scholarly journals Beta-Bungarotoxin. Separation of two discrete proteins with different synaptic actions

1978 ◽  
Vol 175 (1) ◽  
pp. 271-279 ◽  
Author(s):  
J MacDermot ◽  
R H Westgaard ◽  
E J Thompson
Keyword(s):  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Ion Exchanger ◽  
Phospholipase A ◽  
Major Protein ◽  
Subunit Structure ◽  
Crude Venom ◽  
Single Class ◽  
Physiological Properties ◽  
Polypeptide Chains

beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however, in this preparation of the toxin less than 50 muunits of phospholipase A activity were detected (1 unit of activity is the amount of enzyme forming 1 mumol of L-alpha-phosphatidylcholine/min per mg of protein).

scholarly journals Studies on the structure and activity of rabbit C1q (a subcomponent of the first component of complement)

1974 ◽  
Vol 143 (2) ◽  
pp. 265-272 ◽  
Author(s):  
Diane M. Lowe ◽  
Kenneth B. M. Reid
Keyword(s):  
Amino Acids ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Haemolytic Activity ◽  
Subunit Structure ◽  
Carboxypeptidase A ◽  
Rapid Loss ◽  
Collagenase Digestion ◽  
Polypeptide Chains ◽  
Structure And Activity

1. The subunit structure of rabbit subcomponent C1q was examined in a previous publication (Reid et al., 1972). The present paper describes some aspects of the structure of the polypeptide chains derived from the molecule. 2. The three polypeptide chains, produced by performic oxidation, of rabbit subcomponent C1q were isolated by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 3. Each chain was found to contain 15–18% glycine and significant amounts of the amino acids hydroxyproline and hydroxylysine. 4. By means of collagenase digestion it was shown that all three chains of rabbit subcomponent C1q contain collagen-like sequences of amino acids which constitute about 40% of each chain. 5. By use of carboxypeptidase A it was established, indirectly, that the collagen-like sequences, in one of the chains, are probably located near, or at, the N-terminal end of the chain. 6. Collagenase digestion and heating at 52°C (but not at 49°C) caused rapid loss of native rabbit subcomponent C1q haemolytic activity.

scholarly journals Molecular and structural characterization of NADPH-dependent d-glycerate dehydrogenase from the enteric parasitic protist Entamoeba histolytica

2003 ◽  
Vol 375 (3) ◽  
pp. 729-736 ◽  
Author(s):  
Vahab ALI ◽  
Yasuo SHIGETA ◽  
Tomoyoshi NOZAKI
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Entamoeba Histolytica ◽  
Reductase Activity ◽  
Phylogenetic Analyses ◽  
Kinetic Studies ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Subunit Structure ◽  
Protein Alignment

Putative NADPH-dependent GDH (d-glycerate dehydrogenase) of the protozoan parasite Entamoeba histolytica (EhGDH) has been characterized. The EhGDH gene encodes a protein of 318 amino acids with a calculated isoelectric point of 6.29 and a molecular mass of 35.8 kDa. EhGDH showed highest identities with GDH from ε-proteobacteria. This close kinship was also supported by phylogenetic analyses, suggesting possible lateral transfer of the gene from ε-proteobacteria to E. histolytica. In contrast with the implications from protein alignment and phylogenetic analysis, kinetic studies revealed that the amoebic GDH showed biochemical properties similar to those of mammalian GDH, i.e. a preference for NADPH as cofactor and higher affinities towards NADPH and β-hydroxypyruvate than towards NADP+ and d-glycerate. Whereas the amino acids involved in nucleotide binding and catalysis are totally conserved in EhGDH, substitution of a negatively charged amino acid with a non-charged hydroxy-group-containing amino acid is probably responsible for the observed high affinity of EhGDH for NADP+/NADPH. In addition, the amoebic GDH, dissimilar to the bacterial and mammalian GDHs, lacks glyoxylate reductase activity. Native and recombinant EhGDH showed comparable subunit structure, kinetic parameters and elution profiles on anion-exchange chromatography. We propose that the GDH enzyme is likely to be involved in regulation of the intracellular concentration of serine, and, thus, also in controlling cysteine biosynthesis located downstream of serine metabolic pathways in this protist.

