Membrane fragments

Ribosomes and microsomes consisting of endoplasmic reticulum, Golgi, and plasma membrane fragments... 

It can be concluded that MD is a very powerful tool to refine structures of proteins and polypeptides in solution, based on 2D NMR data. This combination of techniques emerges as an important means to determine the 3 D structure of macromolecules up to a molecular weight of 20,000 in solution or in micelles or membrane fragments. 

It is necessary to note that the initial conditions of the samples in solution were absolutely different. RC was extracted from the membranes by detergent (lauryldimethy-lamineoxide—LDAO) the solution contains the individual protein molecules surrounded by a detergent belt shielding the hydrophobic areas of the protein surface. In the case of BR the situation is different. BR is the main part of purple membranes (about 80%) and is already close packed in it. It is difficult to extract BR in the form of individual molecules, for they are very unstable (Okamura et al. 1974). Thus, the initial solution of BR was in reality the solution of sonicated membrane fragments. 

Such differences in the secondary structure behavior with respect to temperature can be explained by suggesting that molecular close packing of proteins in the film is the main parameter responsible for the thermal stability. In fact, as in the case of BR, we have close packing of molecules even in the solution (membrane fragments) there are practically no differences in the CD spectra of BR solution at least tiU 75°C (denaturation takes place only for the sample heated to 90°C). RC in solution begins to be affected even at 50°C and is completely denatured at 75°C, for the solution contains separated molecules. 

The fact that CD spectra of BR in LB and self-assembled films show similar behavior with respect to temperamre is also not strange. Because the basic block of the fihn in both cases is the membrane fragment, which is already closely packed, there is no principal difference between these samples with regard to packing. The difference in the distribution of these fragments cannot be critical for thermal stability. 

The Langmuir-Blodged (LB) technique allows one to form a monolayer at the water surface and to transfer it to the surface of supports. Formation of the BR monolayer at the air/water interface, however, is not a trivial task, for it exists in the form of membrane fragments. These fragments are rather hydrophilic and can easily penetrate the subphase volume. In order to decrease the solubility, the subphase usually contains a concentrated salt solution. The efficiency of the film deposition by this approach (Sukhorukov et al. 1992) was already shown. Nevertheless, it does not allow one to orient the membrane fragments. Because the hydrophilic properties of the membrane sides are practically the same, fragments are randomly oriented in opposite ways at the air/water interface. Such a film cannot be useful for this work, because the proton pumping in the transferred film will be automatically compensated i.e., the net proton flux from one side of the film to the other side is balanced by a statistically equal flux in the opposite direction. 

Therefore, the following method was suggested and realized (the scheme is shown in Fig. 17). A 1.5 M solution of KCl or NaCl (the effect of preventing BR solubility of these salts is practically the same) was used as a subphase. A platinum electrode was placed in the subphase. A flat metal electrode, with an area of about 70% of the open barriered area, was placed about 1.5-2 mm above the subphase surface. A positive potential of +50 -60 V was applied to this electrode with respect to the platinum one. Then BR solution was injected with a syringe into the water subphase in dark conditions. The system was left in the same conditions for electric field-induced self-assembly of the membrane fragments for 1 hour. After this, the monolayer was compressed to 25 mN/m surface pressure and transferred onto the substrate (porous membrane). The residual salt was washed with water. The water was removed with a nitrogen jet. 

The dependence of the surface pressure upon the time with and without applied electric field is shown in Figure 18. It is clear that the electric field strongly improves the ability of the membrane fragments to form a monolayer at the water surface. 

Palytoxin (PTX) is one of the most potent marine toxins known and the lethal dose (LD q) of the toxin in mice is 0.5 Mg/kg when injected i.v. The molecular structure of the toxin has been determined fully (1,2). PTX causes contractions in smooth muscle (i) and has a positive inotropic action in cardiac muscle (4-6). PTX also induces membrane depolarization in intestinal smooth (i), skeletal (4), and heart muscles (5-7), myelinated fibers (8), spinal cord (9), and squid axons (10). PTX has been demonstrated to cause NE release from adrenergic neurons (11,12). Biochemical studies have indicated that PTX causes a release of K from erythrocytes, which is followed by hemolysis (13-15). The PTX-induced release of K from erythrocytes is depress by ouabain and that the binding of ouabain to the membrane fragments is inhibited by PTX (15). 

