Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments. Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity.
Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments. Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity.
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胰蛋白酶灭活 A 型肉毒梭菌神经毒素并纯化两个胰蛋白酶片段。
DOI：
10.1111/j.1432-1033.1985.tb09070.x
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Jack Melling
中科院分区：
文献类型：
--
作者：
C. Shone;P. Hambleton;Jack Melling
用胰蛋白酶有限地处理A型肉毒梭菌神经毒素，导致大约在中间位置的重亚基（95000 Da）断裂，毒性活性丧失。毒性损失速率明显快于中链裂解速率；因此，超过90%的毒性损失只伴随着30-35%的重亚基中链断裂。一项关于125i标记的神经毒素与大鼠脑突触体结合的研究表明，胰蛋白酶治疗毒性的丧失与毒素与大鼠脑突触体结合的丧失是平行的，这表明在95000-Da结合亚基上至少存在两个胰蛋白酶作用位点。用胰蛋白酶长期处理神经毒素导致重亚基的46000-Da片段完全消化，留下约105000 Da的可溶性片段，其中包含与重亚基剩余（49000-Da）部分相连的轻亚基。该片段的毒性低于原始毒性的0.01%，并产生与天然毒素难以区分的免疫沉淀反应。重链的49000-Da部分从毒素的105000-Da片段中纯化出来，并确定了前35个氨基酸的序列。发现前10个残基的序列与先前报道的重亚基相同，表明49000-Da片段代表重链的nh2末端部分，该区域对色氨酸作用具有抗性。这表明，A型肉毒杆菌神经毒素重亚基的胰蛋白酶作用的主要位点靠近COOH末端，该区域多肽链的断裂导致毒性活性的丧失，这是由神经毒素结合位点的破坏介导的。
Limited treatment of Clostridium botulinum type A neurotoxin with trypsin resulted in the cleavage of the heavy (95000 Da) subunit at approximately the mid-position and a loss of toxic activity. The rate of toxicity loss was considerably faster than that of mid-chain cleavage; thus a loss of toxicity in excess of 90% was accompanied by only 30-35% mid-chain cleavage of the heavy subunit. A study of the binding of 125I-labelled neurotoxin to rat brain synaptosomes showed the loss of toxicity on trypsin treatment to be paralleled by a loss of toxin binding to rat brain synaptosomes suggesting the presence of at least two sites of tryptic action on the 95000-Da binding subunit. Prolonged treatment of the neurotoxin with trypsin resulted in the complete digestion of a 46000-Da fragment of the heavy subunit, leaving intact a soluble fragment of approximately 105000 Da containing the light subunit linked to the remaining (49000-Da) portion of the heavy subunit. This fragment exhibited less than 0.01% of the original toxicity and gave immunoprecipitation reactions indistinguishable from the native toxin. The 49000-Da portion of the heavy chain was purified from the 105000-Da fragment of the toxin and the sequence of the first 35 amino acids determined. The sequence of the first 10 residues was found to be identical to that previously reported for the heavy subunit showing that the 49000-Da fragment represents the NH2-terminal portion of the heavy chain and that this region is resistant to tryptic action. It is suggested that the primary site(s) of tryptic action on the heavy subunit of botulinum type A neurotoxin is close to the COOH terminus and that cleavage of the polypeptide chain in this region results in a loss of toxic activity mediated by the destruction of the neurotoxin-binding site.
E型肉毒杆菌神经毒素重链和轻链的部分氨基酸序列。
DOI：
10.1016/s0006-291x(85)80009-7
发表时间：
1985
影响因子：
3.1
作者：
Sathyamoorthy,V;DasGupta,BR
通讯作者：
DasGupta,BR
肉毒杆菌毒素的起源、结构和药理活性。
影响因子：
21.1
作者：
Simpson,LL
通讯作者：
Simpson,LL