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Biomedical subjects

F Hucho

Publications and source records attributed to F Hucho.

At least 127 records · Page 7Linked to original sources
PubMed1978

The acetylcholine receptor as part of a protein complex in receptor-enriched membrane fragments from Torpedo californica electric tissue.

The acetylcholine receptor from Torpedo californica electric tissue consisting of polypeptide chains of molecular weight 42000 (+/- 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270000 (+/- 30000). Besides the receptor subunit (alpha-chain), at least three further classes of polypeptide chains are part of the complex: beta (Mr 48000), gamma (Mr 62000) and delta (Mr 68000). This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent: From the 270000 molecular weight particle the four predominant polypeptide chains of the membrane, alpha, beta, gamma, and delta, can be obtained. The gamma-polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge.

Acetylcholine↗
PubMed1978

Membranes rich in acetylcholine receptor: characterization and reconstitution to excitable membranes from exogenous lipids.

Characterization of acetylcholine-receptor-enriched membranes from Torpedo californica electric tissue by negative-staining electron-microscopy and by lipid analysis is described. The protein/lipid ratio is 70%/30%. The lipids consist of 70% phospholipids (46% phosphatidylcholine, 31% phosphatidylethanolamine, 14% phosphatidylserine, 7% sphingomyelin, 2% phosphatidylinositol of the phospholipids determined) and 20% cholesterol. The acetylcholinesterase-enriched membranes show a similar composition. The only differences are a lower protein/lipid ratio (45%/55%) and a lower phosphatidylcholine/sphingomyelin ratio of 39%/14% as compared to 46%/7% for the receptor-enriched membranes. A method of preparing single-walled phosphatidylcholine vesicles by gel filtration on Sephadex G50 according to Brunner et al. (Biochem. Biophys. Acta, 455, 322--331, 1976) is used to recombine the lipid-depleted receptor complex with artificial lipid vesicles. Starting from a lipid mixture of 46% phosphatidylcholine, 31% phosphatidylethanolamine, 14% phosphatidylserine, 7% sphingomyelin, 2% phosphatidylinositol and 15% cholesterol we obtained vesicles associated with the acetylcholine receptor complex. These receptor vesicles are chemically excitable by 10 micrometer carbamoylcholine as measured by efflux of 22Na+ from the vesicles. The excitability is blocked by preincubation with 0.5 mM alpha-toxin from Naja naja siamensis venom and by reduction with 5 mM dithioerythritol.

Acetylcholine↗
PubMed1976

Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica.

Membrane fragments from electric tissue of Torpedo californica containing nicotinic acetylcholine receptor are composed of four different polypeptide chains with molecular weights of 40,000 (alpha), 48,000 (beta), 62,000 (gamma), and 66,000 (delta). The alpha and beta chains are still present in all and gamma and delta in some of the receptor preparations after Triton X-100 extraction and purification by affinity chromatography. All components of the receptor react covalently with the photoaffinity label 4-azido-2-nitrobenzyltrimethylammonium fluoroborate, the delta chain incorporating less of the reagent as compared to the alpha and beta chains. Agonists and antagonists containing a quaternary ammonium group protect all chains against the label; the principal neurotoxin from Naja naja siamensis protects the alpha chain only. We conclude that the alpha chain binds the neurotoxin from Naja naja, the alpha and beta chains are involved in the binding of ligands with quaternary ammonium groups, and the function of the gamma and delta chains remains to be determined.

Affinity Labels↗
PubMed1975

Investigation of the symmetry of oligomeric enzymes with bifunctional reagents.

The symmetry of proteins composed of identical polypeptide chains has been investigated by means of cross-linking with bifunctional reagents and subsequent sodium dodecylsulfate-polyacrylamide gel electrophoresis. The majority of the investigations were performed with diimidates of different chain lengths (C3-C12), which react exclusively with amino groups. Aldolase, catalase, fumarase, pyruvate kinase, tetrameric proteins with identical polypeptide chains, reveal a D2 symmetry, i.e. they appear to be composed of two pairs of polypeptide chains. The validity of this conclusion is demonstrated with lactate dehydrogenase. This enzyme, shown by X-ray analysis to have a D2 symmetry, yields after cross-linking and subsequent polyacrylamide electrophoresis the band pattern expected for a protein with this quaternary structure and similar to the pattern obtained with the above enzymes. 2. The influence of the experimental conditions on the cross-linking reaction has been investigated. The selectivity of the bifunctional reagent for the different contact domains within the quaternary structure of a protein depends on the reaction time, the chain length and on the concentration of the reagent. In general the D2 symmetry becomes more obvious with increasing chain length and with increasing concentration of the diimidate. Diethylpyrocarbonate showed very little selectivity.

Binding Sites↗
PubMed1975

Studies of glutamate dehydrogenase: analysis of functional areas and functional groups.

1. It is shown by limited tryptic digestion of beef liver glutamate dehydrogenase under native conditions that the amino terminus of the polypeptide chain is located at the surface of the molecule. End-group analysis after trypsin treatment yields aspartic acid as the new N-terminal amino acid while the C-terminal threonine remains unchanged. 2. NADH, especially in the presence of 2-oxoglutarate, protects the enzyme against tryptic degradation. In the absence of the coenzyme, glutamate dehydrogenase is rapidly inactivated. 3. The regulatory effects of ADP and GTP are only slightly altered by trypsin. A small shift of the pH dependence of the activation by ADP is observed. 4. The quaternary structure of the unimer of the enzyme is not affected by limited tryptic digestion indicating that the N-terminal part of the polypeptide chain is not located in the contact domains between the polypeptide chains. The association of the hexamer to large associated particles is reduced but not abolished. 5. It is shown by treatment of the enzyme with iodo[2(-14)C]acetic acid as well as with Ellman's reagent that the six - SH groups of the polypeptide chain are buried and not accessible to these reagents in phosphate buffer. In Tris buffer they become exposed and react in the order 89, 55, 197, 115, 270, 319. This together with the result that in Tris buffer the rat of inactivation caused by trypsin is higher than in phosphate buffer indicates that Tris buffer changes drastically the properties of the enzyme. 6. Cross-linking of the enzyme molecule with bifunctional reagents and subsequent dodecylsulfate-polyacrylamide electrophoresis shows that the six identical polypeptide chains are arranged in two groups of three. 7. The implications of these results for the tertiary and quaternary structure of beef liver glutamate dehydrogenase are discussed.

Animals↗