scholarly journals The effect of tunicamycin on the glycosylation of lactating-rabbit mammary glycoproteins

1979 ◽  
Vol 180 (3) ◽  
pp. 481-489 ◽  
Author(s):  
Brian K. Speake ◽  
David A. White
Keyword(s):  
Acid Hydrolysis ◽  
Paper Chromatography ◽  
Gel Electrophoresis ◽  
Protein Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Strong Acid ◽  
Polyacrylamide Gel ◽  
Control Value ◽  
Acid Hydrolysate ◽  
Hydrolysis Of

1. Tunicamycin inhibited the incorporation of d-[2-3H]mannose into dolichol-linked oligosaccharide and glycoprotein of lactating-rabbit mammary explants by approximately the same extent (approx. 30% of control value), suggesting that lipid-linked intermediates are involved in the mannosylation of mammary glycoproteins. 2. The incorporation of radioactivity from N-acetyl-d-[1-14C]glucosamine into dolichol-linked oligosaccharide was inhibited by tunicamycin to 32% of the control value, whereas the incorporation of the radiolabel into glycoprotein was only inhibited to 72% of the control value. 3. Considerable redistribution of label from N-acetylglucosamine to N-acetylgalactosamine was found to occur in the explants. In the presence of tunicamycin approx. 76% of the radioactivity incorporated into glycoprotein from N-acetyl-d-[1-14C]glucosamine was present as N-acetylgalactosamine, compared with approx. 61% in the absence of the inhibitor. Thus tunicamycin selectively inhibits the incorporation of N-acetylglucosamine into glycoprotein. 4. Radioactivity from N-acetyl-d-[1-14C]glucosamine was incorporated into a glycoprotein that was identified as casein by the use of a casein-specific antiserum, and also into a group of glycopolypeptides with apparent mol.wts. ranging between 40000 and 80000. N-Acetylgalactosamine was the only radioactive sugar released on strong-acid hydrolysis of the immunoprecipitated casein, whereas N-acetylglucosamine was the major radioactive residue present in the non-casein glycoproteins. Glucosamine and galactosamine were the only radiolabelled sugars detected by paper chromatography of the strong-acid hydrolysate of the protein fraction. 5. Tunicamycin inhibited the incorporation of radioactivity from N-acetyl-d-[1-14C]glucosamine into the glycopolypeptides with mol.wts. between 40000 and 80000 as described by polyacrylamide-gel electrophoresis, but did not affect the incorporation of label into casein. It appears that tunicamycin inhibits the incorporation of mannose and N-acetylglucosamine into a number of mammary glycoproteins by inhibiting the formation of lipid-linked intermediates, but does not inhibit the incorporation of N-acetylgalactosamine into casein.

Photoactivated linkage of catechol O-methyltransferase and S-adenosyl-L-methionine

1984 ◽  
Vol 62 (10) ◽  
pp. 964-969 ◽  
Author(s):  
Peter H. Yu
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Ultraviolet Light ◽  
Paper Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Comt Inhibitors ◽  
Isoelectric Focussing

The formation of a stably linked complex of tritiated S-adenosyl-L-methionine (AdoMet) and catechol O-methyltransferase (COMT) has been achieved by irradiating the enzyme and ligand in Tris–HCl buffer (pH 7.5) with ultraviolet light at 254 nm. The reaction is specific as shown by a number of criteria. COMT inhibitors such as S-adenosylhomocysteine can block this photoactivated linkage. The [3H]AdoMet–COMT adduct has been shown to be a homogeneous protein by Sephadex gel filtration, sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and isoelectric focussing. After extensive proteolysis of the [3H]AdoMet–COMT adduct with pronase P, one major labelled product was released. This fragment could be separated by paper chromatography and was shown to be chromatographically identical to that released from the [3H]AdoMet – phenylethanolamine N-methyltransferase adduct.

scholarly journals The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland

1978 ◽  
Vol 170 (3) ◽  
pp. 479-486 ◽  
Author(s):  
D A White
Keyword(s):  
Mammary Gland ◽  
Acid Hydrolysis ◽  
Alkaline Hydrolysis ◽  
Gel Electrophoresis ◽  
Incubation Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Fractions ◽  
Exogenous Substrate ◽  
Major Species ◽  
Hydrolysis Of

1. A lactating rabbit mammary-gland microsomal system catalysed the incorporation of mannose from GDP-[U-14C]mannose into three endogenous acceptors, (i) polyprenyl phosphate mannose, (ii) lipid-linked oligosaccharide and (iii) protein. 2. Synthesis of polyprenyl phosphate mannose was stimulated by addition of dolichol phosphate to the incubation medium and was reversed by addition of GDP. The product had properties identical with those of authentic dolichol phosphate mannose. 3. The oligosaccharides derived from acid hydrolysis of the lipid-linked oligosaccharide fraction were of six, eight and nine to ten monosaccharide units, the octasaccharide being the major species formed. The oligosaccharide appeared to be attached to the lipid via a pyrophosphate bridge, since strong alkaline hydrolysis liberated an oligosaccharide phosphate. 4. Polyprenyl phosphate mannose served as a mannose donor to lipid-linked oligosaccharides and protein. When added as exogenous substrate it gave rise to a lipid-linked oligosaccharide of about six units. 5. Incorporation of radioactivity in protein was low, but polyacrylamide-gel electrophoresis of the protein fractions indicated that polypeptides of mol.wts. 115000, 75000 and 33000 were labelled.

