Biomedical subjects

D A Beneski

Publications and source records attributed to D A Beneski.

7 recordsLinked to original sources

PubMed1984

Differential labeling of the alpha and beta 1 subunits of the sodium channel by photoreactive derivatives of scorpion toxin.

The separation of two photoreactive derivatives of the alpha-scorpion toxin from Leiurus quinquestriatus is described. When the two photoreactive derivatives were photolyzed separately in the presence of brain membranes containing voltage-sensitive sodium channels, one labeled the alpha subunit preferentially while the other labeled beta 1 more intensely than alpha. Batrachotoxin enhanced the efficiency of covalent labeling by the photoreactive derivatives of scorpion toxin. In all the labeling experiments, the specific incorporation of radioactive scorpion toxin was eliminated by an excess of nonradioactive scorpion toxin. The alpha polypeptide labeled in synaptosomes by photoreactive scorpion toxin was demonstrated by immunological techniques to be the same large polypeptide identified in sodium channels purified by their saxitoxin binding activity. The alpha and beta 1 subunits were detected by rapid photoaffinity labeling of a freshly prepared brain homogenate in the presence of a mixture of nine protease inhibitors, indicating that they are components of the sodium channel in intact brain tissue. The association of the covalently labeled polypeptides with the membrane was investigated by treatment of labeled synaptosomes with various agents known to remove proteins only indirectly attached to the lipid bilayer via a membrane-bound protein. In all cases, both the alpha and the beta 1 polypeptides remained in the membrane fraction following extraction. This confirms earlier proposals that the alpha polypeptide has a portion of its mass embedded within the lipid bilayer and suggests that the beta 1 polypeptide does as well.

Affinity Labels↗

PubMed1984

Human retinal S-antigen. Isolation, purification, and characterization.

S-antigen, a highly pathogenic agent for the inducation of experimental autoimmune uveitis ( EAU ), was obtained in a pure state from human retina by conventional salt fractionation, molecular sieve and ion exchange chromatography. The physical and chemical properties of the purified protein including the molecular weight, isoelectric point and amino acid composition were determined. The physical properties of the purified human retinal S-antigen were similar, but not identical, to those previously reported for bovine retinal S-antigen (J Immunol 119:1949, 1977). Following the injection of 50 micrograms of human retinal S-antigen into the footpads of guinea pigs, EAU was documented both clinically and histopathologically. The relationship between S-antigen, uveitis, and the pineal gland is discussed.

Animals↗

PubMed1982

Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a phosphocarrier protein HPr from wild type and mutants of Salmonella typhimurium.

HPr, the histidine-containing phosphocarrier protein of the phosphotransferase system, has been isolated and purified from wild type Salmonella typhimurium and from two mutants of this organism. Comparison of the sequences of these three forms of HPr indicates that the two mutants contain substitutions at residue 4 in the polypeptide chain; in place of glutamine in the wild type, one mutant (SB3899) contained serine and the other (SB3093) contained lysine. The substitution of lysine for glutamine resulted in increased positive charge of the molecule which is reflected in an expected decreased mobility on polyacrylamide gel electrophoresis and its behavior on isoelectric focusing. However, these changes had no effect on phosphocarrier activity in the phoshoenolpyruvate:glycose phosphotransferase system as measured by the kinetics of the interaction on the mutant HPr proteins with both Enzyme I and Enzyme II. These results have important implications for potential chemical modification of HPr. The HPr from wild type cells was crystallized.

Amino Acid Sequence↗

PubMed1982

Sugar transport by the bacterial phosphotransferase system. Preparation and characterization of membrane vesicles from mutant and wild type Salmonella typhimurium.

