Biomedical subjects
Publications and source records attributed to F Hucho.
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The activity of the multienzyme pyruvate dehydrogenase complexes, isolated from mitochondria of beef kidney, beef heart, and pork liver, is regulated by phosphorylation and dephosphorylation. Phosphorylation and concomitant inactivation of each of the three complexes are catalyzed by an ATP-specific kinase, and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase. The phosphatase has been separated from the other component enzymes of each pyruvate dehydrogenase complex, and the three phosphatases are functionally interchangeable. The kinase has been isolated from the beef kidney complex, and it is functional with the beef heart and pork liver complexes. ADP is competitive with ATP, and the ADP effect is more pronounced with the kidney kinase than with the liver and heart kinases. Pyruvate protects strongly the heart and liver pruvate dehydrogenase complexes and, to a lesser extent, the kidney complex against inactivation by ATP. Pyruvate apparently exerts its effect on the pyruvate dehydrogenase component of the complex, rather than on the kinase.
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Several wasp venoms contain philanthotoxins (PhTXs) that act as noncompetitive inhibitors (NCIs) on cation-selective ion channels including the nicotinic acetylcholine receptor (nAChR). In the search for a ligand with high affinity and specificity for the nAChR we tested a series of newly developed PhTX analogues. Modulation of the structural elements of PhTXs can significantly influence their binding affinities. This approach resulted in the development of the photolabile compound MR44. In photoaffinity labelling studies 125I-MR44 was used to map the ligand-binding site at the Torpedo californica nAChR. Upon UV irradiation of the receptor-ligand complex, 125I-MR44 was mainly incorporated into the receptor alpha-subunit. Proteolytic mapping and microsequencing identified the site of 125I-MR44 cross-linking within the sequence alphaHis-186 to alphaLeu-199 that in its C-terminal region partially overlaps with the agonist-binding site. Since bound agonists had only minor influence on 125I-MR44 photocrosslinking, the site where the hydrophobic head group of 125I-MR44 binds must be located outside the zone that is sterically influenced by agonists bound at the nAChR. A possible site of interaction of 125I-MR44 would be the N-terminal region of the labelled sequence, in which aromatic amino-acid residues are accumulated. We suggest that the polyamine moiety of 125I-MR44 interacts with the high affinity non-competitive inhibitor site deep in the ion channel, while the aromatic ring of this compound binds in the vestibule of the nAChR to a hydrophobic region on the alpha-subunit that is located close to the agonist binding site.
Amino acid sequences of several fragments of the 25 k protein (molecular mass 24,953 Da) previously isolated from cobra Naja kaouthia (Kukhtina et al. Bioorg. Khim., 2000, vol. 26, pp. 803-807) were determined. Their comparison with the primary structures of known proteins showed that the 25 k protein belongs to the CRISP family and is the first protein of this type identified in cobra venoms.
By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom alpha-neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of alpha-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions, derived from arylazide or diazirine photolabels, that alpha-neurotoxins and alpha-conotoxins bind at the subunit interfaces. Detecting the labeled alpha-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and alpha-conotoxin molecule.
Photoactivatable derivatives of the alpha-neurotoxin II from Naja naja oxiana are useful tools for investigating the three dimensional architecture of the extra-membrane part of the nicotinic acetylcholine receptor from the electric tissue of Torpedo californica. Three derivatives, carrying an azidobenzoyl group in position Lys-15, Lys-26, and Lys-46, respectively, are shown to react differently within the receptor's quaternary structure. Especially the Lys-26 and Lys-46 derivatives can be used for differentiating between the two nonequivalent alpha-subunits. The Lys-26 derivative is applied for probing the receptor subunits next to the alpha-subunit: the gamma-subunit is shown to be located next to the alpha-subunit binding d-tubocurarine with high affinity. The delta-subunit is the neighbor of the low affinity alpha-subunit. We radioiodinated the toxin derivatives and localized the 125I at the His-31 residue of the toxin. Very little label was found in position Tyr-24, the only tyrosine residue of the toxin, or in position His-4, the only other histidine residue. This result is important for the cleavage experiments necessary in attempts to identify the receptor sequence which reacted with the photolabel.