scholarly journals MURINE AMYLOID FIBRIL PROTEIN: ISOLATION, PURIFICATION AND CHARACTERIZATION

1971 ◽  
Vol 19 (1) ◽  
pp. 16-28 ◽  
Author(s):  
G. G. GLENNER ◽  
D. PAGE ◽  
C. ISERSKY ◽  
M. HARADA ◽  
P. CUATRECASAS ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Amyloid Fibril ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Structure Identification ◽  
Major Protein ◽  
Amyloid Protein ◽  
Molecular Weights ◽  
Sepharose 4B

Murine amyloid has been produced by four different induction methods ( Mycobacterium butyricum, casein, casein plus Freund's adjuvant and endotoxin-induced mouse amyloidosis) in several strains and obtained from mice with "spontaneous" amyloidosis. The amyloid fibrils have been concentrated from spleen and liver. Electron microscopy of all of these preparations reveals the amyloid fibril to be 100 Å in width and composed of two parallel filaments, each measuring 35-40 Å in width and having the appearance of a twisted ribbon. X-ray diffraction of all preparations reveals a "backbone" spacing at 4.75 Å and a "side chain" spacing at 11 Å indicating a "β-pleated sheet" structure. Identification of the major protein component of amyloid fibril concentrates was made by combined use of sodium dodecyl sulfate polyacrylamide disc electrophoresis, labeling of the protein with 3H-tryptophan and Sepharose 4B gel filtration. Purification of the amyloid protein from spontaneous amyloidosis liver was accomplished by sequential gel filtration with 5 M guanidine in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns. The material is a unique glycoprotein with a molecular weight of 7200, a high content of dicarboxylic and short chain amino acids, a significant amount of tryptophan and an unreactive NH2-terminal amino acid, tentatively identified as pyrrolid-2-one-5-carboxylic acid. There are no methionine, half-cystine, hydroxylysine or hydroxyproline residues. Murine amyloid protein, therefore, has striking similarities to many human amyloid protein preparations. It differs from the human proteins in the similarity of molecular weights of different preparations.

scholarly journals HUMAN AMYLOID PROTEIN: CHEMICAL VARIABILITY AND HOMOGENEITY

1971 ◽  
Vol 19 (1) ◽  
pp. 1-15 ◽  
Author(s):  
M. HARADA ◽  
C. ISERSKY ◽  
P. CUATRECASAS ◽  
D. PAGE ◽  
H. A. BLADEN ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amyloid Fibril ◽  
Gel Filtration ◽  
Protein S ◽  
Amyloid Protein ◽  
X Ray Diffraction ◽  
Amyloid Proteins ◽  
X Ray ◽  
Chemical Variability ◽  
Sepharose 4B

The morphology of the fibril of amyloid derived from different individuals is similar, but occasionally significant differences are noted. All human amyloid filaments have a "β-pleated sheet" conformation as revealed by x-ray diffraction, and those examined after orientation show a "cross-β" pattern. All amyloid fibril concentrates studied so far can be fractionated to obtain the major amyloid protein component(s) by sequential gel filtration with 5 M guanidine-HCl in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns with the removal of over 28% of proteins representing minor constituents. The major amyloid protein(s) obtained from the spleen and/or liver of six patients is found to contain tryptophan, to be deficient in hydroxylysine and hydroxyproline and usually at least one commonly occurring amino acid and to have a high content of dicarboxylic acid and short chain amino acids and unreactive (blocked) NH2-terminal groups or aspartic acid-asparagine (Asx). However, the amyloid protein(s) from each individual differs from that of the others in molecular weight, in amino acid composition and in the presence or absence of specific tryptic peptides. Amyloid protein(s) from the liver and spleen of the same individual is identical. No chemical characteristics distinguish amyloid proteins derived from cases classified clinically as "primary" from those classified as "secondary." There is a striking chemical similarity between amyloid proteins and the NH2-terminal variable fragment of the light and heavy chain of immumoglobulin proteins.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals PHYSICAL AND CHEMICAL PROPERTIES OF AMYLOID FIBERS II. ISOLATION OF A UNIQUE PROTEIN CONSTITUTING THE MAJOR COMPONENT FROM HUMAN SPLENIC AMYLOID FIBRIL CONCENTRATES

1969 ◽  
Vol 17 (12) ◽  
pp. 769-780 ◽  
Author(s):  
G. G. GLENNER ◽  
P. CUATRECASAS ◽  
C. ISERSKY ◽  
H. A. BLADEN ◽  
E. D. EANES
Keyword(s):  
Column Chromatography ◽  
Amyloid Fibril ◽  
Sodium Dodecyl ◽  
Chemical Properties ◽  
Disc Electrophoresis ◽  
Protein Component ◽  
Major Protein ◽  
Human Spleen ◽  
Amyloid Fibers ◽  
Physical And Chemical

A major protein component (B protein) obtained from concentrated fibrils of each of two human amyloidotic spleens has been isolated by a series of fractionation methods using extraction in buffered 8 M urea, complete denaturation in buffered 6 M guanidine and column chromatography on Sepharose 4 B (Pharmacia). The major protein obtained by column chromatography was found to be also the major protein component on sodium dodecyl sulfate polyacrylamide disc electrophoresis. This protein was shown to have a molecular weight of 21,000-22,500. Immunologic identity was demonstrated for the B protein derived from the amyloid fibril concentrates of each spleen. No evidence of antibody cross-reaction to native or denatured components of normal human spleen or serum was discernible. It would appear, therefore, that the B protein derived from the two splenic amyloid fibril preparations, in addition to being the major protein component of the fibril, is not found in significant quantity in normal tissue or serum.

