scholarly journals SYNTHESIS OF GLYCOPROTEINS IN A SINGLE IDENTIFIED NEURON OF APLYSIA CALIFORNICA

1974 ◽  
Vol 61 (3) ◽  
pp. 649-664 ◽  
Author(s):  
Richard T. Ambron ◽  
James E. Goldman ◽  
Elizabeth Barnes Thompson ◽  
James H. Schwartz
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Mild Acid Hydrolysis ◽  
Giant Neuron ◽  
Electrophoretic Patterns ◽  
Identified Neuron ◽  
Hydrolysis Of ◽  
Mild Acid

Incorporation of L-[3H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [3H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [3H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [3H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [3H]fucose, gel profiles of particulate fractions labeled with [14C]valine were much more complex.

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 ).

scholarly journals Studies of the polysaccharide fraction from the lipopolysaccharide of Pseudomonas alcaligenes

1974 ◽  
Vol 139 (3) ◽  
pp. 633-643 ◽  
Author(s):  
James A. Lomax ◽  
George W. Gray ◽  
Stephen G. Wilkinson
Keyword(s):  
Gel Filtration ◽  
Periodate Oxidation ◽  
Side Chain ◽  
Partial Hydrolysis ◽  
Methylation Analysis ◽  
Mild Acid Hydrolysis ◽  
Pseudomonas Alcaligenes ◽  
Crude Polysaccharide ◽  
Hydrolysis Of ◽  
Mild Acid

Studies of the lipopolysaccharide of Pseudomonas alcaligenes strain BR 1/2 were extended to the polysaccharide moiety. The crude polysaccharide, obtained by mild acid hydrolysis of the lipopolysaccharide, was fractionated by gel filtration. The major fraction was the phosphorylated polysaccharide, for which the approximate proportions of residues were; glucose (2), rhamnose (0.7), heptose (2–3), galactosamine (1), alanine (1), 3-deoxy-2-octulonic acid (1), phosphorus (5–6). The heptose was l-glycero-d-manno-heptose. The minor fractions from gel filtration contained free 3-deoxy-2-octulonic acid, Pi and PPi. The purified polysaccharide was studied by periodate oxidation, methylation analysis, partial hydrolysis, and dephosphorylation. All the rhamnose and part of the glucose and heptose occur as non-reducing terminal residues. Other glucose residues are 3-substituted, and most heptose residues are esterified with condensed phosphate residues, possibly in the C-4 position. Free heptose and a heptosylglucose were isolated from a partial hydrolysate of the polysaccharide. The location of galactosamine in the polysaccharide was not established, but either the C-3 or C-4 position appears to be substituted and a linkage to alanine was indicated. In its composition, the polysaccharide from Ps. alcaligenes resembles core polysaccharides from other pseudomonads: no possible side-chain polysaccharide was detected.

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

scholarly journals Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes

1975 ◽  
Vol 151 (3) ◽  
pp. 685-697 ◽  
Author(s):  
M Letarte-Muirhead ◽  
A N Barclay ◽  
A F Williams
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Lentil Lectin ◽  
Polypeptide Chains

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.

Binding specificity and reactivity studies on a broad-specificity β-glycosidase from porcine kidney

1988 ◽  
Vol 66 (8) ◽  
pp. 830-838 ◽  
Author(s):  
R. E. Huber ◽  
R. L. Brockbank
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Porcine Kidney ◽  
Hydrolysis Of ◽  
Broad Specificity ◽  
Native Molecular Weight

A broad-specificity β-glycosidase from porcine kidney was purified to homogeneity. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis showed that it had a monomeric molecular weight of 55 000–60 000. Gel filtration showed a native molecular weight of about 115 000. These data imply that the native enzyme is a dimer. The enzyme can catalyze the hydrolysis of β bonds between glycosides and 4-methylumbelliferone or nitrophenol yielding D-fucopyranose, D-galactopyranose, D-glucopyranose, D-xylopyranose, and D-mannopyranose and of α bonds to yield L-arabinopyranose. This is the first study that shows a mammalian broad-specificity cytosolic β-glycosidase carrying out a reaction with a β-D-mannopyranoside. The nature of the broad specificity was studied with inhibitors. Similar inhibitor constants were found regardless of whether the substrate was a β-D-glucopyranoside or a β-D-galactopyranoside, so the enzyme probably has only one binding site with a broad specificity. The enzyme prefers to bind compounds with an axial hydroxyl at the 2 position and an equatorial hydroxyl at the 4 position; the 3 position does not affect binding significantly. The hydroxyl at the 6 position affects binding, but binding at that position depends on the configurations at the 2 and 4 positions. Thus, there must be some interactions between these three positions (2, 4, and 6). Lactones are also good inhibitors and this may relate to strain effects.

Rat intestinal angiotensin-converting enzyme: purification, properties, expression, and function

1992 ◽  
Vol 263 (4) ◽  
pp. G466-G473 ◽  
Author(s):  
R. H. Erickson ◽  
Y. Suzuki ◽  
A. Sedlmayer ◽  
I. S. Song ◽  
Y. S. Kim
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Maximal Velocity ◽  
Converting Enzyme ◽  
And Function

Angiotensin-converting enzyme [ACE (peptidyl-dipeptidase A, EC 3.4.15.1)] was purified from a total cell membrane fraction of rat intestinal mucosa. A 4,500-fold purification was achieved after affinity chromatography with lisinopril-Sepharose and gel filtration. The final preparation was judged to be homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 160,000. The purified protein is a glycoenzyme containing 12% N-linked carbohydrate. Purified ACE had a specific activity of 65 U/mg protein with benzoyl-Gly-His-Leu as substrate. A kinetic analysis showed that the enzyme had the maximal velocity with substrates containing proline at the COOH-terminal end. Inhibitor studies indicated that the enzyme is a metalloprotein. Along the proximal-distal axis of the small intestine, ACE activity is most predominant in the proximal to middle portions, decreasing toward the distal end. This pattern was also observed for ACE mRNA and protein, suggesting that ACE expression is controlled at the level of mRNA. Perfusion of benzoyl-Gly-His-Leu in vivo through a segment of intestinal jejunum demonstrated that ACE is an important intestinal dipeptidyl carboxypeptidase, participating in the digestion and assimilation of dietary peptides.

Identification of high-affinity calmodulin-binding proteins in rat liver

1987 ◽  
Vol 252 (3) ◽  
pp. C277-C284 ◽  
Author(s):  
R. M. Hanley ◽  
J. R. Dedman ◽  
S. Shenolikar
Keyword(s):  
Rat Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Apparent Molecular Weight ◽  
Convincing Evidence ◽  
Calmodulin Binding ◽  
High Affinity

The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or “acceptor” proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding “subunit” of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

scholarly journals A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification

2021 ◽  
Vol 22 (2) ◽  
pp. 701
Author(s):  
Tomasz K Gozdziewicz ◽  
Anna Maciejewska ◽  
Alona Tsybulska ◽  
Czeslaw Lugowski ◽  
Jolanta Lukasiewicz
Keyword(s):  
Current Knowledge ◽  
Membrane Integrity ◽  
Gel Filtration ◽  
Common Antigen ◽  
Mild Acid Hydrolysis ◽  
Enterobacterial Common Antigen ◽  
Lipopolysaccharide Endotoxin ◽  
Lipid Moiety ◽  
Detailed Composition ◽  
Mild Acid

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide − endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

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