scholarly journals The Nature of the Muscle-Relaxing Factor

1963 ◽  
Vol 46 (5) ◽  
pp. 893-904 ◽  
Author(s):  
Franklin Fuchs ◽  
F. Norman Briggs
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Gel Filtration ◽  
Active Agent ◽  
Calcium Deficiency ◽  
Chelating Resin ◽  
Prolonged Heating ◽  
Relaxing Factor ◽  
Chelex 100 ◽  
High Molecular Weight Material

High speed centrifugal fractionation of homogenates of rabbit skeletal muscle has led to the discovery of a soluble muscle-relaxing factor in the homogenate. Assay of the relaxing activity with deoxycholate-treated myofibrils and reconstituted actomyosin systems has established that the activity is not produced by the presence of contaminants. Relaxing activity could be removed or destroyed by charcoal, dialysis, prolonged heating, and treatment with the chelating resin, chelex-100, making it improbable that the effect is due simply to calcium deficiency. Many of the characteristics of this muscle-relaxing factor suggest that it is very similar to or the same as the factor formed by the incubation of muscle granule fractions and ATP. Evidence is presented that some soluble protein component is involved in the stabilization of the factor. The relaxing activity could be separated from the high molecular weight material in the supernatant by the technique of gel filtration. On the basis of the gel used, the molecular weight of the active agent should be less than 4000.

scholarly journals High Molecular Weight Factor VIII Coagulant Activity in Cryoprecipitate and Polyethylene Glycol Precipitates

1977 ◽  
Author(s):  
K. A. Rickard ◽  
T. Exner ◽  
H. Kronenberg
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Human Antibody ◽  
High Molecular Weight Material ◽  
Coagulant Activity ◽  
Molecular Weight Form

Gel filtration of human plasma cryoprecipitate on Sepharose 2B indicated the molecular weight of factor VIII coagulant activity (VIIIc) to be significantly greater than that found in antihaemophilic concentrate. Polyethylene glycol at 3% concentration precipitated approximately half of the VIIIc from cryoprecipitate. This activity eluted as high molecular weight material on gel filtration. The addition of more polyethylene glycol to a concentration of 8% precipitated most of the remaining VIIIc from cryoprecipitate. This activity appeared to be of significantly lower molecular weight, approximately corresponding in elution volume to that observed for antihaemophilic concentrate. The possibility that an antibody to VIIIc generated in a patient treated with cryoprecipitate might be directed against the higher molecular weight form of factor VIII was investigated. However, no significant differences between the higher and lower molecular weight forms of factor VIII either in stability or in reactivity with human antibody to factor VIII were found.

Characterization Of Soluble DES-A And DES-AB Fibrin In Human Plasma At 37°C By Agarose-Gel Filtration

1981 ◽  
Author(s):  
G Müller-Berghaus ◽  
J-C Bernhard ◽  
I Mahn
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Void Volume ◽  
Agarose Gel ◽  
High Molecular Weight Material ◽  
Different Temperatures ◽  
Lower Temperature

In two consecutive steps, thrombin cleaves the fibrinopeptides A and B from fibrinogen producing des-A fibrin and des-AB fibrin. Labeled des-A fibrin was prepared by batroxobin and labeled des-AB fibrin by clotting of 125I-fibrinogen with thrombin. Fibrin solubilized in buffered urea was mixed with plasma containing 131I-fibrinogen (fibrin:fibrinogen ratio = 1:20). These fibrinfibrinogen mixtures were applied to sepharose CL- 6B columns eq ui librated with buffered plasma (0.0025 M EDTA, 0.1 M NaCl, 0.05 M tris, 0.005 M EACA, 2 AT U hirudin/ml, 500 KIU a protinin/ml, 0.003 M NaN3, pH 7.4). Plasma was used as an equilibration and elution medium to prevent precipitation of fibrin in the columns. At 20°C, labeled des-A fibrin as well as des-AB fibrin were eluted in the void volume as high-molecular weight aggregates (peak A) and separated from m onomeric labeled fibrinogen (peak B). At 37°C, however, des- A fibrin was eluted at the same position as monomeric fibrinogen (peak B), whereas des-AB fibrin was eluted in the void volume as at 20°C. Rechromatography of isolated fractions of peak A and peak B at different temperatures showed that monomeric fibrin isolated at 37°C formed high molecular weight material at 20°C, and high-molecular weight fibrin isolated at 20°C dissociated at 37 ° C. The results suggest that des-A fibrin solubilized in plasma in the absence of calcium ismonomeric at 37°C but forms high-molecular weight aggregates at lower temperature, whereas des-AB fibrin is oligomeric at 20°C as well as at 37°C.