Modifications of published procedures (reviewed by Kaback, H. R. (1974) Science (Wash. D. C.) 186, 882-892) were developed for preparing membrane vesicles from Salmonella typhimurium. The preparations consisted largely of closed, unilamellar structures and contained inner membrane with little to no contamination by outer membrane or cell wall. A variety of cytoplasmic proteins was assayed in the membrane preparations, and they were found to be present at low to trace levels, whereas other proteins known to be associated with membranes were found at high levels (with respect to specific activities) in the vesicle preparations. At least 90% of the vesicles appeared to be oriented right-side-out; we do not know whether the remaining 10% represents closed vesicles oriented inside-out or "leaky" right-side-out vesicles. The vesicle preparations were impermeable to both low and high molecular weight solutes, for example, to both intra- and extravesicular sucrose. In double label experiments, the vesicle volumes were found to be about 6 microliters/mg of protein for preparations isolated from the wild type strain, and about 4.5 microliters/mg of protein for vesicles isolated from a mutant, SB2950, deleted in ptsH, ptsI, and crr genes (proteins HPr, Enzyme I, and IIIGlc, respectively). One advantage of S. typhimurium over Escherichia coli for these studies is that the former can be induced to take up phosphoenolpyruvate. This may be the reason that S. typhimurium vesicles transported methyl alpha-glucoside at 4- to 100-fold the rates reported for vesicles from E. coli, while uptake rates of proline were comparable in the two types of preparations. Vesicles from strain SB2950 were unable to take up methyl alpha-glucoside, but the transport (and phosphorylating) system was reconstituted in the vesicles by trapping the soluble purified proteins inside the vesicles during preparation of the latter. All three proteins were required for reconstruction. Studies with intra- and extravesicular soluble proteins of the phosphoenolpyruvate:glucose phosphotransferase system showed that the IIMan complex, which phosphorylates glucose, 2-deoxyglucose, and other sugars, is symmetrically oriented in the membranes. That is, this complex could phosphorylate 2-deoxyglucose when supplemented with Enzyme I and HPr either inside or outside of the membranes, and the sugar phosphate was found on the same side of the membranes as the soluble phosphotransferase system proteins. The integral membrane protein, II-BGlc, which phosphorylates glucose and methyl alpha-glucoside, showed contrasting behavior. Methyl alpha-glucoside phosphate was formed (intravesicularly) only when the soluble proteins (Enzyme I, HPr, and IIIGlc) were located inside the vesicles. Thus, II-BGlc appears to be asymmetrically oriented in the membranes.

Biological Transport↗

PubMed1982

Molecular Properties of neurotoxin receptors sites associated with sodium channels from mammalian brain.

Neurotoxins that act at specific receptor sites on voltage-sensitive sodium channels have been used as molecular probes to identify and purify protein components of sodium channels from mammalian brain. Photoreactive derivatives of scorpion toxin have been prepared and used to covalently label sodium channels in intact synaptosomes. Two polypeptides, alpha with Mr approximately 270,000 and beta with Mr approximately 38,000, are specifically labeled indicating that they are components of the scorpion toxin receptor site on the sodium channel. The sodium channel can be solubilized with retention of specific binding of [3H] saxitoxin using nonionic detergents such as Triton X-100. The solubilized saxitoxin receptor has molecular weight of 316,000 +/- 63,000 and binds 0.9 g of Triton X-100 and phospholipid per g of protein. The solubilized receptor can be purified 750-fold by ion exchange chromatography, wheat germ lectin/Sepharose chromatography and sucrose gradient sedimentation to a final specific activity of 1488 pmol/mg. Analysis of the polypeptide chain composition of the most highly purified fractions indicates that alpha and beta comprise 65% of the protein of these fractions and are only the polypeptides whose presence correlates with saxitoxin binding activity. These studies lead to a working hypothesis of sodium channel structure in which the intact channel is comprised of a complex with Mr of approximately 316,000 containing one mole of alpha (Mr approximately 270,000) and one to three moles of beta (Mr approximately 38,000).

Animals↗

PubMed1980

Interaction of polypeptide neurotoxins with a receptor site associated with voltage-sensitive sodium channels.

Anthopleurin A, a polypeptide toxin from the Pacific sea anemone Anthopleura xanthogrammica, enhances persistent activation of voltage-sensitive sodium channels by the alkaloid toxins veratridine and batrachotoxin with K0.5 = 20 nM. This effect is inhibited by depolarization. There is a close correlation between enhancement of sodium channel activation and block of [125I]scorpion toxin binding by unlabeled scorpion toxin, sea anemone toxin II from Anemonia sulcata, and Anthopleurin A, indicating that these three polypeptide toxins interact with a common receptor site in modifying sodium channel function. Photo-activable derivatives of scorpion toxin label a single Mr approximately 250,000 polypeptide chain at the polypeptide toxin receptor site. Labeling is blocked by unlabeled scorpion toxin or depolarization and is not observed in variant neuroblastoma clones, which lack sodium channels. These results identify a protein component of the polypeptide toxin receptor site of voltage-sensitive sodium channels.

Affinity Labels↗

PubMed1980

Covalent labeling of protein components of the sodium channel with a photoactivable derivative of scorpion toxin.

Azidonitrobenzoyl mono[125I]iodo scorpion toxin can be covalently attached to its receptor site in electrically excitable neuroblastoma cells or synaptosomes by photolysis. A polypeptide of Mr approximately 250,000 is specifically labeled in neuroblastoma cells. Labeling is blocked by unlabeled scorpion toxin and by depolarization. This polypeptide is not labeled in a variant neuroblastoma clone lacking voltage-sensitive sodium channels. Polypeptides of Mr approximately 250,000 and Mr approximately 32,000 are specifically labeled in synaptosomes. This labeling is also blocked by unlabeled toxin and by depolarization. These results identify specific polypeptides of Mr approximately 250,000 and Mr approximately 32,000 that are components of the sodium channel.

Affinity Labels↗