The Nature of Rapidly Extracted Phycocyanin from the Blue-Green Alga Plectonema Boryanum

1971 ◽  
Vol 29 ◽  
pp. 366-367
Author(s):  
M. Kessel ◽  
R. MacColl
Keyword(s):  
Self Assembly ◽  
Analytical Ultracentrifugation ◽  
Uranyl Acetate ◽  
The Self ◽  
Major Protein ◽  
Subunit Structure ◽  
Blue Green Alga ◽  
Thin Sections ◽  
Blue Green Algae ◽  
Cacodylate Buffer

The major protein of the blue-green algae is the biliprotein, C-phycocyanin (Amax = 620 nm), which is presumed to exist in the cell in the form of distinct aggregates called phycobilisomes. The self-assembly of C-phycocyanin from monomer to hexamer has been extensively studied, but the proposed next step in the assembly of a phycobilisome, the formation of 19s subunits, is completely unknown. We have used electron microscopy and analytical ultracentrifugation in combination with a method for rapid and gentle extraction of phycocyanin to study its subunit structure and assembly.To establish the existence of phycobilisomes, cells of P. boryanum in the log phase of growth, growing at a light intensity of 200 foot candles, were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer, pH 7.0, for 3 hours at 4°C. The cells were post-fixed in 1% OsO4 in the same buffer overnight. Material was stained for 1 hour in uranyl acetate (1%), dehydrated and embedded in araldite and examined in thin sections.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

gamma-Tubulin in mammalian cells: the centrosomal and the cytosolic forms

1996 ◽  
Vol 109 (4) ◽  
pp. 875-887 ◽  
Author(s):  
M. Moudjou ◽  
N. Bordes ◽  
M. Paintrand ◽  
M. Bornens
Keyword(s):  
Mammalian Cells ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Cellular Distribution ◽  
Cell Fractionation ◽  
Two Dimensional Gel Electrophoresis ◽  
Gamma Tubulin

The centrosome is one of the cellular organelles for which the mechanism by which it operates still remains to be unlavelled. The finding of the association with the centrosome of gamma-tubulin, a protein which belongs to the tubulin superfamily, has provided a long sought after biochemical tool with which to address centrosome function. We have generated a specific anti-gamma-tubulin polyclonal antibody to study the biochemical properties and the cellular distribution of the human lymphoblastic gamma-tubulin. Using cell fractionation and mass isolation of centrosomes, we observed that in contrast to the figures suggested by immunofluorescence, a minimum figure of 80% of total gamma-tubulin exists as a cytosolic form. The centrosomal form, for which at least half is not strongly associated with the centrosome, behaves in two-dimensional gel electrophoresis identically to the soluble form (as at least two spots of a pI of around 6). Post-embedding immunolocalization reveals that gamma-tubulin is distributed in the pericentriolar matrix but is also closely associated with centrioles. Using a combination of gel filtration, ion exchange chromatography, equilibrium sucrose gradient centrifugation and immunoprecipitation, we show that the major part of cytosolic gamma-tubulin might be involved in complexes heavier than the Tcp1 particle. We further demonstrate, by co-immunoprecipitation of gamma-tubulin and Tcp1 with either anti-Tcp1 or anti-gamma-tubulin antibodies, that a small part of gamma-tubulin participates in Tcp1-gamma-tubulin particles. Interestingly, the soluble form of gamma-tubulin co-purifies with taxol-stabilized microtubules and its association with microtubules resisted salt, ATP and GTP treatments. The existence of a centrosomal form and a large pool of cytosolic gamma-tubulin-containing complexes in somatic cells suggests that the overall gamma-tubulin cellular distribution does not seem to be as straightforward as it was drawn earlier.

scholarly journals Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni)

Toxins ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 54 ◽  
Author(s):  
Supeecha Kumkate ◽  
Lawan Chanhome ◽  
Tipparat Thiangtrongjit ◽  
Jureeporn Noiphrom ◽  
Panithi Laoungboa ◽  
...  
Keyword(s):  
Snake Venom ◽  
Protein Composition ◽  
Treatment Method ◽  
Cross Reactivity ◽  
Major Protein ◽  
Dependent Manner ◽  
Crude Venom ◽  
Monocytic Cells ◽  
Protein Components ◽  
Species Specific

The two venomous pit vipers, Trimeresurus macrops and T. hageni, are distributed throughout Thailand, although their abundance varies among different areas. No species-specific antivenom is available for their bite victims, and the only recorded treatment method is a horse antivenom raised against T. albolabris crude venom. To facilitate assessment of the cross-reactivity of heterologous antivenoms, protein profiles of T. macrops and T. hageni venoms were explored using mass-spectrometry-based proteomics. The results show that 185 and 216 proteins were identified from T. macrops and T. hageni venoms, respectively. Two major protein components in T. macrops and T. hageni venoms were snake venom serine protease and metalloproteinase. The toxicity of the venoms on human monocytes and skin fibroblasts was analyzed, and both showed a greater cytotoxic effect on fibroblasts than monocytic cells, with toxicity occurring in a dose-dependent rather than a time-dependent manner. Exploring the protein composition of snake venom leads to a better understanding of the envenoming of prey. Moreover, knowledge of pit viper venomics facilitates the selection of the optimum heterologous antivenoms for treating bite victims.

scholarly journals NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis: Isolation, characterization and biochemical properties of the enzyme

