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B R DasGupta

Publications and source records attributed to B R DasGupta.

At least 73 records · Page 4Linked to original sources
PubMed1988

Botulinum neurotoxin types A, B & E: pH induced difference spectra.

The alkaline pH induced difference spectra (270-310 nm) of three antigenically distinct forms of the botulinum neurotoxin (NT) types A, B and E were examined. When isolated from the cultures of Clostridium botulinum, type A NT is a fully toxic dichain (nicked) protein, type E is a mildly toxic single chain (unnicked) protein, and type B NT is a mixture of single and dichain proteins and near fully toxic. Trypsin nicks the single chain protein to the dichain and increases its toxicity (up to about 100 fold in type E). A strong difference spectrum peak at approximately 296 nm was found when types A, B or E NT were in the alkaline pH region. This peak was not observed at pH 4.0. For types A and B NT plots of difference absorptivity vs. pH were simple sigmoidal curves. The pK of phenolic moieties of tyrosine residues in both proteins were 10.9. Nearly all tyrosine residues in both proteins were ionized. The single chain type E, unlike type A and B NT, yielded a two step titration curve and pK values 11.3 and less than 7.5; about 60% of the total tyrosine residues present were ionized. The two step titration curve was not observed when the single chain protein was nicked with trypsin to the dichain type E NT. The titration curve of dichain type E NT, although complex, was more like those of type A and B NT.

Botulinum Toxins↗
PubMed1988

Reductive methylation of lysine residues of botulinum neurotoxin types A and B.

Reductive methylation of botulinum neurotoxin (NT) serotypes A and B at various ratios of protein to reagent modified up to 75% of the lysine residues. Amino acid analysis of the modified proteins (HCl hydrolysed) confirmed selective modifications of lysine. The derivative N,N-dimethyl lysine was more abundant than monomethyl lysine; trimethyl lysine was not detected. Distribution of modified lysine residues among the heavy and light chains (Mr approximately 100,000 and approximately 50,000, respectively) of the dichain type A NT (Mr approximately 150,000) was approximately proportional to the lysine contents of the two subunit chains of the NT. Toxicity (mouse lethality) and serological reactivity (polyclonal antibody) of serotype A NT were not (or insignificantly) damaged following methylation of up to 72 lysine residues. Modification of 3 additional residues caused precipitous loss in toxicity. Toxicity of serotype B NT, unlike type A, appeared more sensitive to lysine modification. The large number of lysine residues that can be methylated without damaging toxicity of type A NT can be exploited to a) radiolabel the dichain protein exclusively in one chain keeping the other chain unlabelled, b) restrict the number of tryptic cleavage sites of the NT, and c) tag the protein with various markers or reactive ligands.

Animals↗
PubMed1988

Circular dichroic and fluorescence spectroscopic study of the conformation of botulinum neurotoxin types A and E.

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum in any of seven antigenically distinct forms, called types A through G. Protease(s) endogenous to the bacteria, or trypsin, nicks the single chain protein to a dichain molecule which generally is more toxic. The conformation of dichain type A (nicked by endogenous protease), single chain type E, and dichain type E NT (nicked by trypsin) have been determined using circular dichroism (CD) and fluorescence spectroscopy. The high degree of ordered secondary structure (alpha helix 28%, beta sheet 42%, total 70%) found in type A NT at pH 6.0 was similar to that found at pH 9.0 (alpha 22%, beta 47%, total 69%). The secondary structure of the single chain type E NT at pH 6.0 (alpha 18%, beta 37%, total 55%) differed somewhat from these values at pH 9.0 (alpha 22%, beta 43%, total 65%). The dichain type E NT at pH 6.0 assumed a secondary structure (alpha 20%, beta 47%, total 67%) more similar to that of dichain type A than the single chain type E NT. Examination with the fluorogenic probe toluidine napthalene sulfonate revealed that the hydrophobicity of the type A and E NTs were higher at pH 9.0 than at pH 6.0. Also, the hydrophobicity of the dichain type E NT was higher than its precursor the single chain protein and appeared similar to that of the dichain type A NT. The CD and fluorescence studies indicate that conversion of the single chain type E NT to the dichain form (i.e. nicking by trypsin) induced changes in conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Botulinum Toxins↗
PubMed1987

The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers.

The heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayer membranes. Channel activity is confined to the N-terminal half of this chain; the C-terminal half is inactive. Channel activity is stimulated by low pH (4.5-5.5) on the cis side (the side to which protein is added), neutral pH on the opposite (trans) side, and cis positive voltages. These findings are strikingly similar to those previously reported for analogous fragments of diphtheria and tetanus toxins.

Botulinum Toxins↗
PubMed1987

Role of the heavy and light chains of botulinum neurotoxin in neuromuscular paralysis.

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum in any of seven antigenically distinct forms called types A-G. NT, when fully active, is a dichain protein, composed of two polypeptides, a heavy (H) and a light (L) chain (approximately 100,000 and approximately 50,000 Da, respectively) that are held together by noncovalent bonds and at least one disulfide bond. Two types of dichain NT, A and B, and their respective H and L chains were applied to nerve-muscle (NM) preparations (phrenic nerve-hemidiaphragm of the mouse), in order to develop a broader, comparative understanding of the neuroparalytic actions of NT types. It was found that the paralysis induced by dichain NT was delayed or antagonized if NM preparations were incubated with isolated and purified H chain prior to, or during, incubation with the parent, dichain NT. NM preparations preincubated with H chain and then washed free of unbound H chain became paralyzed after subsequent incubation with L chain. Paralysis did not occur if NM preparations were incubated first with L chain, washed, and then incubated with H chain. These observations suggest that the H chain binds with specific sites on the nerve terminal. This binding appears to permit the L chain, or some combination of the L and H chain, to bring about neuroparalysis through a mechanism very similar to that of the parent, dichain NT.

Animals↗
PubMed1987

The plantar nerves-lumbrical muscles: a useful nerve-muscle preparation for assaying the effects of botulinum neurotoxin.

The plantar nerves-lumbrical muscles (PL) of the hindpaw of the mouse is a preparation that is particularly useful for assaying the effects of botulinum neurotoxin (NT) on the mammalian neuromuscular junction. Each mouse provides a minimum of 4 and a maximum of 8 such preparations. The muscles are thin enough so that neuromuscular junctions can be easily located with Nomarski interference contrast optics for impaling with microelectrodes. The preparations are small enough so they can be sectioned transversely or longitudinally, in their entirety, for light and electron microscopy, or just the regions containing end-plates can be used. An examination of the responses of this preparation to types A and E NT reveal its exceptional suitability for experiments with NT. The small number of fibers and their arrangement permit assays of low concentrations of NT to proceed to complete and total paralysis in a convenient period of time.

Animals↗
PubMed1985

Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E.

Clostridium botulinum produces botulinum neurotoxin (NT) in antigenically distinct forms. When isolated from bacterial cultures type E is a single chain, type B is a mixture of single and two-chain molecules, and type A is essentially a two-chain molecule (Mr approximately 150,000). Protease(s) in the cultures or trypsin nick single-chain NT to the two-chain form. The heavy (Mr approximately 100,000) and light (Mr approximately 50,000) chains of the two-chain molecule remain held together by -S-S-bond(s). The two chains are presumed to have different functions. NT binds to nerve cells via the heavy chain and then light chain enters the cell and blocks release of acetylcholine (Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188). We nicked single-chain NT to form the two-chain form with trypsin, minimizing secondary cleavages, then separated and purified the heavy and light chains using ion-exchange chromatography. The technique, with minor modifications, is a generalized method for types A, B, and E. These subunit chains (each a single band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis) were analyzed for their complete amino acid compositions. The amino acid contents of the heavy and light chains agreed well with the parent two-chain molecule. This affirms that NT is composed of two chains. The two subunit chains are now usable for amino acid sequence and other studies. Comparison of the amino acid contents indicates more similarity among the light chains than the heavy chains of the three NT types, a similarity that agrees with our published partial amino acid sequences (first 13-18 residues) of these chains. Several (up to 9) different amino acid residues of the heavy chain (which is twice the size of the light chain) are present in double the number of corresponding residues in the light chain.

