Muscarinic toxin-like proteins from cobra venom.
Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM.
Kukhtina
V V
VV
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Weise
C
C
Muranova
T A
TA
Starkov
V G
VG
Franke
P
P
Hucho
F
F
Wnendt
S
S
Gillen
C
C
Tsetlin
V I
VI
Utkin
Y N
YN
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D018678:Cholinergic Agents / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000494:pharmacology; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides; D004546:Elapid Venoms / Q000737:chemistry*; D017815:Elapidae; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D011976:Receptors, Muscarinic / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018678:Cholinergic Agents; D018076:DNA, Complementary; D004220:Disulfides; D004546:Elapid Venoms; D008024:Ligands; D011976:Receptors, Muscarinic; C418967:muscarinic toxin-like protein 1; C418968:muscarinic toxin-like protein 2; C418969:muscarinic toxin-like protein 3; D002918:Chymotrypsin; C117608:alpha-chymotrypsin