Characterisation of phosphatidylcholine/phosphatidylinositol sonicated vesicles. Effects of phospholipid composition on vesicle size

Phosphatidylinositol (PI), dipalmitoylphosphatidylcholine (DPPC) and mixed lipid (DPPC plus PI) sonicated vesicles have been prepared covering a range of composition. The vesicles were characterised by gel filtration, electron microscopy and photon correlation spectroscopy. The dimensions of the vesicles as measured by electron microscopy were in good accord with those obtained from photon correlation spectroscopy measurements. The number average diameters of the vesicles increase on increasing the PI content and range from approx. 30–80 nm as the weight % of PI is increased from 0 to 100. Gel filtration on Sepharose 4B columns gave anomalous results indicating that PI-containing vesicles were retarded on the gel possibly due to an interaction between the inositol headgroup and the gel matrix. Electrophoretic measurements on multilamellar vesicles show that the surface charge density increases with the PI content of the vesicles upto 50 weight % PI and remains constant thereafter. The radii of sonicated vesicles also increase with PI content which reflects a decreasing liposome curvature with increasing surface charge density.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Discrimination of flash-induced fast and slow electric potential components

This work aimed at the resolution of the multi-component electric potential changes induced by single-turnover flash illumination of Photosystem-I-enriched subchloroplast vesicles. If supplemented with ferredoxin and under carefully adjusted redox poising, these vesicles show a pronounced slow-rising and -decaying electric potential component, as monitored by endogenous and exogenous field-sensitive probes, carotenoids and oxonol VI, respectively. The fast and slow potential components can be easily discriminated without the need for computer-assisted deconvolution after selective presaturation of the slow component by preillumination or a transmembrane ΔpH, after selective suppression of the slow component by low valinomycin or uncoupler concentrations or in the absence of ferredoxin. The slow electric potential component, as compared to the fast one, is relatively sensitive to low concentrations of ionophores and uncouplers, detergent, ageing and lower temperatures (4–12°C), is associated with electrogenic proton displacements and is interpreted to respond to a field that is more located on the membrane-bulk interface. Temperature effects show transition temperatures around 20°C for both the rise and decay of the slow potential component. The results provide further evidence that the carotenoids and oxonol VI sense the same (slow) electric field, but may be differently located in the thylakoid membrane.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

Resonance Raman resolution of a-, b- and c-type cytochromes in membrane vesicles of alkalophilic bateria

Resonance Raman spectroscopy has been used to obtain complete spectra of each individual cytochrome type — a, b and c — in the reduced state within membrane vesicle preparations from two species of obligately alkalophilic bacteria: Bacillus alcalophilus and Bacillus firmus RAB. The vibrational spectra, in the range 250–1700 cm^?1, were obtained with tunable dye laser excitation in the wavelength range 550–600 nm tuned to resonance with the appropriate reduced alpha band maximum for the cytochrome type of interest. The spectra reveal details which serve to characterize the specific type of cytochrome as well as to confirm the similarity of the heme prosthetic group to previously well-characterized cytochromes of the the a- b- or c-type. Preliminary evidence in support of heterogeneity of b-type, and possibly a-type cytochromes, or of heme-heme interaction within the membrane is presented.     

Author index

Using dynamic light scattering we have been able to determine precisely the hydrodynamic radius of l-α-dimyristoylphosphatidylcholine (DMPC) vesicles as a function of temperature. We have detected a sharp, thermally reversible change in the vesicle radius at a phase transition temperature 24°C, corresponding to an approximate 11% increase in surface are. In the range 10–20°C, the change in radius is less than 1%.     

