[11] Methods for investigating the essential groups for enzyme activity

Fraenkel‐Conrat, H.

doi:10.1016/0076-6879(57)04059-8

Cited by 298 publications

(127 citation statements)

References 69 publications

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“…Surprisingly, the isoelectric point was not changed, indicating that the lysine residues were not modified. It is possible, therefore, that no acetylation actually occurred in Bosmann's preparation since, under the conditions employed, acetylation takes place preferentially on the free amino group (Fraenkel-Conrat, 1957). The radioactivity of his preparation might have come from incomplete desalting.…”

Section: Discussionmentioning

confidence: 99%

“…The yield was determined by the appearance of absorbancy at 245 nm and found to be quantitative. The Fraenkel-Conrat (1957). To 10 mg a-BuTX, dissolved in 0-5 ml 25% (w/v) sodium acetate, was added 12 mg (50 mCi) [3H] acetic anhydride at 0-2' C. Acetylation was stopped at 10 min or 40 min by diluting the reaction mixture with 5 ml cold H20; it was then dialyzed in a cellophane bag against 100 ml H20 in a refrigerator.…”

Section: N-[3h] Acetyl Imidazolementioning

confidence: 99%

“…It has previously been shown that six free amino groups in the original toxin disappear under comparable conditions of acetylation (Chang, 1970). Thus acetylation takes place exclusively at free amino groups under these conditions (Fraenkel-Conrat, 1957). The products of acetylation for 10 and 40 min will be henceforth designated as tri-N-acetyl and hexa-N-acetyl a-BuTX, respectively.…”

Section: N-[5h] Acetylationmentioning

confidence: 99%

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N,O‐di andN,N,O‐tri [³H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

Chang

Chen

Chuang

1973

British J Pharmacology

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Summary α‐Bungarotoxin isolated from the venom of Bungarus multicinctus was acetylated with [3H] acetic anhydride and N‐[3H] acetyl imidazole. Tri‐N‐acetyl and hexa‐N‐acetyl derivatives were obtained from the former, and N,O‐di, N,N,O‐tri and N,N,N,O‐tetraacetyl derivatives from the latter reaction, respectively. There were parallel decreases in both neuromuscular blocking action in the phrenic nerve—diaphragm preparation of rats and depression of acetylcholine response of the rectus abdominis muscle of frogs with increased acetylation. Also, a parallel but greater decrease of toxicity in mice was found. N,O‐Di and N,N,O‐triacetyl toxins were localized mostly in the motor endplate region of the rat diaphragm, whereas a slight nonspecific binding along the whole muscle fibre in addition to the peak in the endplate region was observed with N,N,N,O‐tetraacetyl and tri‐N‐acetyl toxins. In contrast, there was a marked nonspecific binding with hexa‐N‐acetyl toxin and no peak was observed at the endplate zone. The specific binding was saturable and irreversible. The number of toxin‐receptive sites in one endplate was 1·9–2·2 × 107 for all of the labelled toxins irrespective of their potency. (+)‐Tubocurarine protected effectively against the binding as well as the irreversible neuromuscular blocking effect of the toxins. Denervation of the rat diaphragm caused an increase of toxin‐receptive sites beginning from the endplate zone at 1–2 days and then along the whole muscle fibre, reaching the maximum at about 18 days. The total receptive sites increased by about 30‐fold. The significance of the findings is discussed and it is concluded that N,O‐di and N,N,O‐tri‐[3H] acetyl α‐bungarotoxins are specific and irreversible labelling agents for the cholinergic receptors of skeletal muscle.

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Section: Discussionmentioning

confidence: 99%

Section: N-[3h] Acetyl Imidazolementioning

confidence: 99%

Section: N-[5h] Acetylationmentioning

confidence: 99%

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N,O‐di andN,N,O‐tri [³H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

Chang

Chen

Chuang

1973

British J Pharmacology

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“…The acetylation of LDL (acLDL) was performed using saturated sodium acetate and acetic anhydride according to the method previously described (Fraenkal-Conrat, 1957). After the acetylation and subsequent dialysis, the acLDL protein content was determined and filtered through a 0.22 µm filter (Millex; Millipore, USA).…”

Section: Acetylation Of Ldlmentioning

confidence: 99%

Modified Apolipoprotein (apo) A-I by Artificial Sweetener Causes Severe Premature Cellular Senescence and Atherosclerosis with Impairment of Functional and Structural Properties of apoA-I in Lipid-Free and Lipid-Bound State

Jang

Jeoung

Cho

2011

Molecules and Cells

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Long-term consumption of artificial sweeteners (AS) has been the recent focus of safety concerns. However, the potential risk of the AS in cardiovascular disease and lipoprotein metabolism has not been investigated sufficiently. We compared the influence of AS (aspartame, acesulfame K, and saccharin) and fructose in terms of functional and structural correlations of apolipoprotein (apo) A-I and high-density lipoproteins (HDL), which have atheroprotective effects. Long-term treatment of apoA-I with the sweetener at physiological concentration (3 mM for 168 h) resulted in loss of antioxidant and phospholipid binding activities with modification of secondary structure. The AS treated apoA-I exhibited proteolytic cleavage to produce 26 kDa-fragment. They showed pro-atherogenic properties in acetylated LDL phagocytosis of macrophages. Each sweetener alone or sweetener-treated apoA-I caused accelerated senescence in human dermal fibroblasts. These results suggest that long-term consumption of AS might accelerate atherosclerosis and senescence via impairment of function and structure of apoA-I and HDL.

