scholarly journals The non-phospholipase A2 subunit of β-bungarotoxin plays an important role in the phospholipase A2-independent neurotoxic effect: characterization of three isotoxins with a common phospholipase A2 subunit

1994 ◽  
Vol 303 (1) ◽  
pp. 171-176 ◽  
Author(s):  
C C Chu ◽  
S T Chu ◽  
S W Chen ◽  
Y H Chen
Keyword(s):  
Phospholipase A2 ◽  
Terminal Sequence ◽  
Protein Fluorescence ◽  
Linear Gradient ◽  
Sds Page ◽  
Neurotoxic Effects ◽  
Beta 2 ◽  
Chick Biventer Cervicis Muscle ◽  
Derivatives Of

Three isotoxins (SP I-III) of the beta-bungarotoxin family were purified to homogeneity via a series of isolation procedures including a final step of h.p.l.c. on an SP column washed with a linear gradient of 0.2-0.6 M sodium acetate at pH 7.4. Their proportions varied greatly with the batch of venom. Each isotoxin was demonstrated by SDS/PAGE to contain a phospholipase A2 subunit and a non-phospholipase A2 subunit. The three proteins were reductively alkylated with 4-vinylpyridine and the alkylated derivatives of the two subunits of each isotoxin were separated. N-Terminal sequence analysis of the alkylated derivatives revealed that the three isotoxins probably share a common phospholipase A2 subunit but differ in their non-phospholipase A2 subunits. The non-phospholipase A2 subunits of SP II and SP III were identical with those of beta 2- and beta 1-toxin respectively, except that there was an additional valine inserted between Thr-18 and Val-19 in beta 2-toxin and Pro-18 and Val-19 in beta 1-toxin. The non-phospholipase A2 subunit of SP I differed greatly from that of SP III but was almost identical with that of SP II, except that Lys-14 and Ala-29 in SP II were replaced by Arg-14 and Glu-29 in SP I. Analysis of the effect of CaCl2 on protein fluorescence showed the existence of a low- and a high-affinity site on the different domains of each isotoxin for Ca2+ binding. The three isotoxins showed no great difference in their ability to bind Ca2+ on both the high- and low-affinity site. They had slightly different phospholipase A2 activities but differed to a great extent with respect to their neurotoxic effects. LD50 values increased in the order SP I > SP II > SP III. In contrast, the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle was in the order SP III > SP II > SP I.

Purification and Characterization of Cytochrome c6 from the Unicellular Green Alga Scenedesmus obliquus

1997 ◽  
Vol 52 (11-12) ◽  
pp. 740-746 ◽  
Author(s):  
Röbbe Wünschiers ◽  
Thomas Zinn ◽  
Dietmar Linder ◽  
Rüdiger Schulz
Keyword(s):  
Cytochrome C ◽  
Green Alga ◽  
Scenedesmus Obliquus ◽  
Terminal Sequence ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Unicellular Green Alga ◽  
Sds Page ◽  
Monoraphidium Braunii

Abstract Purification of a soluble cytochrome c6 from the unicellular green alga Scenedesmus obliquus by a simple and rapid method is described. The purification procedure includes ammonium sulfate precipitation and non-denaturating PAGE. The N-terminal sequence of the first 20 amino acids was determined and shows 85% similarity and 75% identity to the sequence of cytochrome c6 from the green alga Monoraphidium braunii. The ferrocyto-chrome shows typical UV/VIS absorption peaks at 552.9, 521.9 and 415.7 nm. The apparent molecular mass was estimated to be 12 kD a by SDS-PAGE. EPR-spectroscopy at 20K shows resonances indicative for two distinct low-spin heme forms.

scholarly journals Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies1

1998 ◽  
Vol 331 (2) ◽  
pp. 513-519 ◽  
Author(s):  
Alberto VITALI ◽  
Bruno BOTTA ◽  
Giuliano DELLE MONACHE ◽  
Sabrina ZAPPITELLI ◽  
Paola RICCIARDI ◽  
...  
Keyword(s):  
Cell Wall ◽  
Culture Medium ◽  
Gel Filtration ◽  
Cell Suspension Cultures ◽  
High Specificity ◽  
Coniferyl Alcohol ◽  
Terminal Sequence ◽  
Sds Page ◽  
Plant Peroxidases

