

Monomer-oligomer equilibrium of bacteriorhodopsin in reconstituted proteoliposomes. A freeze-fracture electron microscope study.

An improved freeze-fracture electron microscope procedure has been developed and applied to the study of the association of bacteriorhodopsin in large proteoliposomes reconstituted by reverse-phase evaporation with egg lecithin. Due to the improved accuracy and resolution of this procedure, intramembrane particles, the diameter of which (4.5 nm) closely matched that of bacteriorhodopsin monomer, could be observed at high lipid to protein ratios (greater than or equal to 40 w/w). At lower lipid to protein ratios, larger particles (diameter 7.5 nm) progressively appeared, resulting in bimodal particle size distributions up to a lipid to protein ratio of 1, where the large particles were the sole species present. These large particles were interpreted as corresponding to bacteriorhodopsin oligomers. Because of the large size and homogeneity of proteoliposomes, accurate particle density measurements could be performed. These confirmed the occurrence of a lipid to protein ratio-dependent bacteriorhodopsin monomer-oligomer equilibrium and further allowed us to identify the oligomer as a trimer or a tetramer. In complementary experiments, it was found that the bacteriorhodopsin monomer and oligomer had identical visible CD spectra and light-induced proton pumping rates. However, a large increase of the proton passive leak rate of proteoliposomes was found to be associated with oligomer formation. The appearance of these oligomers may be important as the first step in the formation of two-dimensional crystals of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1987

Effect of calcium on the calf lens cytoplasm.

Opacification was induced in calf lens cytoplasmic extracts by addition of calcium. The sample turbidity was shown to increase with calcium molarity, incubation time and temperature and to decrease with the protein cytoplasmic concentration. Although this turbidity was enhanced when membrane fragments were left over in the cytoplasmic extracts, it did show up in the absence of any detectable vesicular fragment. Scattering techniques (X-ray and light) showed that the calcium-induced opacification is linked to enhanced light scattering, which results from the formation of additional scatterers, a few tens of nm in diameter. Additional structures were indeed visualized by freeze-fracture electron microscopy (FFEM): they appear as molecular clusters with diameters ranging from 20 to 90 nm, made of densely packed particles, with heterogeneous sizes. The turbidity expected from these clusters, as well as the expected variation of turbidity with cytoplasmic concentration, was calculated to be in agreement with the measurements. When compared with cold cataract, these results illustrate that similar opacities may result from completely different biophysical mechanisms.

Animals↗
PubMed1987

The crystal lattice of Paramecium trichocysts before and after exocytosis by X-ray diffraction and freeze-fracture electron microscopy.

Paramecium trichocysts are unusual secretory organelles in that: (a) their crystalline contents are built up from a family of low molecular mass acidic proteins; (b) they have a precise, genetically determined shape; and (c) the crystalline trichocyst contents expand rapidly upon exocytosis to give a second, extracellular form which is also an ordered array. We report here the first step of our study of trichocyst structure. We have used a combination of x-ray powder diffraction, freeze-etching, and freeze-fracture electron microscopy of isolated, untreated trichocysts, and density measurements to show that trichocyst contents are indeed protein crystals and to determine the elementary unit cell of both the compact intracellular and the extended extracellular form.

Animals↗
PubMed1987

Effect of cetiedil on acetylcholine release and intramembrane particles in cholinergic synaptosomes.

The release of acetylcholine (ACh) from instantly frozen Torpedo electric organ synaptosomes in the course of stimulation is systematically associated with an increase in the number of large intramembrane particles counted on freeze-fracture replicas. The drug cetiedil, which is a potent inhibitor of ACh release, also blocks the increase in the number of large particles. The blockage was studied either after ionophore A 23187 or Glycera neurotoxin action in the presence of calcium.

Acetylcholine↗
PubMed1986

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles.

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, Tm. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T greater than Tm, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. At low melittin content, phospholipid-to-peptide molar ratios, Ri greater than 30, heterogeneous systems are observed, the new structures coexisting with the original ones. For lipids in the fluid phase and Ri greater than 12, the complexes formed are large unilamellar vesicles of about 1300 +/- 300 A diameter and showing on freeze-fracture images rough fracture surfaces. For lipids in the gel phase, T less than Tm after passage above Tm, and for 5 less than Ri less than 50, disc-like complexes are observed and isolated. They have a diameter of 235 +/- 23 A and are about one bilayer thick; their composition corresponds to one melittin for about 20 +/- 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. For high amounts of melittin, Ri less than 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide co-micelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: vesicularization of multibilayers; fusion of small lipid vesicles; fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.

Bee Venoms↗
PubMed1986

Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles.