Physicochemical properties of PM-factor, a surface-active agent produced by Pseudomonas marginalis

1996 ◽  
Vol 42 (3) ◽  
pp. 243-251 ◽  
Author(s):  
G. Burd ◽  
O. P. Ward
Keyword(s):  
High Speed ◽  
Surface Active Agent ◽  
Gel Filtration ◽  
Active Agent ◽  
Aromatic Amino Acids ◽  
Polymyxin B ◽  
Culture Broth ◽  
Ultraviolet Absorption Spectrum ◽  
Emulsifying Activity ◽  
Surface Active

An extracellular surface-active agent, PM-factor, was obtained by high-speed centrifugation from the culture broth of Pseudomonas marginalis PD-14B. PM-factor exhibited emulsifying activity on a broad spectrum of hydrocarbon liquids, including aromatics, aliphatics, crude oil, and creosote. The factor appeared as ball-shaped particles of varying diameter when examined by electron microscopy (0.16–1.4 μm). Gel filtration chromatography demonstrated a high molecular mass of the factor (> 106 Da). The ultraviolet absorption spectrum manifested a peak in the region 200 nm rather in the region 260–280 nm. Amino acid analysis showed a very low amount of aromatic amino acids residues in the protein moiety of PM-factor. The presence of 3-deoxy-D-mannooctulosonic acid, heptose, hexosamine, phosphorus, and 3-hydroxy fatty acid indicated that PM-factor contained lipopolysaccharide. The emulsifying activity of PM-factor was inhibited strongly by mercuric chloride and moderately by EDTA. Polymyxin B, Ca2+, and Mg2+ markedly stimulated the factors emulsifying activity. Roles of the bioemulsifier in the functioning of P. marginalis as a plant pathogen and in bioremediation are discussed.Key words: bioemulsifier, Pseudomonas marginalis, polycyclic aromatic hydrocarbons, plant pathogenesis, bioremediation.

Studies on soil polysaccharides. II. The composition and properties in soils under pasture and under a fallow-wheat rotation

Soil Research ◽  
1968 ◽  
Vol 6 (2) ◽  
pp. 225 ◽  
Author(s):  
GD Swincer ◽  
JM Oades ◽  
DJ Greenland
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Extraction Procedure ◽  
Gel Filtration ◽  
Wheat Crop ◽  
Low Molecular Weight ◽  
Neutral Sugar ◽  
Sugar Composition ◽  
High Molecular Weight Material ◽  
Neutral Sugars

After the removal of light fraction from soils under old pasture and under continuous fallow-wheat rotation, carbohydrates were extracted using IN HC1 followed by 0.5N NaOH and finally an acidic acetylation procedure, or by a single extraction with 0.2N NaOH only. The sequential extraction procedure removed 70-80 % of the carbohydrate from the soil under both agronomic systems. 0.2N NaOH removed a larger proportion of the carbohydrates from soil under fallow-wheat rotation (43-52%) than from soil under old pasture (35-38%). The composition of the carbohydrates in a given extract from the soil under pasture or fallow-wheat was similar. This similarity extended even to the neutral sugar composition of fractions obtained by gel filtration of the purified extracts. Generally, low molecular weight materials were rich in amino acids and compounds such as glucose, ribose, and glycerol. Polymers of molecular weight 4000-100,000 contained relatively high proportions of uronic acids and amino acids. Least amino acids were present in materials of molecular weight greater than 100,000 which contained appreciable quantities of deoxyhexoses (up to 20% of the total neutral sugars) indicative of their microbial origin. Against this background of similarity, certain differences between the carbohydrates from soils under pasture and fallow-wheat rotation were apparent. 1N HCl extracts contained more high molecular weight material from the old pasture soils than from the cultivated soil. The composition of these extracts indicated that they comprised the easily extractable recently synthesized microbial polysaccharides. The proportion of such polymers was lower in the cropped soil. A higher proportion of materials of small size was present in soils under a wheat crop. Maximum amounts of these compounds were present during periods of maximum plant and microbial activity. Extracts from soils under fallow-wheat rotation contained a higher proportion of uronic and amino acids and less ribose, arabinose, and deoxysugars than the extracts from soils under pasture. Based on relative deoxysugar contents it was calculated that the pasture soil contains about four times as much microbial polysaccharide as the soil under fallow-wheat.