1997 ◽  
Vol 326 (3) ◽  
pp. 683-692 ◽  
Author(s):  
Wilfried NEUHAUSER ◽  
Dietmar HALTRICH ◽  
Klaus D. KULBE ◽  
Bernd NIDETZKY
Keyword(s):  
Aldose Reductase ◽  
Binding Constants ◽  
Catalytic Efficiency ◽  
Anion Exchange Chromatography ◽  
Competitive Inhibitor ◽  
Biochemical Properties ◽  
Product Inhibition ◽  
Exchange Chromatography ◽  
Binding Studies ◽  
Coenzyme Binding

During growth on D-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists of a single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in D-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 μM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuisis not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5′-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.

scholarly journals Partial Chemical Characterization of S-Carboxymethylated Chains of Rabbit Fibrin(OGEN): Evidence for Separate Chain Biosynthesis

1977 ◽  
Author(s):  
B. Alving ◽  
G. Murano ◽  
D. Walz
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Chemical Characterization ◽  
Molecular Size ◽  
Exchange Chromatography ◽  
12 Steps ◽  
Polypeptide Chains ◽  
Sds Electrophoresis ◽  
Derivatives Of

The purpose of this study was twofold: 1) chemically characterize the isolated polypeptide chains of rabbit fibrin(ogen), and 2) explore their mode of biosynthesis. The three S-carboxy-methyl polypeptide chain derivatives of rabbit fibrin (α, β and γ) were isolated by cation exchange chromatography. Their amino acid composition was similar to the human with a methionine distribution (mole/mole) as follows: γ = 9; β = 14, α = 14. Their molecular size, (SDS electrophoresis) was estimated as follows: γ = 46,000; β = 54,000; α = 63,500. The N-terminal amino acid sequence (12 steps) of the β derivative was:Gly-His-Arg-Pro-Ile-Asp-Arg-Arg-Arg-Glu-Glu-Leu-. To determine whether the three chains are synthesized sequentially (one continuous chain, later split into three) or in parallel, turpentine-stimulated male New Zealand rabbits were given ~40 μCi of [75Se] selenomethionine (SeM) and its incorporation into fibrinogen (F) was followed. F was clotted from plasma samples, washed, reduced, and constituent chains separated by gel electrophoresis in the presence of SDS-urea. The radioactivity of each chain (expressed as percent of total F radioactivity) was determined, and the specific methionine radioactivity calculated for each chain isolated at 20, 25, and 30 min after SeM injection. During this interval the specific activity of the α and the γ chains was essentially the same (within 3%) while that of the β chain was 42 to 97% greater than that of the α chain. The similar activity of the α and γ chains during the early phase of SeM incorporation suggests that these two chains are not synthesized sequentially, rather they are synthesized in parallel.

scholarly journals The 47-kD fragment of talin is a substrate for protein kinase P

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3343-3349 ◽  
Author(s):  
PC Simons ◽  
L Elias
Keyword(s):  
Protein Kinase ◽  
Ion Exchange ◽  
Sequence Data ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Myelogenous Leukemia ◽  
Blue Staining ◽  
Major Protein

Abstract This laboratory has been characterizing protein serine/threonine kinase reactions of hematopoietic tissues, whose most distinguishing characteristics in vitro are stimulation with vesicular phosphatidyl glycerol, and the ability to function using Mn2+ as the sole divalent cation. The major protein substrates are a 73-kD protein and a protein migrating near ovalbumin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 47-kD protein was partially purified from cells harvested by leukapheresis from a patient with acute myelogenous leukemia, using ammonium sulfate precipitation and ion exchange chromatography. This partially purified ion-exchange fraction contained an endogenous kinase activity with characteristics similar to those we previously described of protein kinase P (protein kinase, phospholipid- stimulable: PK-P), but not typical of any form of protein kinase C (PK- C). With longer phosphorylation, the 47-kD band showed increasingly lower mobility demonstrable both by Coomassie blue staining and autoradiography, suggesting both that it was multiply phosphorylated, and that the excisable band was pure. The protein was thus eluted from preparative gel slices and digested with endoproteinase lys C. Sequence data from the fragments identified the protein as the 47-kD calpain fragment of talin, a protein found in focal adhesion plaques and some cell-cell contacts. PK-C phosphorylated the 47-kD protein, as has been reported previously, and phosphopeptide mapping disclosed a similar pattern of phosphorylation using either PK-C or the endogenous activity. The 47-kD protein labeled with the endogenous kinase contained predominantly phosphoserine, with some phosphothreonine and a trace of phosphotyrosine. Intact, purified talin was also phosphorylated by PK-P in a phospholipid-stimulable manner, but at 1/20 the rate of the 47-kD fragment.

Sign in / Sign up

Export Citation Format

Share Document