The rate assay of alpha-toxin assembly in membrane

Abstract A rapid and easy method to determine the 'rate' of the assembly of α-toxin from Staphylococus aureus in erythrocyte membrane was described. Upon addition of a small amount of α-toxins into erythrocyte suspension, absorbance at 700 nm decreased linearly after a short period of lag time. From the linear portion of the record the rate of the assembly of α-toxin was calculated. An optimum temperature and an optimum pH for the assembly of the toxin on erythrocyte membranes were found to be 25–30°C and pH 5.     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Kinetic analyses of the membrane-bound alkaline phosphatase activity of hymenolepis diminuta (Cestoda: Cyclophyllidea) in relation to development of the tapeworm in the definitive host

The specific activities of the alkaline phosphatase (APase), type I phosphodiesterase and 5'-nucleotidase activities associated with the brush-border plasma membrane of the tapeworm, Hymenolepis diminuta, decrease significantly as the tapeworm grows and matures. Kinetic analyses of the APase activity associated with membrane preparations from whole 6-, 12-, and 18-d-old H diminuta, and individual pieces of 18-d-old H diminuta cut into ten pieces of equal length, failed to demonstrate qualitative changes in the APase activity. Therefore, the decreased specific activities are apparently due to changes in the ratios of enzymatically active to enzymatically inactive membrane proteins (ie, quantitative changes in the membrane proteins) which occur as the tapeworm grows.     

B-cells of the synovial membrane. II. Differentiation during development of the synovial cavity in the mouse

Summary Study of pre- and postnatal development of the metatarsophalangeal joint of the mouse shows that the synovial cavity (SC) forms before any differentiation of the synovial mesenchyme. The primitive cleft results from degradation of a thin vascular mesenchymal layer in direct contact with the chondrogenic layers. Differentiation of the synovial membrane coincides with clarification of the SC (3rd to 6th day of postnatal life). When dilatation of the SC occurs (6th to 8th day), the two intimal cells types (A- and B-cells) are well identified. The B-cells already show typical features at day 6; their content of typical dense secretory vesicles is comparable to that of the adult B-cells at day 13. The specific secretory function of B-cells could be correlated with the particular structure of the intimal interstitial tissue and could account for the origin of some protein(s) of the synovial fluid.ERA 178 (Neuroendocrinologie Comparée) du CNRS et INSERM     

The permeability coefficient of the wall of a villous membrane

Summary The equations hitherto used to correct the permeability coefficient for the unstirred layer influence are valid only for flat membranes. Therefore, appropriate equations for membranes with a villous surface (e.g., small intestine) have been derived. They take into account the non-linear concentration gradient in the intervillous part of the unstirred layer. Quantitative information about the geometry of the villous surface and the unstirred layer thickness are needed to calculate the permeability coefficient of the membrane wall (e.g., intestinal epithelium). The concentration of highly permeable substances drops sharply already in the upper part of the intervillous space, so that the tips of the villi function as effective absorbing area. The intervillous concentration gradient of a substance with a low permeability coefficient is so small, that such a substance is absorbed by the total surface area of the villous membrane. The effective absorbing area of substances with intermediate permeability coefficient lies between the described limits.     

Electrical properties of the rabbit urinary bladder assessed using gramicidin D

Summary Recently, antibiotics have enjoyed widespread usage as tools in studies of epithelial transport. In the present study we assess the usefulness of the pore-forming antibiotic gramicidin D as a means for probing the electrical properties of the tight epithelium rabbit urinary bladder. Addition of 50 M gramicidin to the mucosal bath (either a NaCl or KCl Ringer's solution) led to a large irreversible increase in the transepithelial conductance (G _T ) within 800 sec.G _T increased by approximately 1200% and 500% in KCl and NaCl Ringer's solutions, respectively. Microelectrode measurements of the resistance ration (the ration of apical membrane resitance to basolateral membrane resistance) showed that apical membrane resistance is dereased by the drug. Measurements of the basolateral membrane resistance (R _bl ) and tight junctional resistance (R _j ) using a new and independent method (based on the perturbation of basolateral membrane electrogenic Na⁺ pump) demonstrated thatR _bl andR _j were unaffected, suggesting that the effects of gramicidin are restricted to the apical membrane for periods of at least 2 hours after drug addition. The selectivity of the gramicidin-induced permeability in the apical membrane was calculated from measurements of the apical membrane potential after ion substitutions using a modified version of the constant field equation. The selectivity sequence for cations was Cs⁺>K⁺>Na⁺>Li⁺>choline. Unlike the commonly used polyene antibiotics nystatin and amphotericin B, gramicidin did not induce a significant Cl^– permeability. In addition, the dose-response curve had a slope of 1. A method is described for calculating membrane resistances directly from transepithelial measurements under some conditions of gramicidin use, without requiring the use of microlectrode measurements.     