It is also interesting to note that only a fraction of PS II membrane protein forms a stable monolayer structure and the rest of them fall into the water subphase. This can be seen directly by the naked eye during the compression. Furthermore, if we use the total amount of PS II membrane protein to calculate the average particle size from the n-A curve, we obtain an area of about 200 nm. This value is very small when compared with that of the PS II core complex (320 nm, as discussed in the subsequent section), which is a smaller subunit of the PS II membranes. A PS II membrane fragment contains PS II core complex and several LHC II proteins, and is much larger in size than a PS II core complex... 

Compression of the PS II membrane monolayer shows that the monolayer collapses at a relatively low surface pressure, at around 20mN/m. This can be attributed to the formation of a multilayered structure [8] and some of PS II membrane fragments diffuse into the subphase. This observation further indicates that PS II membranes can only marginally stay at the air-water interface and one must be very careful in choosing the experimental parameters. 

Heidmann, T. Oswald, R.E. and Changeux, J.-P. Multiple sites of action for noncompetitive blockers on acetylcholine receptor rich membrane fragments from Torpedo marmorata. Biochemistry 22 3112-3127, 1983. 

The application of whole-cells or enzyme-based catalysts was protected in two different bioprocess patents ([56] and [57], respectively). The patent specifies the process [57] involving a sulfur-specific reactant with membrane fragments, an enzyme, or a composition of enzymes having the ability to selectively react with sulfur by cleavage of organic C—S bonds, derived from R. rhodochrous strain ATCC No. 53968 or B. sphaericus strain ATCC No. 53969. 

EP0445896 26 A membrane fragments extract from R. rhodochrous strain ATCC No. 53968 (and/or B.sphaericus strain ATCC No. 53969) Extract and/or enzymes Enzymes associated with membrane [52]... 

Other claimed matter DBT for enrichment, biocatalyst preparation contacting process Enzymes contacting process Pure compounds as feedstock Membrane fragments and extracts Cell-free extract (envelope and its fragments + associated enzyme) reversible emulsion microemulsion reverse micelles Cell-free enzyme preparation microemulsified process RR and derivatives and other biocatalyst concepts + any known microorganism active for C—S bond cleavage... 

Godia, F., Adler, H. I., Scott, C. D., and Davison, B. H., Use of Immobilized Microbial Membrane Fragments to Remove Oxygen and Favor the Acetone-Butanol Fermentation, Biotechnol. Prog., 6 210 (1990)... 

Fig. 8.19 SEM images of mesolamellar thin films produced by intercalation of nanosheets of (A) aminopropyl-functionalized silica or (B) AMP between stacked purple membrane fragments containing bacteriorhodopsin (scale bars= 10pm).

Wilhelnova N. Thylakoid membranes fragmentation by means of different detergents during leaf antogeny. Photosynthetica 1994 30 415-424. 

Disintegration of Ligaments into Primary Membranous Fragments and Spheroid Droplets... 

Although this correlation uses rather crude data it gives a clear indication that the actual antagonist/receptor binding is the same at both the cardiac and bronchial sites and indicates that cardioselectivity is a function of distribution to the micro-environment of the receptor site. Some experimental evidence supporting this conclusion is now available from biochemical studies on isolated membrane fragments derived from heart and lung. ... 

Table 3 shows pA values for practolol and propranolol against isoprenaline induced tachycardia in the guinea pig atria, relaxation of the tracheal chain, and adenylate cyclase activity of purified membrane fragments prepared from guinea pig heart and lung. While practolol is clearly cardioselective in the tissue preparations it becomes non-selective on the membrane fragments. It is conceivable that in the purification of the membranes the integrity of the micro-environment which controls access to the receptors in the different tissues, is destroyed. 

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