Mode of interaction of polyoxyethyleneglycol detergents with membrane proteins

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.     

Ion movement through gramicidin A channels. Studies on the diffusion-controlled association step

The permeability characteristics of gramicidin A channels are generally considered to reflect accurately the intrinsic properties of the channels themselves; i.e., the aqueous convergence regions are assumed to be negligible barriers for ion movement through the channels. The validity of this assumption has been examined by an analysis of gramicidin A single-channel current-voltage characteristics up to very high potentials (500 mV). At low permeant ion concentrations the currents approach a voltage-independent limiting value, whose magnitude is proportional to the permeant ion concentration. The magnitude of this current is decreased by experimental maneuvers that decrease the aqueous diffusion coefficient of the ions. It is concluded that the magnitude of this limiting current is determined by the diffusive ion movement through the aqueous convergence regions up to the channel entrance. It is further shown that the small-signal (ohmic) permeability properties also reflect the existence of the aqueous diffusion limitation. These results have considerable consequences for the construction of kinetic models for ion movement through gramicidin A channels. It is shown that the simple two-site-three-barrier model commonly used to interpret gramicidin A permeability data may lead to erroneous conclusions, as biionic potentials will be concentration dependent even when the channel is occupied by at most one ion. The aqueous diffusion limitation must be considered explicitly in the analysis of gramicidin A permeability characteristics. Some implications for understanding the properties of ion-conducting channels in biological membranes will be considered.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

Migration Inhibition Studies on Peripheral Leukocytes and Lymph Node Cells from Ruminants with Fascioliasis

Delayed type hypersensitivity against antigens of Fasciola hepatica has been repeatedly documented in infected hosts. Evidence has been presented to suggest that the delayed reactivity may develop earlier in the regional lymph nodes of the parasitized organ than in other lymph nodes of the body (Soulsby 1971).     

Growth of Mycobacterium lepraemurium in Cultures of Mouse Peritoneal Macrophages

Successful growth of Mycobacterium lepraemurium was observed in cultures of mouse peritoneal macrophages. The optimal host cell maintenance medium was composed of 40% horse serum, 50% of the chemically defined medium NCTC 109, and 10% of a 1:5 dilution of beef embryo extract, supplemented with both liver extract and ferric nitrate. Multiplication of the bacilli was observed in 1 week and maximal growth in 6 to 7 weeks. All macrophages were filled with tens to hundreds of the organisms in cultures showing maximal growth. Glycerol caused an increase in the normal length of M. lepraemurium, without a corresponding increase in the number of the bacilli. Elongation of M. lepraemurium was observed 3 or 4 days after infection. Rapid and uniform growth of M. lepraemurium was achieved in serially transferred cultures (subcultures). The cumulative increase of the number of intracellular bacilli was 1.4 x 10(20)-fold in 14 transfers over a period of 68 weeks in one series, and 10(17)-fold in 12 transfers over a period of 56 weeks in another series. The generation time of M. lepraemurium was 7 days, a growth rate which approximates the fastest growth of the organisms in vivo. Organisms harvested from cultures at various stages of growth produced murine leprosy in mice, but showed no growth in bacteriological media. The present model offers an opportunity for studies on the host-parasite relationship without the complication of extracellular growth of the parasites.     

A Monitor for Airborne Bacteria

A unique agar drum sampler is described which indicates, continuously, the number of viable, bacterial particles per unit volume of air at the time and point of sampling. By selection of the timer and the sampling rate the sampler is suitable for quite a wide range of concentration and time. An impaction line of 484 in. greatly increases the capacity of this device over slit samplers and other instruments designed to give time-concentration data for viable airborne particles. This sampler should prove useful for: (i) monitoring airborne bacteria in hospitals, public places, and food and industrial plants; (ii) decay rate studies of bacterial aerosols; (iii) evaluation of aerial germicides; (iv) determination of effectiveness of air conditioning systems in removing airborne bacteria; and (v) many other studies in aerobiology.