scholarly journals Occurrence of an endo-1,3-β-glucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity

1979 ◽  
Vol 177 (1) ◽  
pp. 107-114 ◽  
Author(s):  
T G Villa ◽  
V Notario ◽  
J R Villanueva
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Candida Utilis ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Beta Glucanase ◽  
Hydrolysis Of

The endo-1,3-beta-glucanase (EC 3.2.1.6) secreted into the culture medium by cells of Candida utilis was isolated and purified to homogeneity on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (s20,w = 1.97S). The purified enzyme represented only 0.001% of the total 1,3-beta-glucanase activity, the remainder being due to an exo-1,3-beta-glucanase enzyme, and behaved as an acidic glycoprotein (pI 3.3) in isoelectric-focusing experiments. The mol.wt. was estimated to be 21 000 by gel filtration and polyacrylamide-gel electrophoresis. Studies on the hydrolysis of different substrates showed that the enzyme was only able to break down (1 leads to 3)-beta-linkages, by an endo-splitting mechanism. Glucono-delta-lactone, D-glucoronolactone and heavy metal ions such as Hg2+ were inhibitors of the enzyme activity. The function of this endo-beta-glucanase in C. utilis is discussed.

scholarly journals Purification of an exo-β-D-glucanase from cell-free extracts of Candida utilis

1976 ◽  
Vol 159 (3) ◽  
pp. 555-562 ◽  
Author(s):  
V Notario ◽  
T G Villa ◽  
J R Villanueva
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Candida Utilis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Acidic Amino Acids ◽  
Sepharose 4B ◽  
Hydrolysis Of

β-Glucanase present in cell-free extracts from Candida utilis was isolated and purified 562-fold by procedures that include adsorption on DEAE-Sephadex A-50 and filtration through columns of Sephadex G-50, G-100 and G-200, Bio-Gel P-10, and Concanavalin A-Sepharose 4B. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (S20,w = 1.74S). The enzyme behaved as an acidic glycoprotein (pI4.1) with 68% carbohydrate and a high content of acidic amino acids. The mol.wt. was estimated to be 20000 from gel filtration and polyacrylamide-gel electrophoresis and 36000 from sedimentation experiments. Studies on the hydrolysis of different substrates showed that the enzyme is an unspecific β-glucanase able to break down both (1 leads to 3)-eta- and (1 leads to 6)-β-linkages by an exo-splitting mechanism. Glucono-δ-lactone, Zn2+ and Hg2+ inhibited the enzyme activity.

scholarly journals Human liver alkaline phosphatase purified by affinity chromatography, ultracentrifugation and polyacrylamide-gel electrophoresis

1976 ◽  
Vol 159 (3) ◽  
pp. 697-705 ◽  
Author(s):  
A L Latner ◽  
A W Hodson
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of

A method is presented for the preparation of human liver alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). The method gives a purification factor of 12.5 × 10(3) over the initial aq. butan-1-ol extract, a recovery of 6.0% and a specific activity for the preparation of 1450-1550 units/mg of protein, 1 unit being defined as the amount of enzyme catalysing the hydrolysis of 1mumol of p-nitrophenyl phosphate/min at 35 degrees C in 0.1 M-2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10mM-p-nitrophenyl phosphate. Homogeneity was studied by ultracentrifugation, by immunoelectrophoresis and by polyacrylamide-gel electrophoresis. A single contaminating protein was present which was less than 5% of the total. Ultracentrifugation and equilibrium-gradient-pore electrophoresis techniques indicated a mol.wt. of 156000 and 160000 respectively. Equilibrium-gradient-pore electrophoresis indicated that the alkaline phosphatase molecule is possibly a dimer, comprising two subunits of about 80000 mol.wt. Amino acid analysis proved remarkably similar to that for alkaline phosphatase from other sources, regardless of species.

Altered amounts of hemoglobin synthesis in livers of dystrophic hamsters

1977 ◽  
Vol 55 (8) ◽  
pp. 894-900 ◽  
Author(s):  
K. Wrogemann ◽  
N. M. Quilliam ◽  
E. G. Nylen ◽  
M. D. Johnson ◽  
J. F. Clancy Jr.
Keyword(s):  
Gel Electrophoresis ◽  
Protein Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Leucine Incorporation ◽  
Major Protein ◽  
Hemoglobin Synthesis ◽  
Histological Sections ◽  
Age Dependent ◽  
Different Levels

In liver supernatants similar amounts of leucine are incorporated into proteins of normal and dystrophic hamsters. However, using a dual labeling technique, a protein fraction is detected which shows different levels of leucine incorporation between normal and dystrophic animals in an age-dependent fashion. The major protein in this fraction comigrates with hemoglobin or its subunits under various conditions of polyacrylamide gel electrophoresis. In animals younger than 3 days there is more synthesis of this protein fraction in dystrophic animals, while at 6 days there is considerably less synthesis of this protein. These changes are paralleled by differences in the number of erythroid foci in histological sections of the livers. There is no evidence for structurally altered hemoglobin in dystrophic hamsters. The significance of this finding and its possible relation to the dystrophy process are not known at present.

Ultrastructural localization of specific nucleoprotein fractions

1978 ◽  
Vol 36 (2) ◽  
pp. 204-205
Author(s):  
G. L. Brown
Keyword(s):  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultrastructural Localization ◽  
Polyacrylamide Gel ◽  
Acid Extraction ◽  
Acid Solutions ◽  
Low Salt ◽  
Liver Nuclei ◽  
Phosphate Groups

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

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