Immunocytochemical Localization of Amyloid Protein AA in the “Dense Fibrillar Inclusions” of the Reticuloendothelical Cells

1977 ◽  
Vol 35 ◽  
pp. 438-439
Author(s):  
T. Shirahama ◽  
M. Skinner ◽  
A.S. Cohen
Keyword(s):  
Amyloid Fibril ◽  
Close Association ◽  
Gel Filtration ◽  
Fibril Formation ◽  
Amyloid Protein ◽  
Electron Microscopic ◽  
Amyloid Deposits ◽  
Amyloid Fibril Formation ◽  
Electron Microscopic Technique ◽  
Lysosomal System

A1thought the mechanisms of amyloidogenesis have not been entirely clarified, proteolysis of the parent proteins may be one of the important steps in the amyloid fibril formation. Recently, we reported that "dense fibrillar inclusions" (DFI), which had the characteristics of lysosomes and contained organized fibrillar profiles as well, were observed in the reticuloendothelial cells in close association with the foci of new amyloid deposits. We considered the findings as evidence for the involvement of lysosomal system in amyloid fibril formation (l). In the present study, we attempted to determine the identity of the contents of the DFI by the use of antisera against the amyloid protein (AA) and an immuno-electron microscopic technique.Amyloidosis was induced in CBA/J mice by daily injections of casein (l). AA was isolated from amyloid-laden spleens by gel filtration and antibody to it was produced in rabbits (2). For immunocytochemistry, the unlabeled antibody enzyme method (3) was employed.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

The outer membrane of Haemophilus influenzae type b: cell envelope associations of major proteins

1983 ◽  
Vol 29 (2) ◽  
pp. 280-287 ◽  
Author(s):  
J. W. Coulton ◽  
D. T. F. Wan
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Haemophilus Influenzae Type ◽  
Major Protein ◽  
Haemophilus Influenzae Type B ◽  
Type B ◽  
Molecular Weights ◽  
Triton X

Membrane proteins fom the cell envelope of Haemophilus influenzae type b ATCC 9795 were examined by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. When envelopes were extracted with a phosphate-based buffer containing 2% Triton X-100, a major protein of molecular weight 43 000 was detected in fractions containing cytoplasmic membrane proteins. The cell wall material which was Triton X-100 insoluble contained six major proteins of molecular weights 46 000, 40 000, 36 000, 30 000, 27 000, and 16 000. One of these proteins showed a shift in molecular weight from 27 000 to 36 000 when it was heated over a temperature range from 50 °C to 100 °C in buffer containing 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol. This alteration in mobility could be demonstrated either by the membrane-bound form of the protein or by a detergent-soluble form of the protein. Enriched preparations of the 36 000 molecular weight form were obtained by a series of purification steps. Extraction of the Triton X-100 insoluble material with buffer containing 2% Triton X-100, 5.0 mM EDTA yielded chiefly one major protein molecular weight 30 000 and many minor protein species. Pretreatment of the Triton X-100 insoluble fraction with lysozyme followed by extraction with buffer containing 2% Triton X-100, 5.0 mM EDTA released two proteins of molecular weights 16 000 and 27 000 and few minor proteins. By these operational manipulations, the proteins of molecular weights 16 000 and 27 000 may be considered as peptidoglycan-associated proteins.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

Isomerization of Peroxidizing Thiadiazolidine Herbicides Is Catalyzed by Glutathione S-Transferase

1996 ◽  
Vol 51 (5-6) ◽  
pp. 342-354 ◽  
Author(s):  
Beate Nicolaus ◽  
Yukiharu Sato ◽  
Ko Wakabayashi ◽  
Peter Böger
Keyword(s):  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Total Activity ◽  
Naphthalic Anhydride ◽  
Molecular Weights ◽  
Isomerase Activity ◽  
Ammonium Sulfate Precipitate ◽  
Catalyzed Reactions

Abstract Thiadiazolidine-converting activity (isomerase), detected in a 45-75% ammonium sulfate precipitate from corn seedlings extracts, was purified by chromatography on hydroxyapatite and by anion exchange on Mono Q Sepharose. Two fractions 1 and 2 with isomerase activity were separated on Mono Q by combination of a stepwise elution and continuous salt gradient; fraction 2 eluting at higher salt concentrations was found the most active. Total activity could be enhanced by treatment of seedlings with naphthalic anhydride. Both fractions containing isomerase activity were further purified by glutathione-(GSH) agarose affinity chromatography and characterized by their specificity for different thiadiazolidines. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration revealed that the isomerase of fraction 2 consists either of a homodimer or a heterodimer of two proteins with apparent molecular weights of 28 and 31 kDa, respectively. The protein pattern as well as the strict dependence of activity on thiol groups (GSH or dithiothreitol) suggested a glutathione Stransferase (GST) catalyzing the thiadiazolidine conversion. Further evidence was obtained by measuring reactions specific for GSTs in both purified fractions, namely the conjugating activity for l-chloro-2,4-dinitrobenzene (CDNB ). atrazine and metazachlor. While no atrazine turnover was found, metazachlor and CDNB conjugation occurred rapidly. Both fractions differed in their activities to several GST substrates with fraction 2 being more effective in metazachlor but less active in C DN B conjugation. Inhibitors specific for GST-catalyzed reactions also inhibited thiadiazolidine conversion confirming that isomerizing activity is attributed to a GST form. We conclude that GST isoforms with different affinities towards thiadiazolidines have been isolated. CDNB activity, molecular weight, the protein pattern on SDS-PAGE as well as the amino acid sequence of one of its polypeptides suggest that fraction 1, less active in thiadiazolidine isomerization, is identical to GST I. The second peptide of this fraction was resistant to Edman degradation probably due to N-terminal blockage. The properties of the high isomerase activity found in fraction 2 are in agreement with characteristics of a GST previously termed as isoform II.

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