Tetanus toxin: biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis.
Single-chain tetanus toxin (toxin S) was prepared from short-term cultures by lysis under protection with protease inhibitors, precipitation with 40% ammonium sulfate, gel filtration, and chromatography on DEAE ion exchanger. Its limited proteolysis by trypsin, post-arginine cleaving enzyme from mouse submaxillary gland and clostripain led to bichainal derivatives (BT, BA, BCl) consisting of a heavy chain and a larger version of the light chain. The latter was then converted by trypsin into a small version which comigrated with the light chain of bichainal extracellular toxin (BE). The light chain produced by chymotrypsin (BC) and elastase (BE1) was of intermediate size. The nick region serves as substrate for all esteroproteases investigated and comprises between one and two kDa. Limited proteolysis increased the hydrophilicity (BT greater than BE greater than S) in hydrophobic interaction HPLC, and anionic behaviour (BC greater than BE greater than BT greater than S) in DEAE ion exchanger HPLC. The bichainal toxins assessed (BC, BE or BT) were about two times more toxic than toxin S (LD50, mouse s.c. 2 ng/kg vs. 4 ng/kg). They were five to twelve times more potent than toxin S in three in vitro assays measuring the prevention of neurotransmitter release, i.e. on the phrenic nerve-hemidiaphragm preparation of the mouse (acetylcholine, with toxin BE and BT), on primary brain cell cultures from the mouse ([3H]noradrenaline, with toxin BE and BT), and on brain homogenate from rats ([3H]noradrenaline, with toxin BA, BC, BE and BT). Thus single-chain toxin is a less potent precursor of, or protoxin for, various bichainal isotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)
Weller
U
U
Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Mauler
F
F
Habermann
E
E
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D006868:Hydrolysis; D066298:In Vitro Techniques; D051379:Mice; D009469:Neuromuscular Junction / Q000187:drug effects; D009638:Norepinephrine / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D010447:Peptide Hydrolases; D051381:Rats; D013744:Tetanus Toxin / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity
D010446:Peptide Fragments; D013744:Tetanus Toxin; D010447:Peptide Hydrolases; D009638:Norepinephrine