Asymmetry of the Na+-succinate cotransporter in rabbit renal brush-border membranes

We have investigated the symmetry of Na⁺-succinate cotransport in rabbit renal brush-border membrane vesicles. Succinate influx and efflux kinetics were measured under voltage-clamped conditions using [¹⁴C]succinate and a rapid filtration procedure. Both influx and efflux were Na⁺-dependent, saturable, temperature-sensitive, and influenced by the trans Na⁺ and succinate concentrations. The system was judged to be asymmetric, since the maximal velocity for influx was 3-fold higher than that for efflux, and trans Na⁺ inhibited influx more than efflux. This may be due to the asymmetrical insertion of the transporter in the brush-border membrane, which leads to differences in either the forward and backward translocation rates of the fully loaded carrier or the Na⁺ and succinate binding constants at the inner and outer faces of the membrane.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

Comparison of chemical properties of purified plasma membranes and secretory vesicle membranes from the bovine adrenal medulla

Summary A highly enriched fraction of plasma membranes from the bovine adrenal medulla has been isolated by differential and sucrose gradient centrifugation. The membranes were found to occur as 0.1–0.5 diameter vesicles and to equilibrate at a density of 1.13–1.14 g/ml. This fraction was characterized by 4-fold elevated levels of adenylate cyclase and 20-fold elevated levels of 5-nucleotidase. Secretory vesicle membranes, isolated by repeated hypotonie and hypertonic shocks of whole vesicles, were found to equilibrate between d = 1.08 and d = 1.12 on a sucrose density step gradient. These membranes were highly enriched in cytochrome b₅₆₂ and dopamine--hydroxylase. Proteins in the two membranes were compared by SDS gel electrophoresis. All protein size classes found in the vesicle membrane fraction were also represented in the plasma membrane fraction, though in different proportions on the basis of staining intensity. The plasma membrane fraction contained prominent bands co-migrating with the - and -bands of tubulin, as well as a component co-migrating with actin. These bands were absent from the vesicle membranes. Fingerprint analysis of stained bands from the membrane fraction demonstrated that the components were indeed tubulin and actin. The plasma membranes contained twice as much sialic acid residues as did the chromaffin granule membranes, but had only half the cholesterol content on a weight basis. The cholesterolphospholipid ratio in the plasma membranes was 0.63, while in the secretory vesicle membranes it was 1.04. These results show that plasma membranes and secretory vesicle membranes are functionally and structurally different.Supported, in part, by a stipend to O.Z. from The Grant Foundation, New York     

Metacercarial dispersion and intracellular parasitism in a strigeid trematode

Combes C. and Nassi H. 1977. Metacercarial dispersion and intracellular parasitism in a strigeid trematode. International Journal for Parasitology7: 501–503. The life cycle of Apatemon graciliformis Szidat, 1928 (Trematoda, Strigeidae), a parasite of Biorrphalaria glabrata in Guadeloupe, involves a novel mode of transmission, experimentally demonstrated, between the second intermediate host and the definitive host. The furcocercariae penetrate gravid females of the ovoviviparous fish, Poecilia reticulata, and develop into metacercariae in vitelline vesicles of the embryos where they encyst a short time before parturition. The young guppies are born infected with 1–3 metacercariae. It is considered that young infected fish are more prone to predation by the definitive host, thereby increasing the probability of the cycle being completed. Domestic ducks have been experimentally infected with these metacercariae. If cercariae penetrate non-gravid P. reticulata, they enter the oocytes; this represents a phase of intracellular parasitism.     

II. Phospholipid exchange and size enlargement in sonicated vesicles induced by a ‘critical’ fatty acid concentration

Size enlargement of dipalmitoyl phosphatidylcholine vesicles was greatly accelerated in the range of the phase-transition temperatures, when fatty acid concentration was above a threshold level (‘critical’ concentration). This ‘critical’ concentration varied with the length of the fatty acid chain. The size enlargement process had second-order kinetics dependent on the vesicle concentration. Alkaline pH and low ionic strength inhibited the rate of size enlargement.Phospholipid exchange between dimyristoyl and dipalmitoyl phosphatidylcholine vesicles increased abruptly above a ‘critical’ fatty acid concentration. The donor vesicles were those vesicles in which fatty acids reached the ‘critical’ concentration. The phospholipid exchange occurred both in fluid- and in solid-state vesicles. The ‘critical’ fatty acid concentration accelerating the phospholipid exchange process was lower than that accelerating the size enlargement process.The phospholipid exchange process explained in terms of a diminished hydrophobic attraction among the phospholipid molecules of the bilayer occurs via a free phospholipid molecule transfer through the aqueous phase. The size enlargement process is interpreted in terms of high fatty acid concentration in the membrane fluid domains. The membrane structure is locally perturbed inducing vesicle sticking after collision.