A study of electrokinetic properties of reconstituted sarcoplasmic reticulum was undertaken to determine the nature of the groups bearing the negative charge of the membrane. After incorporation of phosphatidylcholine into the bilayer, it was found that the Ca2+-ATPase embedded in functional vesicles bore 3e- per mole. When the surface charge density of the hydrodynamic particles became more negatively charged by incorporation of phosphatidylserine molecules, the reconstituted vesicles had a tendency to build large structures resulting from vesicle-vesicle interaction and containing large amounts of divalent cations. These aggregated structures may partially explain the discrepancy observed between the expected value of the surface charge density and the data obtained by electrophoretic mobility measurements. This work emphasizes the importance of a renewal of the classical interpretation of electrophoretic mobility data in order to analyze the results obtained with biological material. To explain the energy transduction process which takes place in the sarcoplasmic reticulum membrane, it was of interest to determine whether or not variations of the surface electrical properties affect the calcium ion translocation upon ATP hydrolysis. Relatively significant modifications of the bilayer composition and surface charge density did not appreciably affect the calcium transport activity.

Biological Transport↗
PubMed1978

The use of low temperature X-ray diffraction to evaluate freezing methods used in freeze-fracture electron microscopy.

Two methods of freezing samples for freeze-fracture electron microscopy have been compared using X-ray diffraction and freeze-fracture results from lipid-water model systems. Perturbations of the molecular organization of hydrocarbon chains and the extent of ice crystal formation have been evaluated for lamellar phases of egg lecithin containing 16% water and egg lecithin-phosphatidylinositol containing 55% water, both freeze quenched in liquid Freon-22 near its melting temperature (113 K). Very thin samples sandwiched between copper sheets separated by an electron microscope grid show much less freezing induced structural rearrangement than small smaples contained on conventional Balzers-type gold planchettes. These results show that the rate of freezing in the very thin preparations is greater than in the conventional ones, which is probably due in part to the improved dissipation of heat from a poorly conductive sample through highly conductive copper sheets.

Freeze Fracturing↗
PubMed1976

Correlated x-ray diffraction and freeze-fracture studies on membrane model systems. Perturbations induced by freeze-fracture preparative procedures.

Lipid-water and protein-lipid-water phases have been examined by X-ray methods before and after freezing. Frozen samples have been subsequently fractured and replicated, thus permitting an evaluation of the nature of structural perturbations in samples examined by freeze-fracture electron microscopy. Important results are summarized: (1) Freezing low water content (approx. less than 25%) phases causes perturbations in the packing of hydrocarbon chains. The results suggest that freezing liquied paraffin chains produces a condensed "glass-like" packing. (2) Additional perturbations occur in high water content samples. After freezing, much smaller lamellar repeat distances, intense ice reflections, and extensive perturbation of fracture faces are consistant with the expulsion of water from between lamellae. Presence of glycerol generally relieves these perturbations but in some cases introduces additional lattice disorder. (3) Surprisingly, cooling by a stream of cold N2 gas (-140 degrees C) produces qualitatively the same results as rapid cooling in liquid Freon-22 (-160 degrees C). (4) Complex perturbations occur in phases containing integral membrane proteins. Interesting results have been obtained with cytochrome b5-lecithin lamellar associations which display both smooth and rough fracture faces without clearly defined particles.

Binding Sites↗
PubMed1976

Lipid molecular motion and enzyme activity in sarcoplasmic reticulum membrane.

In biochemically active sarcoplasmic reticulum vesicles (SR) the physical state of the membrane lipids was studied by high angle x-ray diffraction and proton nuclear magnetic resonance (NMR) at 220 MHz, and related to thermal effects observed in SR functional parameters. It is shown by high angle x-ray diffraction that even at temperatures as low as 1 degree C nearly all the SR lipid hydrocarbon chains are in a disordered conformation and only a very small part (less than 3%) are in rigid crystalline order. Consistent with this observation, the NMR data indicate that the majority of SR phospholipid molecules are in a state of restricted anisotropic motion having no apparent crystalline order at temperatures as low as 5 degrees C. At this temperature most of the resonance signal is contained in a broad feature-less line of 700-Hz half-width. On the other hand, as the temperature is raised, high-resolution NMR signals, representing groups with highly isotropic motion, begin to grow in intensity. It is estimated that by 35 degrees C 90-100% of the phosphatidylcholine N-methyl protons and 35% of the hydrocarbon-chain protons give high-resolution signals. Concurrent studies on functional parameters reveal thermal effects giving rise to nonlinear Arrhenius plots for the rates of calcium transport and calcium activated ATPase. The thermal effects observed on functional parameters and on the character of phospholipid molecular motion exhibit a parallel behavior, suggesting a relationship between enzyme activity and the physical state of the membrane lipids.

Adenosine Triphosphatases↗