The structures of purified rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). ESR spectra were recorded with a 50 gauss field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The hyperfine splittings measured from the "expanded" spectra were significantly more precise than those obtained from the "unexpanded" spectra. Both procedures were used to study the effects of various I(12,3) probe concentrations on the spectra of liver and heart membranes, as well as the effects of temperature and CaCl2 additions on the spectra of liver membranes, and revealed the following: The polarity-corrected order parameters of liver (31 degrees) and heart (22 degrees) membranes were found to be independent of the probe concentration, if experimentally-determined low I(12,3)/lipid ratios were employed. The absence of obvious radical-interaction broadening in the unexpanded spectra indicated that "intrinsic" membrane properties may be measured at these low probe/lipid ratios. Here, "intrinsic" properties are defined as those which are measured when probe-probe interactions are negligible, and do not refer to membrane behavior in the absence of a perturbing spin label. At higher I(12,3)/lipid ratios, the order parameters of liver and heart membranes were found to substantially decrease with increasing probe concentration. The increase in the "apparent" fluidity of both membrane systems is attributed to enhanced radical interactions; however, an examination of these spectra (without reference to "low" probe concentration spectra) might incorrectly suggest that radical interactions were absent. For the membrane concentrations employed in these studies, the presence of "liquid-lines" (or "fluid components") in the unexpanded ESR spectra was a convenient marker of high probe concentrations. A thermotropic phase separation was observed in liver membranes between 19 degrees and 28 degrees. Addition of CaCl2 to liver plasma membrane [labelled with "low" I(12,3) concentrations] increased the rigidity of the membrane at 31 degrees and 37 degrees, without inducing a segregation of the probe in the bilayer. Previously reported data are discussed in relation to these results, and suggested minimal criteria for performing membrane spin label studies are included.

A new neuroactive protein, beta-leptinotarsin-h, has been purified to near-homogeneity from the hemolymph of the beetle Leptinotarsa haldemani by column chromatography. beta-Leptinotarsin-h has a molecular weight of 57 000. Rat brain synaptosomes incubated with appropriate radioactive precursors release acetylcholine (ACh), norepinephrine, and 4-aminobutyrate when exposed to beta-leptinotarin-h, but do not release lactate dehydrogenase. Release of ACh has been examined in some detail. Release of ACh varies with the concentration of beta-leptinotarsin-h in a rectangular hyperbolic fashion. Half-maximal release is stimulated by a concentration of 50 ng/mL. Altering the ionic composition of the bathing solution affects the release in a manner which suggests that neither Na+ channels nor K+ channels are affected by beta-leptinotarsin-h but that the beta-leptinotarsin-h acts to increase permeability to Ca2+. Varying the concentration of Ba2+, Sr2+, Co2+, and Cd2+ indicates that beta-leptinotarsin-h acts to open the voltage-sensitive presynaptic Ca2+ channel. beta-Leptinotarsin-h may be a useful tool for studying the Ca2+ channel associated with the release of neurotransmitters.

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