Anticholinesterase Toxins In Mamba Venoms
Mbugua, Paul Mungai
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Toxins that are potent inhibitors of cholinesterases from various sources have been isolated from three mamba venoms, Dendroaspis angusticeps (Green mamba), Dendroaspis polylepis (Black mamba) and Dendroaspis viridis (Guinea's mamba). The prototypes of the new type of anticholinesterases have been named fasciculins 1 and 2 isolated and purified from Dendroaspis angusticeps venom. The first anticholinesterase toxins have been called fasciculins because after injection into mice (i.p. 0.5 - 3.0 ug/g body weight) they cause severe, generalised and long-lasting (5 - 7 hours) muscle fasciculations (Rodriquez-Ithurralde et al., 1983). Fasciculins are closely homologous proteins of 61 amino acid residues and four disulphide bridges. The molecular weights are 6765 for fasciculin 1 and 6735 for fasciculin 2. Amino acid analysis indicates that fasciculin 1 and 2 differ probaB1.y only by a single amino acid -replacement, a tyrosine residue in fasciculin 1 appears to be replaced by an asparagine or aspartic acid in fasciculin 2. The sequence of fasciculin 2 is known. Most of the positive charges are concentrated in a small section of the central part of the molecule, and most of the negative charges are in the C-terminal region. Therefore, fasciculins appear to have a pronounced dipole character. Fasciculin binds to the peripheral anionic site of acetylcholinesterase rather than the active site, since it can displace propidium, a probe for the peripheral anionic site. Fasciculin. binds with a Hill coefficient of 0.97. That suggests that one fasciculin binds per catalytic subunit. In vitro, in Krebs-Henseleit original Ringer bicarbonate solution containing 2mM NaH2P04 (pH 7.4), fasciculin 2 inhibits acetylcholinesterase from human erythrocytes (K. = 1.1 x 10-10 1 -10 M, 37°) and Electrophorus electricus ( 3 x 10 M, 22°). It also inhibits butyrylcholinesterase, but with a much lower affinity (K. = 3 x 10-6 M, 1 Fasciculins influence the cholinesterase activity in three different ways:- 1) Inhibition of acetilcholinesterases with K.s between l x 10-11 M and 7 x 10-10 M1 and butyrylcholinesterases with K.s 2 - 3 UM.1 2) Partial inhibition by 10 - 30% by low (< 0.5 nM) concentrations and reactivation at 1 nM to a level of 80 - 110% of the initial activity. A further increase of the fasciculin concentration has no effect on the enzyme activity. Fasciculins have no effect on the cholinesterase activity of cobra venom, chick brain and biventer cervicis, rat liver, hen plasma and a minor component in human plasma. Several organs contain fasciculin-sensitive and insensitive enzymes; example are monkey uterus, ratuterus, bovine gall bladder and atrium, ~en atrium and ventricle. Human plasma also contains these two types. The major component of human plasma is a fasciculine-sensitive butylcholinesterase inhibited by a K. of 2.5 UM.1 The enzyme activity in chick brain and in chick biventer cervicis muscle changes from being insensitive to fasciculin in an8 day old chick to sensitive in a 2 month 61d hen. Cobra venom acetylcholinesterase becomes sensitive to fasciculin after storage for about six montps at 200. The fasciculinin sensitive cholinesterase in human plasma had a molecular weight of 71,000 as determined by gel filtration on AcA 34, the same size as the Gl form of cholinesterases. The insensitive acetylcholinesterase from cobra venom had a molecular weight of only 43,000. The great differences in the behaviour between fasciculin and various cholinesterases are believed to reflect structural differences in the peripheral regulatory non-catalytic anionic sites. In mouse phrenic nerve-hemidiaphragm preparations, the fasciculins can increase twitch responses to indirect stimulati6n. Fasciculins facilitate neuromuscular transmission by increasing the amplitude and duration of endplate potentials. Interactions with neostigmine cop f i rm that fasciculins act as anticholinesterases. They have no presynaptic actions on transmitter release or postsynpatic receptor blocking actions. Fasciculins have no fascilitatory effect on chick biventer cervicis nerve:mucsle, anterior latissiumus dorsi (ALD) muscles and skeletal muscle cultures preparations, confirming biochemical findings of species selectivity. Results obtained with guinea pig ileum longitudinal muscle myenteric preparation suggest that fasciculins produce smooth muscle spasms by enhancing release of neurohumoral transmitter at presynaptic level.