Purification et quelques propriétés de l'AMPc phosphodiestérase d'Arthrobacter crystallopoietes

1988 ◽  
Vol 66 (5) ◽  
pp. 454-459 ◽  
Author(s):  
François Schneider ◽  
Gérard Lefebvre ◽  
Michèle Ribolzi-Chery ◽  
Jean-Michel Bertin ◽  
Robert Gay
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Cyclic Nucleotides ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Negative Cooperativity ◽  
Divalent Ions ◽  
Camp Phosphodiesterase ◽  
Hydrolysis Of

cAMP phosphodiesterase was purified 8250-fold from extracts of Arthrobacter crystallopoietes, primarily by hydrophobic chromatography. The molecular weight of this enzyme was estimated as 51 000 by gel filtration and density-gradient centrifugation. The results suggest that the enzyme consists of two subunits with a molecular weight of 25 600. Properties of this enzyme are reported, including its negative cooperativity. This phosphodiesterase specifically catalyzes the hydrolysis of 3′,5′-cyclic nucleotides. Divalent ions either have no effect on activity or are weak inhibitors. Photooxidation of the enzyme with methylene blue and treatment with mercuribenzoates suggest that this enzyme may possess an imidazole group within its active site. The effects of thiols and Fe2+ on activity suggests that this enzyme may be a metalloenzyme.

scholarly journals THE SUBUNITS IN RABBIT ANTI-FORSSMAN IGM ANTIBODY

1968 ◽  
Vol 127 (5) ◽  
pp. 967-982 ◽  
Author(s):  
Michael M. Frank ◽  
John H. Humphrey
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sheep Erythrocyte ◽  
Gel Filtration ◽  
Isolation Procedure ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Forssman Antibody

Rabbit IgM anti-Forssman antibody was highly purified and the subunits obtained on reduction and alkylation were labeled radioactively and isolated by two different and unrelated methods. In both cases the subunits were found to have a molecular weight of about 90,000, based on their behavior on density gradient centrifugation and gel filtration, and evidence is given that they contained one light and one heavy chain. The subunits bound only weakly to sheep erythrocyte stroma, and only half could be shown to possess antigen specific sites. The data are consistent with the concept that each anti-Forssman IgM molecule has five effective binding sites, but it is uncertain whether the ineffectiveness of the remaining five H-L chain pairs is inherent in the structure of the IgM molecule or an artifact due to the isolation procedure. Intact IgM anti-Forssman antibody binds very firmly to structures containing multiple repeating antigen sites, and it appears that this is due to the presence of multiple binding sites on the molecule.

scholarly journals The heterogeneity of the lipoprotein lipase of rat epididymal adipose tissue

1978 ◽  
Vol 171 (2) ◽  
pp. 305-311 ◽  
Author(s):  
P Ashby ◽  
A M Tolson ◽  
D S Robinson
Keyword(s):  
Molecular Weight ◽  
Adipose Tissue ◽  
Lipoprotein Lipase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Particulate Material ◽  
Epididymal Adipose Tissue ◽  
Fat Cell ◽  
Enzyme Preparations

Lipoprotein lipase is heterogeneous, and it was suggested that the enzyme in adipose tissue is transformed from a species of mol. wt. approx. 120000 to forms of much higher molecular weight as it is secreted from the fat-cell. This paper demonstrates that the forms of higher molecular weight are probably artifacts. Enzyme preparations were characterized by gel filtration, by density-gradient centrifugation and by affinity chromatography. The results indicate that the enzyme forms of mol. wt. greater than 120000 result from an association of the enzyme with particulate material. It is therefore necessary to reconsider schemes that have recently been proposed for the synthesis and export of lipoprotein lipase.

scholarly journals SYNTHESIS OF CHLOROPLAST DNA IN ISOLATED CHLOROPLASTS

1968 ◽  
Vol 38 (1) ◽  
pp. 151-157 ◽  
Author(s):  
N. Steele Scott ◽  
Vinod C. Shah ◽  
Robert M. Smillie
Keyword(s):  
Molecular Weight ◽  
Chloroplast Dna ◽  
Euglena Gracilis ◽  
Bacterial Contamination ◽  
Actinomycin D ◽  
Density Gradient Centrifugation ◽  
Tritiated Thymidine ◽  
Gradient Centrifugation ◽  
Acid Stable ◽  
Isolated Chloroplasts

Chloroplasts isolated from Euglena gracilis incorporated both tritiated thymidine 5'-triphosphate and tritiated deoxyadenosine 5'-triphosphate into an acid-stable fraction. The incorporation was dependent on the presence of all four deoxynucleoside triphosphates and was sensitive to treatment with deoxyribonuclease and actinomycin D. It was demonstrated that bacterial contamination could not account for the incorporation of label. Extraction of DNA from the chloroplasts and subsequent density gradient centrifugation of the DNA in CsCl2 showed that the incorporation was into chloroplast DNA (ρ = 1.686) of high molecular weight.

