Purification par séparation de phase et caractérisation du plasmalemme de l'épicotyle de pois

1986 ◽  
Vol 64 (5) ◽  
pp. 448-455 ◽  
Author(s):  
Jacques Rembur ◽  
Pierre Landré ◽  
Arlette Nougarède
Keyword(s):  
Atpase Activity ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Alkaline Ph ◽  
Phase Partition ◽  
Pea Epicotyl ◽  
Glucan Synthetase ◽  
Triton X ◽  
Two Sides ◽  
Slight Contamination

The validity of phase partition to obtain a substantial proportion of vesicles of plasmalemma origin from the microsomal fraction of pea epicotyl has been demonstrated. In the fractions enriched with plasma membranes, N-naphthyl phtalamic acid binding and β-glucan synthetase II activity, showed a yield of about 60% and an enrichment of 2.3 and 2.2, respectively, in comparison with the microsomal fraction. When such plasmalemmic vesicles are permabilized by Triton X-100, an intense Mg2+-ATPase activity is obtained in presence of K+ at acid as well as alkaline pH. Inhibition of Mg2+-ATPase by vanadate in presence of K+ and its variations in relation to pH were shown. Dicyclohexylcarbodiimide and diethylstilbestrol inhibit 40–55% of this enzymatic activity, both at acid and neutral pH. The data show a slight contamination of the plasmalemmic fraction by endomembranes and suggest an asymmetry of the two sides of the plasmalemma.

scholarly journals Characterization and localization of adenylyl cyclase in membrane vesicles and intact boar and human spermatozoa

1980 ◽  
Vol 43 (1) ◽  
pp. 93-102
Author(s):  
R.N. Peterson ◽  
L. Russell ◽  
L. Hook ◽  
D. Bundman ◽  
M. Freund
Keyword(s):  
Electron Microscope ◽  
Adenylyl Cyclase ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Human Sperm ◽  
Human Spermatozoa ◽  
Alkaline Ph ◽  
Plasma Membrane Vesicles ◽  
Triton X ◽  
Boar Spermatozoa

The enzymic properties of adenylyl cyclase in purified membrane vesicles from human and boar spermatozoa are described. Plasma membrane vesicles, which appear to be right-side-out, show a marked increase in activity in the presence of the detergent Triton X-100, manganous ion and alkaline pH. Electron-microscope cytochemical assays indicated the presence of adenylyl cyclase in boar and human sperm plasma membranes and also within the axoneme of intact human spermatozoa. The significance and precautions in the evaluation of the cytochemical data are discussed.

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

Comparative Isolation of Cilia and Flagella from the Lamellibranch Mollusc, Aequipecten irradians

1973 ◽  
Vol 12 (2) ◽  
pp. 345-367
Author(s):  
R. W. LINCK
Keyword(s):  
Atpase Activity ◽  
Cross Sections ◽  
Basal Body ◽  
Electron Dense Material ◽  
The Matrix ◽  
Fine Structures ◽  
Dynein Arms ◽  
Cilia And Flagella ◽  
Triton X ◽  
Ciliary Ultrastructure

Gill cilia and sperm flagella from the lamellibranch mollusc Aequipecten irradians were compared with respect to their ultrastructures and adenosinetriphosphatase activities. Cilia were isolated from excised gills using 3 different solutions: twice-concentrated seawater, 10 % ethanol-10 mM CaCl2 and 60% glycerol. In each case deciliation occurs by the severance of the cilium at the junction of the transition zone and the basal body, and in each case the ciliary ultrastructure is maintained. Sperm flagella were purified by mechanical decapitation. Cilia and sperm flagella have similar fine structures, except that the matrix of the cilia contains substantially more electron-dense material than that of flagella. The ATPase activity of purified cilia is approximately 0.09,µmol P1/min/mg protein; that of flagella is 0.13. Ciliary and flagellar axonemes were prepared by repeated extraction of the membranes with 1% Triton X-100. Ciliary axonemes maintain their 9 + 2 cylindrical orientation, whereas flagellar axonemes often appear as opened or fragmented arrays of the 9 + 2 structure, due to the partial breakdown of the flagellar nexin fibres. A-subfibre arms which were obvious in whole organelles are rarely seen in axoneme preparations. Again the ciliary matrix is considerably more amorphous than in flagellar axonemes. The ATPase activities of ciliary and flagellar axonemes are 0.13 and 0.12 µmol P1/min/mg protein respectively; however, activities of ciliary axonemes may vary by a factor of 2, depending on the method of isolation. The difficulty in observing A-subfibre arms in cross-sections of ciliary and flagellar axonemes is discussed in terms of random, non-reinforcing arrangements of the dynein arms.

