Characterization of tonoplast-enriched vesicles isolated by gradient density fractionation of suspension-cultured cells.
A tonoplast-enriched microsomal fraction was isolated from Catharanthus roseus cells. It was characterized by structural and functional criteria. This fraction presented a homogeneous size distribution as shown by quasi-elastic light scattering and a homogeneous density on self-generated gradients of Percoll. The mean diameter of the vesicles was estimated to be 0.30 micron and the buoyant density around 1.04 g/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of its polypeptide pattern was in good agreement with the one obtained for tonoplast purified from isolated vacuoles. According to enzymatic assays and inhibition tests, this fraction possessed pyrophosphate and ATP-dependent proton pumps and very low contamination by submitochondrial particles, endoplasmic reticula and Golgi membranes. In light of our previous published results on tonoplast purified from isolated vacuoles, the very low extent of AMP hydrolysis by the microsomal fraction is interpreted as supplementary proof in favor of a tonoplast-enriched fraction.
Dupaix
A
A
U.R.A. 1116 du C.N.R.S., Université de Paris-Sud, Centre d'Orsay, France.
Guyen
L
L
Hill
M
M
Arrio
B
B
D000251:Adenosine Triphosphatases / Q000032:analysis; D015415:Biomarkers; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology; D010944:Plants / Q000187:drug effects / Q000378:metabolism*; D017680:Potassium Compounds; D011506:Proteins / Q000378:metabolism*
D015415:Biomarkers; D009566:Nitrates; D017680:Potassium Compounds; D011506:Proteins; C031618:sodium nitrate; D009699:N-Glycosyl Hydrolases; C019599:adenosine nucleosidase; D000251:Adenosine Triphosphatases; C023844:potassium nitrate