Amino Acid Sequence↗
PubMed1985

Partial amino acid sequences of botulinum neurotoxins types B and E.

Clostridium botulinum type E neurotoxin, a single-chain protein of Mr 147,000, was purified and subjected to amino acid sequencing. The same was done for single-chain botulinum type B neurotoxin (Mr 152,000), and for the heavy and light chains (Mr 104,000 and 51,000 respectively) derived from type B by limited trypsin digestion. Twelve to eighteen residues were identified and the following conclusions were drawn: The light chain of the nicked (dichain) type B is derived from the N-terminal one-third of the single-chain (unnicked) parent neurotoxin; sequence homologies are present between single-chain types B and E and the light chain of the nicked type A [J. J. Schmidt, V. Sathyamoorthy, and B. R. DasGupta (1984) Biochem. Biophys. Res. Commun. 119, 900-904]; the N-terminal regions of the heavy chains of types A and B have some structural similarity; and activation of type B neurotoxin cannot involve removal of amino acids or peptides from the N terminus.

Amino Acid Sequence↗
PubMed1985

Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E.

The dichain type E botulinum neurotoxin, a product of nicking the single chain protein by trypsin, is composed of a heavy and light chains. Sequence of the first 13 and 20 N-terminal residues of these two chains were determined. Also, proof is provided here that (i) the light chain of the nicked (dichain) is derived from the N-terminal one-third of the parent single chain neurotoxin, and (ii) molecular events leading to the activation, of the single chain neurotoxin cannot involve tryptic cleavage at or very close to the N-terminal of the single chain protein. The partial amino acid sequence of the light chain of botulinum type E and tetanus neurotoxins show significant similarity between the two clostridial neurotoxins.

Amino Acid Sequence↗
PubMed1985

Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes.

The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as "tunnel proteins" for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

Botulinum Toxins↗
PubMed1984

Partial amino acid sequence of the heavy and light chains of botulinum neurotoxin type A.

The dichain (nicked) type A botulinum neurotoxin is a protein (mol. wt. 145,000) composed of a heavy and a light chain (mol. wt. 97,000 and 53,000, respectively) that are held together by disulfide bond(s). We report here the sequence of the first 17 amino acid residues of the light chain, and the first 10 residues of the heavy chain. The heavy chain was isolated from the neurotoxin by two different methods, while the light chain was isolated by the only available method. The identical amino acid sequence was found in both preparations of heavy chain. Two samples of the light chain isolated from two separately prepared batches of the neurotoxin also had identical sequences.

Amino Acid Sequence↗
PubMed1984

Amino acid composition of Clostridium botulinum type B neurotoxin.

To characterize type B botulinum neurotoxin based on reliable data on the amino acid composition, three batches of the neurotoxin were analyzed. Each batch was isolated from a separate neurotoxin producing bacterial culture (strain Okra). Two batches were purified by the same method and one was purified by a different method. The toxin preparations were comparable in purity (judged by polyacrylamide gel--sodium dodecyl sulfate electrophoresis) and similar in amino acid composition. The best estimate of the number of amino acid residues per toxin molecule (mol. wt 152,000) was: Asp212,Thr54,Ser83,Glu130,Pro46,Gly61++ +,Ala44,Val54,CyS11,Met23,Ile144,Leu107 , Tyr81,Phe77,Lys118,His7,Arg39,Trp18.

Amino Acids↗
PubMed1984

Purification and amino acid composition of type A botulinum neurotoxin.