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“…The reaction was performed as previously described [15]. Several small aliquots of a solution of tritiated acetic anhydride in dioxane were added to the protein in half-saturated sodium acetate, at 0 "C. The acetylated protein was removed from excess reagents and salts by filtration on a Sephadex G-25 column, as described above.…”

Section: Preparation and Radioactive Labelling Of Snake Toxinsmentioning

confidence: 99%

Postsynaptic Effects of Crotoxin and of Its Isolated Subunits

Bon

Changeux

Jeng

et al. 1979

European Journal of Biochemistry

210

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Crotoxin is a potent neurotoxin from the venom of Crotalus durissus terrificus. It is composed of two subunits: a basic phospholipase A2 with low toxicity (component B) and an acidic protein seemingly devoid of intrinsic biological activity (component A). Crotoxin and its isolated phospholipase subunit block the depolarisation caused by cholinergic agonists on the isolated electroplaque from Electrophorus electricus. The other component, which is inactive when applied alone, enhances the pharmacological activity of the phospholipase when the two components are used together. Crotoxin also blocks the increase of z2Na+ efflux caused by carbamylcholine from excitable microsacs prepared from Torpedo marmorata electric organ. Crotoxin therefore acts postsynaptically, but does not interfere with the binding of a-toxin from Nu@ nigricollis to the nicotinic cholinergic receptor site. Instead, like local anesthetics, it stabilizes a desensitized form of the acetylcholine receptor characterized by its high affinity for agonists. The phospholipase component B binds in a nonsaturable manner to receptor-rich membranes. In contrast, component A does not bind to acetylcholine receptor-rich membranes, but completely prevents the non-saturable binding of component B. When the two components are applied together, a saturable binding of the latter is observed with the acetylcholine receptor-rich membranes.Crotoxin, the major component of the brazilian rattle-snake (Crotalus durissus terrificus) venom is a very potent neurotoxin which possesses a phospholipase activity [l]. It is a complex of two distinct protein subunits [2,3] : a strongly basic component (component B, M , 12000) which carries the phospholipase activity and an acidic protein (component A, M , 10000) which has no enzymatic activity and is not toxic by itself. Whereas the isolated subunits are either not or only a little toxic, they recombine into a complex which exhibits the same toxicity as the original material [4,5].The lethal effect of crotoxin has generally been attributed to a presynaptic blockage of the neuromuscular transmission [6]. At this level, it causes a reduction of acetylcholine release by the nerve terminals similar to that observed with /l-bungarotoxin [7], notexin [8,9] and taipoxin [lo]. However, having observed a depression of the response to acetylcholine of the isolated denervated rat diaphragm, VitalBrazil [ l l ] concluded that crotoxin also acts at the postsynaptic level in the mammals. In order to examine a possible postsynaptic action of crotoxin without interference with its presynaptic action, we studied the effect of crotoxin on two postsynapticEnzyme. Phospholipase A2 (EC 3.1.1.4) preparations : the isolated electroplaque from Electrophorus electricus and the acetylcholine receptor-rich membranes from Torpedo marmorata. Crotoxin affects the response of both preparations to cholinergic agonists, and our observations suggest how interactions between the two subunits increase the pharmacological effect of the phospholipase component...

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[11] Methods for investigating the essential groups for enzyme activity

Cited by 298 publications

References 69 publications

N,O‐di andN,N,O‐tri [³H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

N,O‐di andN,N,O‐tri [³H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

Modified Apolipoprotein (apo) A-I by Artificial Sweetener Causes Severe Premature Cellular Senescence and Atherosclerosis with Impairment of Functional and Structural Properties of apoA-I in Lipid-Free and Lipid-Bound State

Postsynaptic Effects of Crotoxin and of Its Isolated Subunits

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[11] Methods for investigating the essential groups for enzyme activity

Cited by 298 publications

References 69 publications

N,O‐di andN,N,O‐tri [3H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

N,O‐di andN,N,O‐tri [3H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

Modified Apolipoprotein (apo) A-I by Artificial Sweetener Causes Severe Premature Cellular Senescence and Atherosclerosis with Impairment of Functional and Structural Properties of apoA-I in Lipid-Free and Lipid-Bound State

Postsynaptic Effects of Crotoxin and of Its Isolated Subunits

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Product

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N,O‐di andN,N,O‐tri [³H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

N,O‐di andN,N,O‐tri [³H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors