(6)(7)(8) and from rabbit muscle (5). All of these purified Na+ channels contain one very large glycoprotein subunit that migrates anomalously in sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). Al-though this large component appears to be the only subunit in the eel channel (9), the mammalian Na+ channel contains in addition one or more smaller subunits (5, 10, 11).Monoclonal antibodies provide powerful tools for probing the molecular architecture of complex proteins, for isolating these proteins from mixtures of cellular components, and for locating the protein of interest in situ. Availability of such tools would be extremely useful in studies of the mammalian The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6227 Na' channel. Monoclonal and polyclonal antibodies have been prepared against the Na' channel from the eel electroplax (12-14); however, these antibodies do not crossreact with the channel from mammalian nerve and muscle (14). A polyclonal antiserum against the rat sarcolemmal Na' channel has been characterized by our group (15). This antiserum will be useful for immunocytochemistry but lacks the fine resolution of monoclonal antibodies for probing channel structure.We report here the development of a panel of monoclonal antibodies raised against the purified rat sarcolemmal Na'channel. These antibodies have been characterized by radioimmunoassay, immunoprecipitation, immunoblot transfers, and immunocytochemistry, and they should provide the basis for new approaches to the relationship between channel structure and function in mammals.MATERIALS AND METHODS Materials were obtained from sources previously identified (3, 5-7, 10, 15) unless otherwise indicated. Saxitoxin was generously provided by E. J. Schantz, University of Wisconsin; the toxin was tritiated and purified as described (3, 16). 125I-labeled rabbit antibodies against mouse a, y, and /I heavy chains and F(ab')2 fragments were prepared by M. Cancro of the Department of Pathology at the University of Pennsylvania.Rat skeletal muscle sarcolemma was isolated in the presence of a number of protease inhibitors by differential centrifugation after extraction of the contractile proteins with LiBr (10,17). The sarcolemma was solubilized in Nonidet P-40 and the Na+ channel was purified as described (3,10
Antibodieswere raised in rabbits against the purified voltage-dependent sodium channel from rat skeletal muscle sarcolemma. The resultant antiserum reacted with the purified channel in a solid-phase radioimmunoassay and precipitated the sodium channel from a crude mixture of solubilized membrane proteins.
Human growth hormone (hGH) bound to specific sites on rat hepatocytes. The time course of hGH dissociation was comprised of more than one component. Dissociation was resolved into rapid (t1/2 = 10.5 min) and slow (t 1/2 = 6.4 h) fractions. The amount of slowly dissociable hormone increased for the first 75 min during which time cells and [125I]hGH associated. Subsequently, the amount of slowly dissociable hGH was constant. The time courses of hGH receptor binding and subsequent retention of slowly dissociable label were similar. The capacity of hepatocytes to accumulate slowly dissociable label was saturated by hGH over the same concentration range as the high-affinity binding site (KD approximately 2 nM). This suggested that a receptor-mediated process was responsible for the accumulation of slowly dissociable hGH. Rapidly dissociable label was intact [125I]hGH and fragments resulting from growth hormone degradation. Slowly dissociable hGH recovered from hepatocytes by acid extraction was intact and immunocompetent. There was a large increase in the extent of [125I]hGH degradation between 23 and 37 degrees C. Over this temperature range, the proportion of hGH not in rapid equilibrium with the medium decreased. High concentrations of hGH decreased the amount of slowly dissociable [125I]hGH retained by hepatocytes by competing for high-affinity sites. The interaction of [125I]hGH with low-affinity degradative systems was favored by the presence of hGH. The temperature and concentration dependencies of hGH retention and degradation distinguished these proceses.
Fluorescent derivatives of cytochrome c were prepared by replacing the heme iron with closed‐shell metals such as zinc or tin. The iron‐free derivatives of cytochrome c bind to yeast lactate dehydrogenase (cytochrome b2) stoichiometrically and with high affinity. Spectral overlap exists between the fluorescence of porphyrin, Zn(II) or Sn(IV) cytochrome c and the absorption of the heme of cytochrome b; therefore dipole‐dipole interaction is possible as predicted by Forster's theory of energy transfer. Changes in the fluorescence yield and the fluorescent decay profile of the cytochrome c derivatives are consistent with the view that the heme distance is sufficiently close for dipolar interactions. The distance calculated from the data depends upon assumptions in the theory for energy transfer and uncertainties in the experiment. It can be argued that due to the symmetry of the metalloporphyrins the relative orientations of the two hemes do not introduce a significant uncertainty in the calculation. However the decay profiles of the iron‐free cytochromes are complex, possibly reflecting structural rearrangement of the polypeptide chain during the fluorescent lifetime. The steady‐state fluorescent yields would indicate that the mean distance is around 1.8 nm
Twenty-one monoclonal antibodies specific for the rat skeletal muscle voltage-sensitive sodium channel have been characterized according to subunit reactivity, recognition of carbohydrates, and mutual binding interactions. All antibodies recognize the 260-kDa alpha-subunit of the sodium channel on immunoblots. N-Acetylneuraminic acid inhibited the binding of five antibodies in a concentration-dependent manner, but five other monosaccharides known to be components of the channel had no effect on antibody binding. Competition studies using biosynthetically labeled antibodies separated these 21 antibodies into groups recognizing at least nine distinct domains. Through common interactions between domains, these could in turn be associated into two larger topologically related regions. One region encompasses seven interacting domains and 16 antibodies. This region is probably extracellular by virtue of the interaction of one subgroup with N-acetylneuraminic acid, and may represent a particularly immunogenic region on this channel protein.
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