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya(wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3´,4´-trihydroxychalcone and 4,3´,4´-trihydroxy-3-methoxychalcone to the corresponding 3,3´-biflavanones, as mixtures of racemic and mesoforms.

scholarly journals Human synovial cells secrete a 39 kDa protein similar to a bovine mammary protein expressed during the non-lactating period

1990 ◽  
Vol 269 (1) ◽  
pp. 265-268 ◽  
Author(s):  
P Nyirkos ◽  
E E Golds
Keyword(s):  
Synovial Cell ◽  
Lung Fibroblasts ◽  
Synovial Cells ◽  
Terminal Sequence ◽  
Reverse Phase ◽  
Cell Monolayers ◽  
Human Synovial Cell ◽  
Sds Page

By SDS/PAGE analysis we have observed that human synovial cell monolayers secrete a prominent 39 kDa protein which could not be detected in skin and lung fibroblasts. This protein was purified to homogeneity by heparin-Sepharose chromatography and reverse-phase h.p.l.c. The N-terminal sequence was found to be almost identical to that of a recently described bovine protein detected in the mammary secretions during the involutionary phase of the lactational cycle. Characterization of this 39 kDa protein may provide a useful marker for classification of connective tissue cells.

scholarly journals Characterization of the 70 kDa polypeptide of the Na/Ca exchanger

1999 ◽  
Vol 338 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Rami I. SABA ◽  
Alex BOLLEN ◽  
André HERCHUELZ
Keyword(s):  
Wild Type ◽  
Terminal Portion ◽  
Terminal Sequence ◽  
Protein Ratio ◽  
Reducing Conditions ◽  
Sds Page ◽  
Cloned Bovine ◽  
Sarcolemmal Vesicles ◽  
Exchange Activity

The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1–21; NCX1, residues 393–406; and Exon F, residues 622–644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same N-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35% from its C-terminal portion as compared with that of the wild-type exchanger.

CHARACTERIZATION OF RECOMBINANT HUMAN SINGLE-CHAIN LOW MOLECULAR WEIGHT UROKINASE (RE-SC-LUK)

1987 ◽  
Author(s):  
W A Günzler ◽  
B Wolf ◽  
L Flohé
Keyword(s):  
Fibrinolytic Activity ◽  
Agar Plate ◽  
Single Chain ◽  
Amidolytic Activity ◽  
Terminal Sequence ◽  
Kringle Domains ◽  
Structural Identity ◽  
Sds Page ◽  
A Chain

RE-SC-LUK obtained from recomoinant b. con Bacteria showed a molecular mass similar to that of recombinant two-chain LUK (RE-TC-LUK) as judged from SDS-PAGE. By “Western“ blot analysis immunoreactivity of RE-SC-LUK was observed with monoclonal antibodies directed against the B chain but not with those against the A1 chain of urokinase. N-terminal sequence analysis c RE-SC-LUK showed identity to the A, chain of RE-TC_LUK and provided evidence for its single-chain nature, i.e. integrity of the Lys-Ile bond which is split in TC-UK. In all other respects structural identity of RE-SC-LUK and RE-TC-LUK was demonstrated by fingerprinting of fragments. Similar to recombinant pro-urokinase (RE-SCU-PA), RE-SC-LUK exhibits only marginal amidolytic activity, which is greatly enhanced by treatment with plasmin, but considerable fibrinolytic activity in a fibrin agar plate test.Thus, RE-SC-LUK is characterized as a fragment (residues 136 -411) of RE-SCU-PA, which lacks the “growth factor” and “kringle” domains. Moreover further evidence is provided that a free N-terminus of the B chain is essential for amidolytic but not for fibrinolytic activity of urokinase in more complex systems.

scholarly journals Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus)

1995 ◽  
Vol 311 (3) ◽  
pp. 895-900 ◽  
Author(s):  
I H Tsai ◽  
P J Lu ◽  
Y M Wang ◽  
C L Ho ◽  
L L Liaw
Keyword(s):  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Cdna Sequencing ◽  
Crude Venom ◽  
Phospholipases A2 ◽  
Sequencing Method ◽  
Sds Page ◽  
Filtration Experiment ◽  
Chick Biventer Cervicis Muscle

