Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA.

This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface. The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface. Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision. Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface. To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate. The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface. Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates. The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer.     

Alterations in high-affinity binding characteristics and levels of opioids in invertebrate ganglia during aging: Evidence for an opioid compensatory mechanism

In Mytilus and Leucophaea the high-affinity binding site density is significantly lower in old animals than in young animals, whereas the low-affinity site density remains unchanged. In Mytilus the estimated met-enkephalin and met-enkephalin-Arg6-Phe7 levels are significantly higher in old than in young animals. In Leucophaea only the met-enkephalin level can be determined, and it is also higher in old animals. The decrease in the high-affinity binding site density and the corresponding increase in endogenous enkephalin levels suggest the existence of an opioid compensatory mechanism associated with the aging process. In Mytilus there is a demonstrated decrease with age in intraganglionic dopamine levels in response to applied opiates. In addition, the inhibition of dopamine-stimulated adenylate cyclase activity by opiates also decreases in older animals. In Leucophaea the sex difference in opioid binding densities diminishes with age.     

Genetic basis of ultraviolet-B effects on contact hypersensitivity

The genetic basis of the effects of ultraviolet B(UVB) radiation on the induction of contact hypersensitivity (CH) to dinitrofluorobenzene (DNFB) has been explored in genetically defined mice. It was found that acute, low-dose UVB radiation produced profound depletion of epidermal Langerhans cells (LC) at UVB-treated sites in all strains of mice tested. However, when DNFB was applied to UVB radiation sites, unresponsiveness developed in some strains of mice, but vigorous contact hypersensitivity was induced in others. The UVB-susceptible phenotype proved dominant or codominant in F₁ hybrids derived from parental strains of the susceptible and UVB-resistant phenotypes. Experiments conducted in one set of F₁ hybrids derived from two UVB-susceptible parental strains displayed UVB resistance, suggesting gene complementation, and showed that more than one genetic locus was involved. Segregant backcross populations, analyzed for the capacity to develop CH after UVB treatment and skin painting with DNFB, revealed that at least two, and probably three, independent genetic loci participate in determining UVB resistance. Results of experiments with H-2 congenic and recombinant mice derived from the B10 background implicated class I genes of the major histocompatibility complex as relevant genetic factors. These results indicate that there is a dissociation between the effects of UVB radiation on epidermal Langerhans cells and the capacity of a cutaneous surface to support the induction of contact hypersensitivity. The data indicate that the induction of CH to haptens is dependent on normal numbers of functional LC at the skin painting site only in some strains of mice. The data imply that in the so-called UVB-resistant strains of mice, alternative (non-Langerhans cell-dependent) mechanisms allow for the induction of CH. Several independent genetic loci, one of which appears to be H-2, govern this UVB-related effect.     

Kinetics of Ca+ adsorption and cationic selectivity with a synaptic membrane protein

A study was conducted on the adsorption of ⁴⁵Ca²⁺ to a surface film of a hydrophobic protein derived from synaptic membranes isolated from bovine cerebellum. A kinetic analysis of Ca²⁺ displacement from the protein by various metal and organic cations could be described by a rate law based on diffusion and displacement. The relative rate constants for the displacement of bound Ca²⁺ were in the order Li⁺ <Na⁺, Rb⁺ <Cs⁺ <K⁺, NH₄⁺. Among the alkaline earth series the sequence was Mg²⁺, Sr²⁺ <Ba²⁺. Ca²⁺ adsorption could be described by a theoretical formulation which takes into account an interfacial energy and potential barrier as well as the diffusional process. An attempt was made to consider the effect of energy of hydration of the cations, surface charge, and the chemical environment at the interface on catonic selectivity. The behavior of the cations in this system significantly resemble their behavior in natural membranes, particularly excitatory ones. The structural and physicochemical environment of the protein at the interface is discussed in relation to Ca²⁺ binding and cationic selectivity.     

Nasal trigeminal responses to toluene presented by an automated delivery system

An apparatus was developed which permits the automated deliveryof volatile chemical stimuli for use in neurophysiology experiments.A computer-controlled olfactometer, incorporating electronicmass flow controllers (EMFCs) and Teflon-lined solenoid valves,generated and delivered clean or odorized air. Neural and respiratorysignals from the animal were amplified and stored, along withtrial information (e.g. odorant concentration) on a chart recorderand video cassette recorder, both of which were controlled bythe computer. This apparatus was used to measure responses totoluene from the rat ethmoid nerve, a part of the ophthalmicdivision of the trigeminal nerve. Multi-unit responses to thiscompound were first observed at 2000 p.p.m. The magnitude ofthe response increased linearly with logarithmic increases inconcentration up to vapour saturation. Changes in respirationin response to toluene also were observed, although neural responsesoften were seen in the absence of respiratory changes.     

Ammonium release by zooplankton in suspensions of heat-killed algae and an evaluation of the flow-cell method

The maximum excretion rate of NH₄ (39 nmol mg dry wt^–1h^–1) was directly measured for Daphnia pulex by measuringNH₄ accumulation in bottles containing D. pulex and dense, satiatingsuspensions of heat-killed algae. Ammonium release rates inthe algal suspensions were compared to those of individual animalsremoved from the suspension and placed in flow cells. Ammoniumrelease rate, R (nmol mg dry wt^–1 h^–1). in the flowcell decreased very rapidly with time, t (min), after removalaccording to the relation R = 26 + 25e^–0.16t. Ammoniumexcretion obtained by the flow cell method after extrapolationto time zero was not significantly different from that obtainedin the bottles. The considerable experiment-to-experiment variationin NH₄ excretion was in large part correlated (r² = 0.73) withthe feeding rate on the algae.