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.

Determination of the Molecular Weight and the Hydrodynamic Properties of a Polypeptide from the Thylakoid Membrane by Sedimentation, Diffusion and Binding Measurements in Dodecyl Sulphate Solutions

1975 ◽  
Vol 30 (9-10) ◽  
pp. 615-621 ◽  
Author(s):  
Hans Craubner ◽  
Friederike Koenig ◽  
Georg H. Schmid
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Partial Specific Volume ◽  
Hydrodynamic Properties ◽  
Lamellar System ◽  
Self Diffusion

The molecular weight and hydrodynamic properties of a polypeptide isolated from the lamellar system of Antirrhinum chloroplasts were determined in sodium dodecyl sulphate solution by measurement of sedimentation velocity, diffusion and effective partial specific volume. The polypeptide fraction exhibits a molecular weight of 25 000 which agrees with the apparent molecular weight found by polyacrylamide gel electrophoresis. The molecular weight of the polypeptidesodium dodecyl sulphate micelle was 54 000, with a friction ratio of 1.6 which indicates an effective asymmetric hydrodynamic shape. For binding measurements self-diffusion equilibrium dialysis with dodecyl [35S] sulphate was used. In this case, dialysis equilibrium was reached within about 10 hours, in contrast to the dialysis with initial concentration differences which requires much longer times. A binding value of δD = 1.15g sodium dodecyl sulphate per g polypeptide was obtained which corresponds to a molar binding ratio of 100 mol dodecyl sulphate bound per mol of polypeptide. After the removal of dodecyl sulphate the polypeptide is present in an aggregated state. In phosphate buffers of pH 6.8 and 7.5 the aggregates preponderantly have sedimentation coefficients of 11.7 and 6.8 Svedberg units respectively. Assuming equivalent spheres the molecular weights were calculated to be 340 000 and 150 000.

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 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.

Localization and Functional Characterization of Three Thylakoid Membrane Polypeptides of the Molecular Weight 66000

1977 ◽  
Vol 32 (9-10) ◽  
pp. 817-827 ◽  
Author(s):  
Friederike Koenig ◽  
Wilhelm Menke ◽  
Alfons Radunz ◽  
Georg H. Schmid
Keyword(s):  
Molecular Weight ◽  
Electron Transport ◽  
Outer Surface ◽  
Thylakoid Membrane ◽  
Functional Characterization ◽  
Apparent Molecular Weight ◽  
Photochemical Reactions ◽  
Antigenic Determinants ◽  
Reaction Centre

Abstract Three polypeptide fractions with the apparent molecular weight 66 000 were isolated from stroma-freed Antirrhinum chloroplasts which were solubilized with dodecyl sulfate. Antisera to these fractions affect electron transport in distinctly different ways. For the characterization of the three antisera photochemical reactions of chloroplast preparations with artificial electron donors and acceptors as well the analysis of fluorescence rise curves were used. Antiserum 66 000 PSI-96 inhibits electron transport apparently on the acceptor side of photosystem I, provided the antibodies are adsorbed onto the outer surface of the thylakoid membrane. Antiserum 66 000 PSI-88 probably acts directly on the reaction centre I or on its immediate vicinity, if the antibodies are adsorbed at the inner surface of the thylakoid membrane. Antiserum 66 000 PSII-42 inhibits electron trans­ port in the region of photosystem II. The antigen towards which the antiserum is directed appears to belong to the reaction centre II, as also in the condition of high inhibition degrees, the fluorescence intensity remains unchanged. The antigenic determinants are located at the outer surface of the thylakoid membrane.

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.

Polypeptides of the Thylakoid Membrane and Their Functional Characterization

1978 ◽  
Vol 33 (9-10) ◽  
pp. 723-730 ◽  
Author(s):  
Georg H. Schmid ◽  
Wilhelm Menke ◽  
Alfons Radunz ◽  
Friederike Koenig
Keyword(s):  
Molecular Weight ◽  
Electron Transport ◽  
Electron Donor ◽  
Photosystem I ◽  
Thylakoid Membrane ◽  
Apparent Molecular Weight ◽  
Variable Fluorescence ◽  
Phenazine Methosulfate ◽  
Cyclic Photophosphorylation ◽  
High Concentrations

Abstract From stroma-freed chloroplasts of Antirrhinum majus polypeptides with the apparent molecular weights 44 000, 26 000 and 20 000 were isolated.The antiserum to a polypeptide with the moleculair weight 44 000 inhibits the photoreduction of anthraquinone-2-sulfonate with dichlorophenol indophenol/ascorbate when the concentration of the electron donor dichlorophenol indophenol is low. The antiserum enhances the rate of phenazine methosulfate-mediated cyclic photophosphorylation. The variable fluorescence yield is increased by the antiserum . It is assumed that this polypeptide plays a role in electron transport between the two photosystems. From two polypeptides with the apparent molecular weight 26 000 one seems to belong to the reaction center of photosystem II as it inhibits the photooxidation of tetramethyl benzidine and diphenyl carbazide with suitable electron acceptors and inhibits electron transport between water and silicomolybdate. Variable fluorescence is not or not too strong decreased by the antiserum . The other polypeptide of the apparent molecular weight 26 000 inhibits the photoreduction of anthraquinone-2-sulfonate with high concentrations of dichlorophenol indophenol as the electron donor. Phenazine methosulfate-mediated cyclic photophosphorylation is also inhibited by the antiserum . Therefore, we should like to associate it with the reaction center of photosystem I. The antiserum to the polypeptide with the apparent molecular weight 20 000 inhibits the photoreduction of anthraquinone-2-sulfonate with low and high concentrations of the electron donor dichlorophenol indophenol. It enhances phenazine methosulfate-mediated cyclic photophosphorylation. The polypeptide, therefore, should be functionally involved on the acceptor side of photosystem I.The results obtained up-to-now on the function and localization of the polypeptides in the thylakoid membrane are summarized.

scholarly journals A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage

1981 ◽  
Vol 197 (2) ◽  
pp. 355-366 ◽  
Author(s):  
D Heinegård ◽  
M Paulsson ◽  
S Inerot ◽  
C Carlström
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Partial Specific Volume ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40-1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.

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.

scholarly journals The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

1977 ◽  
Vol 167 (2) ◽  
pp. 509-512 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Partial Specific Volume ◽  
Triton X

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.

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