Enhancing Effect of Surfactant and Protein on Hydrolysis of Thymolphthalein Monophosphate by Purified Prostatic Acid Phosphatase

1975 ◽  
Vol 21 (12) ◽  
pp. 1761-1765 ◽  
Author(s):  
Andras G Foti ◽  
Harvey Herschman ◽  
J Fenimore Cooper ◽  
Hedi imFeld
Keyword(s):  
Acid Phosphatase ◽  
Serum Proteins ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Prostatic Acid Phosphatase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Brij 35 ◽  
Hydrolysis Of ◽  
Protein Bovine Serum Albumin

Abstract Purified prostatic acid phosphatase catalyzes the hydrolysis of thymolphthalein monophosphate 10-fold faster if an optimal concentration of Brij 35 (a wetting agent) or protein (bovine serum albumin or human serum proteins) is present. Results of gel filtration, dialysis, and sucrose density-gradient centrifugation analysis suggest that the substrate must combine with detergent or protein before the enzyme can catalyze its hydrolysis.

scholarly journals Factor V activity of platelets: evidence for an activated factor V molecule and for a platelet activator

Blood ◽  
1977 ◽  
Vol 49 (5) ◽  
pp. 819-834 ◽  
Author(s):  
B Osterud ◽  
SI Rapaport ◽  
KK Lavine
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Factor V ◽  
Freezing And Thawing ◽  
Early Step ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Native Factor

Abstract This study was prompted by the observation that fresh platelet suspensions--prepared by gel filtration or albumin density gradient centrifugation--possessed only minimal factor V activity, whereas frozen-and-thawed platelet suspensions possessed striking factor V activity. Results of experiments with fresh suspensions suggested that unaltered platelets did not bind plasma factor V. The factor V activity of frozen-and-thawed platelet suspensions was markedly diminished after exposure to a factor V antibody, was not activated by thrombin, and was not associated with an increase in factor V antigen over that found in fresh platelet suspensions. Consequently, disruption by freezing and thawing must have resulted in the appearance of small amounts of an activated factor V molecule in platelet suspensions. Disrupted platelets were shown to activate native factor V, but an interaction between a platelet activator and traces of native factor V in fresh suspensions could not be demonstrated to account for the full activity of frozen-and-thawed suspensions. Apparently, therefore, platelets also contained an activated factor V molecule. Adding collagen, but not adenosine 5′-diphosphate to fresh platelet suspensions increased their factor V activity. Release of an activated platelet factor V molecule after exposure to collagen could represent a physiologically significant early step in hemostasis.

scholarly journals A study of nucleated erythrocytes by density-gradient centrifugation in a zonal rotor

1969 ◽  
Vol 111 (4) ◽  
pp. 583-591 ◽  
Author(s):  
A P Mathias ◽  
D. Ridge ◽  
N. St G. Trezona
Keyword(s):  
Molecular Weight ◽  
Ribosomal Rna ◽  
Base Composition ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Density Gradients ◽  
Avian Erythrocytes ◽  
Nuclear Rna ◽  
Zonal Rotor

1. Several substances of high molecular weight were examined for their suitability as suspension media in the formation of density gradients for the zonal centrifugation of avian erythrocytes. None proved satisfactory. 2. The behaviour of pigeon erythrocytes in rate-sedimentation experiments in a type A zonal rotor with density gradients of sucrose was examined. The mature cells sediment more rapidly than the younger cells and have a lower RNA/DNA ratio. Maturation is accompanied by a greater loss of RNA from the nucleus than from the cytoplasm. 3. The base composition of the nuclear RNA and of the two species of cytoplasmic ribosomal RNA is reported. 4. The RNA of erythrocytes may be labelled in vivo by injection of inorganic [32P]phosphate. The cells most active in the synthesis of RNA sediment less rapidly than the bulk of the cells. 5. Reticulocyte nuclei sediment more slowly than those from erythrocytes. Reticulocyte nuclei have a mean volume of 35μ3 and are isopycnic with sucrose of density 1·2871 (measured at 20°). Maturation of the nuclei causes them to shrink to a volume of 25μ3 and the density to increase to 1·2944.

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