scholarly journals Purification and characterization of a collagenase extracted from rabbit tumours

1975 ◽  
Vol 152 (1) ◽  
pp. 131-142 ◽  
Author(s):  
P A McCroskery ◽  
J F Richards ◽  
E D Harris
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Chelating Resin ◽  
Rabbit Muscle ◽  
Ph Optimum ◽  
Chelex 100 ◽  
Cnbr Cleavage ◽  
Total Enzyme

A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of α1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as μmol of collagen in solution cleaved/h per mg of enzyme at 35°C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1μM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides α2 and α1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [α1 (I)]2α2 and [α1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [α1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4)2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the α1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.

The temperature-dependent dissociation of β-casein from bovine casein micelles and complexes

1970 ◽  
Vol 37 (3) ◽  
pp. 361-372 ◽  
Author(s):  
W. K. Downey ◽  
R. F. Murphy
Keyword(s):  
Molecular Weight ◽  
Low Temperatures ◽  
High Speed ◽  
Gel Filtration ◽  
Skim Milk ◽  
The Other ◽  
Free Form ◽  
Temperature Dependent ◽  
Casein Micelles ◽  
Expected Position

SummaryThe temperature-dependent dissociation of β-casein from the casein micelles of milk and from the soluble casein complexes of colloidal phosphate-free (CPF) milk was investigated by high-speed centrifugation and gel-filtration. The percentage of the total casein in supernatants prepared by high-speed centrifugation of mid-lactation milks increased from approximately 6 to 15% on cooling the milks from 30 to 5 °C; β-casein accounted for about 46% of this increase, while αs-and κ-casein constituted 30 and 23%, respectively. On gel-filtration both of skim-milk and CPF milk on Sepharose 2B at 0, 2, 5, 10 and 25 °C, maximum amounts of free β-casein (c. 60% of total) were obtained at 5 °C. The remainder of the β-casein appeared to be more strongly bound to the αs- and κ-casein and may be involved in the internal cohesion of casein micelles. The free β-casein of both milk preparations appeared to be in equilibrium with the bound β-casein. On Sephadex G-200 columns at 5 °C, approximately 5 and 60% of the β-casein of skim-milk and CPF milk, respectively, was eluted in the free form in the expected position for a globular protein of molecular weight about 200000. At low temperatures, particularly at 5 °C, colloidal phosphate appeared to play an integrating role in the association of over half the total β-casein with the other casein components of native micelles. However, when the equilibrium between micellar and free β-casein was disturbed by gel-filtration on Sepharose 2B, the presence of colloidal phosphate did not prevent the release of most of the β-casein from casein micelles. Some problems encountered in the use of densitometry for the estimation of individual caseins on electropherograms are described.

Separation of Low Molecular Weight RNA Species by High-Speed Gel Filtration

1981 ◽  
Vol 90 (3) ◽  
pp. 643-648 ◽  
Author(s):  
Saburo UCHIYAMA ◽  
Takeyoshi IMAMURA ◽  
Shin-ichi NAGAI ◽  
Katsutoshi KONISHI
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Gel Filtration ◽  
Low Molecular Weight

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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