A method to purify type A botulinum neurotoxin from a 64 liter bacterial culture is reported. The procedure includes cation exchange chromatography at pH 7.0. The final product, essentially homogeneous (according to polyacrylamide gel-sodium dodecylsulfate electrophoresis), is a mixture of two forms of the neurotoxin (mol. wt 145,000); the dichain or nicked form (over 95%) and its precursor the single chain or unnicked form. Two batches of the neurotoxin purified by the method described here and one batch purified according to the method of Sugii and Sakaguchi were similar in purity and amino acid composition. The best estimate of the number of amino acid residues per neurotoxin molecule (mol. wt 145,000) is: Asp200Thr75Ser79Glu114Pro44Gly64Ala53Val70CyS10Met22Ile111Leu104Tyr71 Phe68Lys100His14Arg43Trp17.

Amino Acids↗
PubMed1983

Purification and amino acid composition of type E botulinum neurotoxin.

The procedure we have adopted to purify type E botulinum neurotoxin (mol. wt. approximately 147,000) from bacterial cultures consistently yields a pure protein (a single band in polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate). Our procedure is a modification of one of the five published procedures. Other procedures have failed to yield pure neurotoxin. To develop reliable data on the amino acid composition, three batches of the neurotoxin were analyzed, each batch isolated from a separate neurotoxin producing culture. The best estimate of the number of amino acid residues per neurotoxin molecule was: Asp240 Thr75 Ser98 Glu118 Pro45 Gly58 Ala40 Val62 CyS7 Met17 Ile123 Leu107 Tyr70 Phe62 Lys97 His15 Arg34 Trp16.

Amino Acids↗
PubMed1981

Enhancement of N-hydroxy-2-aminofluorene bacterial mutagenicity by the soluble protein fraction from rat liver and partial purification of the enhancement activity.

Bacterial mutagenesis from 2-aminofluorene mediated by washed rat liver microsomes was elevated 2- to 3-fold by addition of the hepatic soluble protein fraction. Enhancement was observed at 2-aminofluorene concentrations between 1 and 20 micrograms/assay but not at 30 to 50 micrograms/assay. The soluble protein fraction (without added microsomes) did not activate 2-aminofluorene for bacterial mutagenesis. However, mutagenesis by N-hydroxy-2-aminofluorene or 2-nitrosofluorene was enhanced by the soluble protein fraction, but only when reduced nicotinamide adenine dinucleotide phosphate was also present. On the basis of chemical assay, reduced nicotinamide adenine dinucleotide phosphate reduced 2-nitrosofluorene to N-hydroxy-2-aminofluorene and protected the hydroxylamine from oxidation, thus indicating that it was the mutagenicity of N-hydroxy-2-aminofluorene (but not 2-nitrosofluorene) which was enhanced by the soluble protein fraction. Without the added soluble protein fraction, mutagenesis by N-hydroxy-2-aminofluorene or 2-nitrosofluorene was unaffected by reduced nicotinamide adenine dinucleotide phosphate. We succeeded in partially purifying a protein fraction with properties of the enhancement activity. The partially purified fraction, which represents a 14-fold increase in specific activity, was assigned a molecular weight of 33,500 by gel filtration through Sephadex G-100. This fraction was resolved into three components by polyacrylamide gel electrophoresis; the molecular weights of the three components were determined by sodium dodecyl sulfate-polyacrylamide gel (10%) electrophoresis to be 33,000, 27,000, and 16,250. The mechanism of mutagenesis enhancement remains unknown.

Animals↗
PubMed1980

Electrophoretic analysis of Clostridium botulinum types A and B hemagglutinins.

The serologically related hemagglutinins of Clostridium botulinum types A and B were resolved into several bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Reduction of the samples with mercaptoethanol partly changed the band patterns, but with each hemaglutinin the two fastest moving bands in the reduced and unreduced samples were the same. In type A these two smallest units were of molecular weight 14 500 and 19 500; in type B they were of molecular weight 14 500 and 20 000. The experimentally determined molecular weight of the other resolved units agreed with those calculated on the basis that they were aggregations of the two smallest units in varying but whole multiples.

Botulinum Toxins↗