Using gel-filtration chromatography and reverse-phase (RP) HPLC we have purified a presynaptic neurotoxin (designated as trimucrotoxin) from the crude venom of Taiwan habu (Trimeresurus mucrosquamatus). Its complete primary structure was solved by an automated N-terminal sequencing and cDNA sequencing method. The enzyme inhibited the twitch of the chick biventer cervicis muscle at 0.1-1 micrograms/ml and showed lethality in mice (LD50 = 1.2 micrograms/g, when given intravenously). Trimucrotoxin exists mainly as a homodimer of 14 kDa subunits as shown by a gel-filtration experiment, and dissociates into monomers during SDS/PAGE in the absence of Ca2+. However, most of trimucrotoxin migrated as slowly as a trimer during nondenaturing SDS/PAGE in the presence of Ca2+ or Sr2+. Its amino acid sequence identity to crotoxin B and agkistrodotoxin is about 75%, and its cDNA sequence is 82% identical to that of crotoxin B. Rabbit antiserum against trimucrotoxin also cross-reacted with the other crotalid neurotoxic phospholipases A2. Furthermore, the purified acidic subunit of crotoxin potentiated the neurotoxicity of trimucrotoxin. A comparison of the sequences of these crotalid neurotoxins revealed some common features of the possible neurotoxic sites, including residues 6, 11, 76-81 and 119-125.

scholarly journals Purification and characterization of cytosolic and microsomal cyclophilins from maize (Zea mays)

1996 ◽  
Vol 315 (3) ◽  
pp. 965-970 ◽  
Author(s):  
Philip S. SHELDON ◽  
Michael A. VENIS
Keyword(s):  
Cyclosporin A ◽  
Sequence Similarity ◽  
Terminal Sequence ◽  
Purification And Characterization ◽  
Cyclophilin B ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses ◽  
High Level

Methods for the purification and separation of peptidyl prolyl cis–trans isomerase (PPI) from cytosolic and microsomal fractions of etiolated maize are described. On SDS/PAGE, the purified preparations appear as single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa respectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabling purification of the proteins on a much larger scale than previously described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant species on an immunoblot. There is virtually no recognition of the microsomal PPI. The cytosolic and microsomal PPIs are inhibited by cyclosporin A (Ki = 6 nM in both cases), indicating that they are cyclophilins. The cytosolic enzyme is inactivated by 5 mM N-ethylmaleimide and 2 mM phenylglyoxal. N-terminal sequencing of the microsomal PPI indicates a high level of sequence similarity with the N-terminal sequence of mature animal s-cyclophilin (cyclophilin B).

CHARACTERIZATION OF SYNOVIAL AND PLASMA FIBRIN DEGRADATION PRODUCTS IN RHEUMATOID ARTHRITIS

1987 ◽  
Author(s):  
A N Whitaker ◽  
P Masci ◽  
R A Hazelton ◽  
J J Morrison
Keyword(s):  
Mixed Lymphocyte Reaction ◽  
Degradation Products ◽  
Plasma Concentrations ◽  
Joint Damage ◽  
Rheumatoid Disease ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Derivatives Of ◽  
Study Species

