scholarly journals Incorporation of l-[3H]fucose and d-[3H]glucosamine into cell-surface-associated glycoconjugates in epidermis of cultured pig skin slices

1980 ◽  
Vol 190 (1) ◽  
pp. 65-77 ◽  
Author(s):  
I A King ◽  
A Tabiowo ◽  
R H Williams
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Hydrophobic Interactions ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Membrane Glycoproteins ◽  
Molecular Weight Range ◽  
Electron Microscope Autoradiography ◽  
Pig Skin ◽  
Membrane Bound

1. Electron microscope autoradiography indicated that L-[3H]fucose and D-[3H]glucosamine were both incorporated into cell-surface-associated glycoconjugates in the epidermis of cultured pig skin slices. 2. Acid hydrolysis and paper chromatography of skin homogenates confirmed that there was little metabolic conversion of the labeled precursors to other sugars. 3. Epidermis was separated from dermis using CaCl2, and was extracted with 8 M-urea/5% (w/v) sodium dodecyl sulphate and was then analysed by gel electrophoresis. The major component labelled with D-[3H]glucosamine had an apparent molecular weight in excess of 200 000. This material was not labelled with L-[3H]fucose. Lower molecular-weight components were labelled to a similar extent with both L-[3H]fucose and D-[3H]glucosamine. 4. The high molecular-weight material labelled with D-[3H]glucosamine was released into the medium when the epidermal cells were dispersed with trypsin, indicating that it was either surface-associated or was extracellular. It was also labelled with D-[14C]glucuronic acid, 35SO4(2-) and to a small extent with 14C-labelled amino acids indicating that it contained glycosaminoglycans derived from epidermal proteoglycans. This was confirmed by the fact that it was degraded by testicular hyaluronoglucosidase. It was not present in isolated membranes but was recovered in the soluble fraction from epidermal homogenates. It is therefore only very loosely bound at the cell surface or is present in the extracellular spaces. 5. Membrane-bound [3H]glycoproteins were identified after differential centrifugation of epidermal homogenates. The radioactivity profiles of membrane glycoproteins were similar whether L-[3H]fucose or D-[3H]glucosamine were used and both consisted of a major heterogeneous peak in the apparent mol.wt. range 70 000–150 000. [3H]Glycoproteins in this molecular-weight range were also major components of a plasma-membrane-enriched fraction. These glycoproteins were probably bound to the membrane by hydrophobic interactions, since they were only solubilized by treatment with detergent or organic solvent. They contained terminal sialic acid residues, since they were degraded by neuraminidase.

The Activation of Human Factor IX

1975 ◽  
Vol 33 (03) ◽  
pp. 553-563 ◽  
Author(s):  
B Østerud ◽  
K Laake ◽  
H Prydz
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Tissue Thromboplastin ◽  
Native Factor

SummaryThe activation of factor IX purified from human plasma has been studied. Factor XIa and kallikrein separately activated factor IX to factor IXa. In both cases factor IX a had an apparent molecular weight of about 42–45000 in sodium dodecyl sul-phate-polyacrylamide disc gel electrophoresis compared with a molecular weight of about 70000 for the native factor IX. The activation by XIa required Ca2+-ions whereas Ca2+-ions did not influence the activation by kallikrein. A mixture of tissue thromboplastin and factor VII or RusselPs-viper venom alone did not activate factor IX. Trypsin activated and plasmin inactivated factor IX.

scholarly journals Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle.

1977 ◽  
Vol 72 (1) ◽  
pp. 194-208 ◽  
Author(s):  
L D Hodge ◽  
P Mancini ◽  
F M Davis ◽  
P Heywood
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Nuclear Matrix ◽  
Apparent Molecular Weight ◽  
Molecular Weight Range ◽  
Weight Range ◽  
Molecular Weights ◽  
Liver Nuclei ◽  
Hela S3 ◽  
Biochemical Analyses

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS-polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000-75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.

SHEDDING OF SURFACE PROTEINS BY PORCINE THYROID CELLS

1980 ◽  
Vol 85 (2) ◽  
pp. 245-251 ◽  
Author(s):  
A. BRENNAN ◽  
P. M. POVEY ◽  
B. REES SMITH ◽  
R. HALL
Keyword(s):  
Cell Surface ◽  
Sodium Dodecyl Sulphate ◽  
Culture Medium ◽  
Cell Metabolism ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Half Time ◽  
Thyroid Cells ◽  
Peptide Cleavage ◽  
Membrane Bound

Isolated porcine thyroid cells were surface-labelled with 125I using the lactoperoxidase technique. Samples of the cells were then cultured and harvested at various intervals for up to 7 days. The labelled proteins remaining on the cells or shed into the culture medium were analysed by electrophoresis on polyacrylamide gels run in sodium dodecyl sulphate. These studies indicated that the several different surface proteins of the thyroid cells were lost from the cell surface at similar rates (half-time of approximately 28 h) as the result, at least in part, of a process which depended on active cell metabolism. In addition, the gel profiles obtained from analysis of both medium and membrane-bound labelled proteins were similar and this suggested that peptide cleavage was not involved in the shedding of the majority of these proteins.

scholarly journals Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain

1976 ◽  
Vol 156 (1) ◽  
pp. 143-150 ◽  
Author(s):  
R H Quarles
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Weight Class ◽  
Developmental Difference ◽  
Adult Rats ◽  
Neuraminidase Treatment

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

Variation in goat fibre protein

1989 ◽  
Vol 40 (3) ◽  
pp. 675 ◽  
Author(s):  
DJ Tucker ◽  
AHF Hudson ◽  
A Laudani ◽  
RC Marshall ◽  
DE Rivett
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wool Fibre ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Cashmere Goats