scholarly journals The hyaluronate synthase from a eukaryotic cell line

1993 ◽  
Vol 290 (3) ◽  
pp. 791-795 ◽  
Author(s):  
L Klewes ◽  
E A Turley ◽  
P Prehm
Keyword(s):  
Monoclonal Antibodies ◽  
Phase Separation ◽  
Affinity Chromatography ◽  
Cell Line ◽  
Aqueous Phase ◽  
Plasma Membranes ◽  
Eukaryotic Cell ◽  
Molecular Masses ◽  
Triton X ◽  
Hyaluronate Synthase

The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase.

Cholinephosphotransferase activities in microsomes and neuronal nuclei isolated from immature rabbit cerebral cortex: the use of endogenously generated diacylglycerols as substrate

1982 ◽  
Vol 60 (7) ◽  
pp. 724-733 ◽  
Author(s):  
R. Roy Baker ◽  
Huu-Yi Chang
Keyword(s):  
Phospholipase C ◽  
Microsomal Fraction ◽  
Neuronal Development ◽  
Nuclear Fraction ◽  
Neuronal Nuclei ◽  
Nuclear Membranes ◽  
Choline Phosphotransferase ◽  
Rabbit Cerebral Cortex ◽  
Low Levels ◽  
Triton X

A neuronal nuclear fraction (N1) and a microsomal fraction (P3) were isolated from homogenates of cerebral cortices of 15-day-old rabbits. A nuclear envelope fraction (E) was prepared from N1. To assay cholinephosphotransferase, diacylglycerols were first generated in the membranes of these subfractions using a phospholipase C (Bacillus cereus) preincubation. With levels of endogenous diacylglycerols producing maximal specific cholinephosphotransferase activities, an activity ratio of 1:1:5 was found for N1, P3, and E, respectively. An independent neuronal nuclear cholinephosphotransferase, concentrated in nuclear membranes, is indicated. With regard to changes in pH and concentrations of MgCl2 and CDP-choline, N1 and P3 activities responded in a similar manner. However, in contrast to P3, N1 activities were much more profoundly inhibited at low levels of Triton X-100 (0.01–0.02 w/v%) and N1 showed quite significant levels of cholinephosphotransferase activity in the absence of a phospholipase C preincubation. Choline phosphotransferase in N1 and P3 showed Km values for CDP-choline (0.028 and 0.031 mM, respectively) which were much lower than corresponding literature values determined using exogenous diacylglycerols as substrates for this enzyme. The presence of cholinephosphotransferase in neuronal nuclear membranes reflects a rather exceptional nuclear autonomy. This may be related to a need to maintain nuclear phospholipid in the absence of a well-developed endoplasmic reticulum at early stages of neuronal development or to synthesize phospholipid in response to functions unique to the nucleus.

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

1991 ◽  
Vol 32 (7) ◽  
pp. 1067-1075 ◽  
Author(s):  
Tomonori Shiraishi ◽  
Miwa Araki ◽  
Hirofumi Yoshioka ◽  
Issei Kobayashi ◽  
Tetsuji Yamada ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Plasma Membranes ◽  
Mycosphaerella Pinodes
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