The presence of fibrinogen/fibrin derivatives in vasculitic and synovial lesions of rheumatoid disease has long been recognized, although a pathogenetic role for them has not been clearly defined. In this study species of fibrin (ogen) derivatives have been characterized from joint aspirates from 32 patients (40 samples) with rheumatoid disease, and from 12 cases of seronegative arthridities, using immunoadsorption and SDS PAGE (reduced and unreduced gels); and quantitated by immunoassays utilizing the D dimer specific monoclonal antibody DD-3B6/22. The dominant fibrinogen derivatives in synovial aspirates were identifiable as derivatives of the degradation of crosslinked (XL) fibrin and included large quantities of XL high molecular weight degradation products. By enzymeimmunoassays (EIA) levels of XL fibrin derivatives ranged from 12 - 194µg/ml. Elevated levels of XLFDP (0.5 - 11.4 µg/ml) were also found in the majority of plasmas studied (29/38). A significant correlation was demonstrated between synovial and plasma concentrations of XLFDP (r = 0.455, p <0.005). A significant correlation also existed between erythrocyte sedimentation rate and plasma XLFDP (r = 0.554, p< 0.001) but not between ESR and synovial XLFDP. Autologous mixed lymphocyte reaction, measured in 26 patients, did not correlate with XLFDP levels. The synovial fluid data are consistent with the sequential alteration of fibrinogen by thrombin, XIIIa and plasmin (and/or elastase). Although the activation of fibrinolysis in the joint may be protective, the capacity for XLFDP to mediate bone and joint damage or to initiate immune responses is as yet unknown, whether in inflammatory arthritis or in haeraophilic arthropathy. The plasma XLFDP level may provide an index of disease activity in rheumatoid disease, although the extent to which this measures transfer from synovial accumulations to plasma remains to be determined.

scholarly journals Isolation and partial characterization of a D-galactose-binding lectin from the latex of Synadenium carinatum

2005 ◽  
Vol 48 (5) ◽  
pp. 705-716 ◽  
Author(s):  
Maria Aparecida Souza ◽  
Francielle Amâncio-Pereira ◽  
Cristina Ribeiro Barros Cardoso ◽  
Adriano Gomes da Silva ◽  
Edmar Gomes Silva ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Apparent Molecular Weight ◽  
Size Exclusion ◽  
Terminal Sequence ◽  
Native Page ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Polypeptide Chains ◽  
Binding Lectin

A lectin from the latex of Synadenium carinatum was purified by affinity chromatography on immobilized-D-galactose-agarose and shown to be a potent agglutinin of human erythrocytes. The haemagglutination of human red cells was inhibited by 3.0 mM N-acetyl-D-galactopyranoside, 6.3 mM methyl-beta-D-galactopyranoside, 50 mM methyl-alpha-D-galactopyranoside and 50 mM D-fucose but not by L-fucose, demonstrating an anomeric and a conformational specificity. According to SDS-PAGE analysis, the lectin appeared to be a glycoprotein composed of two polypeptide chains of ca. 28 and 30 kDa, but size exclusion chromatography (Sephadex G-100) and native PAGE revealed a protein of apparent molecular weight 120 - 130 kDa made up of 28 and 30 kDa subunits. The lectin was stable in the range pH 6 - 9, and 4 - 56ºC. The N-terminal sequence of the 30 kDa subunit contained the conserved consensus sequence GPN observed in other D-galactose-binding lectins found in latex of members of the Euphorbiaceae.

scholarly journals Purification and partial characterization of rat factor D

1991 ◽  
Vol 279 (3) ◽  
pp. 775-779 ◽  
Author(s):  
B C Baker ◽  
C J Campbell ◽  
C J Grinham ◽  
G Turcatti
Keyword(s):  
Wheat Germ ◽  
Human Factor ◽  
Sequence Similarity ◽  
Terminal Sequence ◽  
Sds Page ◽  
Fast Flow ◽  
Sepharose 4B ◽  
Wheat Germ Lectin ◽  
Human And Mouse

Rat factor D has been purified to homogeneity (10,559-fold) from serum by chromatography on CM-Sepharose Fast Flow, phenyl-Sepharose CL-4B and Mono S and has been shown to resemble its human and mouse counterparts both in substrate specificity and in its susceptibility to inhibition by the organophosphorous inhibitor di-isopropylfluorophosphate. The rat enzyme, however, is heavily glycosylated and binds to wheat-germ lectin-Sepharose 6MB and 5-hydroxytryptamine-agarose, but not to concanavalin A-Sepharose 4B. All of the carbohydrate chains are N-linked. Enzymic removal of this carbohydrate decreased the Mr by approx. 15,000. The deglycosylated rat enzyme had the same mobility as native human factor D on SDS/PAGE, corresponding to an Mr of 24,500. N-Terminal sequence analysis of the first 30 amino acids of rat factor D highlighted the sequence similarity with human factor D (greater than 76%) and, in particular, with mouse adipsin (greater than 93%).

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