The proteins from a range of cashmere, mohair, angoratcashmere crossbred and wool fibre samples were extracted at pH 8 with 8 M urea containing dithiothreitol, and were then radiolabelled by S-carboxymethylation using iodo(2-14C) acetate. The proteins from each sample were examined by two dimensional polyacrylamide gel electrophoresis in which the separation in the first dimension was according to charge at pH 8.9 and in the second dimension according to apparent molecular weight in the presence of sodium dodecyl sulfate. After electrophoresis the proteins were detected by fluorography. Protein differences in keratin samples from some individual goats existed, although the overall protein patterns were similar. None of the differences were consistent with any one goat fibre type. The protein patterns obtained for fibre samples from individual cashmere goats showed some differences when compared to those found for commercial blends from the same country of origin, indicating that blending can mask any animal-to-animal variation. While the electrophoretic technique does not unequivocally distinguish between cashmere, mohair and angora/cashmere crossbred fibres it does differentiate between wool and goat fibres.

Molecular Weight and the Dodecyl Sulphate Binding of a Thylakoid Membrane Polypeptide Involved in a Reaction on the Oxygen-Evolving Side of Photosystem II

1977 ◽  
Vol 32 (5-6) ◽  
pp. 384-391 ◽  
Author(s):  
Hans Craubner ◽  
Friederike Koenig
Keyword(s):  
Molecular Weight ◽  
Thylakoid Membrane ◽  
Phosphate Buffer ◽  
Equilibrium Dialysis ◽  
Sodium Dodecyl ◽  
Spherical Shape ◽  
Apparent Molecular Weight ◽  
Partial Specific Volume ◽  
Experimental Conditions ◽  
Sodium Phosphate Buffer

Abstract The molecular weight of a thylakoid membrane polypeptide with the apparent molecular weight 11 000 was determined by measurement of the sedimentation velocity, the diffusion and the ef­fective partial specific volume. The molecular weight was found to be 6300 and that of the poly-peptide-dodecyl sulphate micelle was found to be 11 200. The frictional ratio was 1.35. In ad­dition, we determined the binding of dodecyl sulphate onto the polypeptide by equilibrium dialysis. We found that 1 g polypeptide binds 0.77 g sodium dodecyl sulphate which corresponds to 17 molecules dodecyl sulphate bound per polypeptide chain. In the absence of dodecyl sulphate the polypeptide aggregates. The molecular weights of the aggregates are in 0.01 м sodium phosphate buffer pH 7.2 150 000 and in a 1 :1 mixture of 0.01 м phosphate buffer and 96% ethanol 365 000. The frictional ratios were 1.07 and 1.16 respectively which points at a spherical shape. The experimental conditions for the determination of the dodecyl sulphate binding were critically scrutinised.

Phosphorylation Of Platelet Actin-Binding Protein During Platelet Activation

1981 ◽  
Author(s):  
Roger C Carroll ◽  
Jonathan M Gerrard
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Time Course ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Platelet Protein ◽  
External Calcium

We have followed the 32P-labelling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples we found 2 to 3 fold labelling increases in actin-binding protein 30 to 60 seconds after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis of purified actin-binding protein as well as cytoskeletal cores prepared by detergent extraction of activated 32p-iabelled platelets. In addition, these cytoskeletal cores indicated that the 32P-labelled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labelling of actin-binding protein over an 8 minute time course revealed that in aggregating platelet samples rapid desphosphorylation to almost initial levels occurred between 3 and 5 minutes. A similar curve was obtained for the 20,000 apparent molecular weight peptide. This rapid dephosphorylation was shown to be dependent on platelet aggregation in the absence of external calcium or in thrombastenic platelets lacking the aggregation response to activation. These results suggest that phosphorylation of actin-binding protein initiates its association with the platelet cytoskeleton during activation.

Characterization of cytosolic female rat liver receptors of the lactogenic type

1990 ◽  
Vol 123 (2) ◽  
pp. 231-237
Author(s):  
M. Emtner ◽  
P. Roos
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Sodium Dodecyl ◽  
Human Growth ◽  
Structural Features ◽  
Female Rat ◽  
Membrane Bound

Abstract. Some properties of cytosolic receptors of the lactogenic type from female rat liver were studied and compared with those of membrane-bound (microsomal) receptors. The association constant between the cytosolic receptors and human growth hormone was 2.2 l/nmol, which was not significantly different from the value obtained for the microsomal receptors (3.6 l/nmol). Since unlabelled hGH and human prolactin, but not bovine growth hormone, displaced [125I]hGH bound to receptors from both sources, the cytosolic receptors, like the microsomal receptors, must be lactogenic. Furthermore, the cytosolic receptors were recognized by a monoclonal antibody raised against microsomal receptors from female rat liver. However, covalent cross-linking of cytosolic receptors to [125I]hGH and subsequent sodium dodecyl sulphate electrophoresis gave a single band corresponding to a molecular weight of 42 200 (after subtraction of the molecular weight of hGH), which differs significantly (p<0.01) from the values determined for the two distinct bands given by the microsomal fraction. Moreover, upon molecular sieve chromatography the receptor activity in the two fractions appeared at significantly (p<0.05) different elution volumes. These results show that the cytosolic and microsomal receptors have some structural features in common but are definitely not identical.

scholarly journals Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels.

1983 ◽  
Vol 31 (6) ◽  
pp. 709-716 ◽  
Author(s):  
M R Green
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Blue Staining ◽  
Polyacrylamide Gels ◽  
Periodic Acid Schiff ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.

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