scholarly journals The formation of lipid-linked sugars as intermediates in glycoprotein synthesis in rabbit mammary gland

1978 ◽  
Vol 170 (2) ◽  
pp. 273-283 ◽  
Author(s):  
Brian K. Speake ◽  
David A. White
Keyword(s):  
Ammonium Acetate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Total Radioactivity ◽  
Glycoprotein Synthesis ◽  
Molecular Weights ◽  
Oligosaccharide Moiety ◽  
Chloroform Methanol ◽  
Mild Acid

1. The incorporation of d-[1-14C]mannose, d-[2-3H]mannose and N-acetyl-d-[1-14C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-3H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-14C]mannose and N-acetyl-d-[1-14C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-14C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000–80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-14C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-14C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-14C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.

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 Purification and properties of nitrite reductase from Escherichia coli K12

1978 ◽  
Vol 175 (2) ◽  
pp. 483-493 ◽  
Author(s):  
K J Coleman ◽  
A Cornish-Bowden ◽  
J A Cole
Keyword(s):  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Specific Activity ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Coli Strain ◽  
Molecular Weights

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2′-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

scholarly journals The Archaeal Small Heat Shock Protein Hsp17.6 Protects Proteins from Oxidative Inactivation

2021 ◽  
Vol 22 (5) ◽  
pp. 2591
Author(s):  
Pengfei Ma ◽  
Jie Li ◽  
Lei Qi ◽  
Xiuzhu Dong
Keyword(s):  
Heat Shock ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Small Heat Shock Protein ◽  
Phase Cell ◽  
Molecular Weights ◽  
Oxidative Inactivation ◽  
Methionine Residues ◽  
Shock Proteins ◽  
And Function

Small heat shock proteins (sHsps) are widely distributed among various types of organisms and function in preventing the irreversible aggregation of thermal denaturing proteins. Here, we report that Hsp17.6 from Methanolobus psychrophilus exhibited protection of proteins from oxidation inactivation. The overexpression of Hsp17.6 in Escherichia coli markedly increased the stationary phase cell density and survivability in HClO and H2O2. Treatments with 0.2 mM HClO or 10 mM H2O2 reduced malate dehydrogenase (MDH) activity to 57% and 77%, whereas the addition of Hsp17.6 recovered the activity to 70–90% and 86–100%, respectively. A similar effect for superoxide dismutase oxidation was determined for Hsp17.6. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis assays determined that the Hsp17.6 addition decreased H2O2-caused disulfide-linking protein contents and HClO-induced degradation of MDH; meanwhile, Hsp17.6 protein appeared to be oxidized with increased molecular weights. Mass spectrometry identified oxygen atoms introduced into the larger Hsp17.6 molecules, mainly at the aspartate and methionine residues. Substitution of some aspartate residues reduced Hsp17.6 in alleviating H2O2- and HClO-caused MDH inactivation and in enhancing the E. coli survivability in H2O2 and HClO, suggesting that the archaeal Hsp17.6 oxidation protection might depend on an “oxidant sink” effect, i.e., to consume the oxidants in environments via aspartate oxidation

Effect of detergents and chemicals on purified vaccinia virus: analysis by SDS polyacrylamide gel electrophoresis and electron microscopy

1977 ◽  
Vol 23 (3) ◽  
pp. 240-252 ◽  
Author(s):  
J. Boisvert ◽  
T. Yamamoto
Keyword(s):  
Electron Microscopy ◽  
Vaccinia Virus ◽  
Gel Electrophoresis ◽  
Alkali Treatment ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ammonium Bromide ◽  
Molecular Weights ◽  
Viral Particles

Vaccinia virus particles were dissociated into their constituent polypeptides and analysed by sodium dodecyl sulfate (SDS) gel electrophoresis. Thirty-three distinct polypeptide bands were identified and their molecular weights ranged between 11 000 and 150 000 daltons.Specific staining of gels containing polypeptides of dissociated virions revealed the presence of eight glycopeptides. No lipopeptides were detected.Analysis of chemical extracts (urea, guanidine hydrochloride, and alkali treatment) of the virus by SDS gel electrophoresis indicated that a total of 10 to 14 different polypeptides ranging in molecular weights from 11 000 to 70 000 daltons were solubilized.Analysis of detergent extracts and of the remains of extracted viral particles has shown that the detergent Nonidet P-40 (NP-40) solubilized a total of 11 polypeptides of which 6 were glycopeptides. The other detergents sodium deoxycholate (SDC) and cetyl trimethyl ammonium bromide (CTAB) were not as selective, both solubilizing more than 25 of the polypeptides composing the virus. Gel electrophoresis results also indicated that most of the small molecular weight (11 000–70 000 daltons) polypeptides were readily solubilized by NP-40, SDC, and CTAB, while those with molecular weights of 70 000 daltons and higher were not well solubilized.The effects of detergents were also analysed by electron microscopy. Evidence was obtained for subpopulations of viral particles having different susceptibility to detergent extraction.

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements

1990 ◽  
Vol 10 (10) ◽  
pp. 5177-5186
Author(s):  
J Zhang ◽  
S T Jacob
Keyword(s):  
Ribosomal Dna ◽  
Core Promoter ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Column ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Molecular Weights ◽  
The Core ◽  
Pol I

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

scholarly journals Chemical synthesis of all-trans-β-retinoyl phosphate

1976 ◽  
Vol 159 (3) ◽  
pp. 799-801 ◽  
Author(s):  
J P Frot-Coutaz ◽  
L M de Luca
Keyword(s):  
Retinoic Acid ◽  
Chemical Synthesis ◽  
Cellulose Acetate ◽  
Silica Gel ◽  
Absorption Maximum ◽  
Ammonium Acetate ◽  
Deae Cellulose ◽  
Thin Layers ◽  
Molar Ratio ◽  
Chloroform Methanol

all-trans-β-Retinoic acid is phosphorylated to retinoyl phosphate by bis(triethylamine) phosphate with yields of 10-15%. The product is soluble in methanol and is eluted from DEAE-cellulose acetate at a concentration of 0.1M-ammonium acetate in 99% (v/v) methanol. Its phosphate/retinoic acid molar ratio is 1. Retinoyl phosphate has an absorption maximum at 360nm in methanol, whereas retinoic acid has a maximum at 350 nm. The compound is hydrolysed at pH2 and pH13 for 20 min at 37 degrees C, but is relatively stable under the same conditions at pH4, 6, 8 and 10. Retinoyl phosphate (RF 0.1) can be separated from retinyl phosphate (RF 0.2) by chromatography on thin layers of silica gel in chloroform/methanol/water (60:25:4, by vol.).

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 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.

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