ABSTRACT
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 100 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.
Subject(s)
Black Widow Spider/metabolism , Gene Expression , Spider Venoms/biosynthesis , Spider Venoms/chemistry , Amino Acid Sequence , Animals , Ankyrins/chemistry , Base Sequence , Cloning, Molecular , Consensus Sequence , DNA Primers , DNA, Complementary , Escherichia coli , Insecta , Lipid Bilayers , Mass Spectrometry , Membrane Potentials/drug effects , Molecular Sequence Data , Molecular Weight , Muscles/drug effects , Muscles/physiology , Oligonucleotide Probes , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Sequence Deletion , Sequence Homology, Amino Acid , Spider Venoms/pharmacologyABSTRACT
The structural gene of delta-latroinsectotoxin was cloned and its nucleotide sequence was determined. The gene contains an open reading frame of 3642 bp. The deduced amino acid sequence is homologous to the sequences of latrotoxins studied earlier.
Subject(s)
Spider Venoms/genetics , Amino Acid Sequence , Animals , Base Sequence , Black Widow Spider , Cloning, Molecular , Molecular Sequence Data , Open Reading FramesABSTRACT
cDNA encoding the putative alpha-latrotoxin precursor was isolated from spider venom glands cDNA library and sequenced. The cDNA contained the 4203 base-pair open reading frame corresponding to the 156,855-Da protein composed of 1401 amino acids. Computer analysis of the deduced primary structure revealed the presence of various internal imperfect repeats mainly in its central and C-terminal regions.
Subject(s)
Spider Venoms/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Gene Library , Molecular Sequence Data , Peptide Fragments/genetics , Poly A/genetics , RNA, Messenger/genetics , Repetitive Sequences, Nucleic Acid , Spider Venoms/chemistryABSTRACT
The primary structure of a gene of the Na+, K+-ATPase multigenic family in the human genome has been determined. The gene corresponds to a hypothetical alpha III-form of the enzyme catalytic subunit. The gene comprises over 25,000 bp, and its protein coding region includes 23 exons and 22 introns. Possible correlation between structural features of the protein and location of introns in the gene are discussed.
Subject(s)
Sodium-Potassium-Exchanging ATPase/genetics , Amino Acid Sequence , Animals , Bacteriophage lambda/genetics , Base Composition , Base Sequence , Binding Sites , Catalysis , DNA/genetics , DNA, Recombinant , Exons , Humans , Introns , Molecular Sequence DataABSTRACT
The neurotoxin Os-1 from the venom of the Central Asian scorpion Orthochirus scrobiculosus possesses a high paralytic activity against mice. This neurotoxin was subjected to tryptic, chymotryptic and BrCN cleavages and its total amino acid sequence was established. It was shown that neurotoxin Os-1 consists of 66 amino acid residues an contains four disulfide bonds.
Subject(s)
Neurotoxins/analysis , Scorpion Venoms/analysis , Amino Acid Sequence , Animals , Cyanogen Bromide , Hydrolysis , Neurotoxins/isolation & purification , Oligopeptides/analysis , TrypsinABSTRACT
Four polypeptide neurotoxins, possessing paralytic activity for mice, were isolated from the venom of the Central Asian black scorpion Orthochirus scrobiculosus. All these toxins, Os-1 - Os-4, were shown to be homogeneous by disc-electrophoresis and N-terminal group analyses. The amino acid composition of the toxins was determined, methionine residues being found in toxin Os-1. The neurotoxin Os-3 was subjected to tryptic and chymotryptic hydrolyses and its total amino acid sequence was established. It was shown that neurotoxin Os-3 consists of 67 amino acid residues with four intramolecular disulfide bonds.