Membrane transport during erythroid differentiation

Summary Transport, unidirectional flux, of a monosaccharide, a nucleoside and three amino acids, all of which enter cells by independent, discrete carriers, was compared at three stages of erythroid maturation, the normal (anucleate) mouse erythrocyte, and in differentiated and undifferentiated Friend erythroleukemia cells. We found specific transport alterations during this developmental program. Transport of 3-O-methylglucose increased with each successive developmental stage. Aminoisobutyrate transport was maintained during Friend cell differentiation, but fell slightly in erythrocytes. Leucine, lysine and uridine transport began to fall two days after dimethylsulfoxide exposure, and diminished further in red cells. These studies of transport are not directly comparable to uptake studies reported by others.Median cell volume and thus surface area decreased more during differentiation than amino acid transport declined, so flux, transport past a unit area of membrane, actually increased. Monosaccharide flux also increased. Only uridine transport fell in parallel to surface area. Perhaps sites for nutrient transport required for energy production are preferentially maintained.     

Electron spin resonance study of the isolated lipid components from Blastocladiella emersonii zoospores

The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T_|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (T_L and T_H) were found to be independent of the external K⁺, Ca²⁺, or Mg²⁺ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (T_M) was markedly influenced by the external Ca²⁺ concentration. Ca²⁺ increased T_M from 12°C (no Ca²⁺ added) to 22°C (10 mM Ca²⁺), i.e., growth temperature. K⁺ reversed this Ca²⁺ effect, downshifting T_M from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in T_M, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.     

Transbilayer distribution of phospholipids in photoreceptor membrane studied with trinitrobenzenesulfonate alone and in combination with phospholipase D

In a further study of the transbilayer distribution of phospholipids in rod disk membranes, the amino group reagent, trinitrobenzenesulfonate, and the phospholipid-hydrolyzing enzyme, phospholipase D, have been used alone and in combination.Under carefully defined conditions (1 mM trinitrobenzenesulfonate, pH 7.4, 20°C, darkness), trinitrobenzenesulfonate yields limited final levels of modification of phosphatidylethanolamine and phosphatidylserine, suggesting only minor reagent penetration and membrane disturbance under these conditions.Treatment of stacked disks with trinitrobenzenesulfonate under these conditions leads to a biphasic modification of the a aminophospholipids. Relatively fast (less than 1 h) modification of 50% phosphatidylethanolamine and 40% phosphatidylserine occurs, slowly rising (approx. 3 h) to 60 and 50%, respectively.Extensive treatment of stacked disks with phospholipase D leads to the hydrolysis of 55% phosphatidylcholine and 50% phosphatidylethanolamine, while phosphatidylserine is hardly attacked by this enzyme.Treatment of stacked disks with trinitrobenzenesulfonate after prior treatment with phospholipase D leads to no further modification than that maximally obtained with either reagent alone: about one-half of the three major phospholipid classes is accessible. Although both reagents differ greatly in molecular size, mode of action and other properties, they apparently see the same pool of phosphatidylethanolamine, their joint substrate. Considering that we start with the original right-side-out configuration, that all phospholipids can in principle be modified (no shielding) and that the membrane remains essentially intact, we conclude that the accessible lipid pool represents the outer face of the disk membranes.These results confirm our earlier conclusions from treatment with three phospholipases that the three major phospholipids are nearly symmetrically distributed over the two faces of the disk membrane.The divergence with the conclusions of other investigators is most likely explained by their use of disk membranes (disk vesicles) in which the original phospholipid distribution had not been maintained and/or of conditions under which trinitrobenzenesulfonate markedly penetrates the membrane.