Characterization and partial purification of a proton translocating ATPase from corn coleoptile tonoplasts
ATP-dependent proton translocating activity in microsomal membranes from corn coleoptiles was characterized. Proton pumping activity, detected by either /sup 14/C-methylamine uptake or quinacrine fluorescence quenching, had a broad optimum at pH 7.5, and was substrate specific for MgATP. N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and protonophores were found to inhibit proton transport, while vanadate and oligomycin had little effect. Proton pumping activity was stimulated 10 fold with Cl/sup -/ but was almost completely inhibited by 50 mM, KNO/sub 3/. Uptake studies with /sup 36/Cl/sup -/ indicated the Cl/sup -/ was transported into the microsomal vesicles in response to the pH gradient. ATP-stimulated proton pumping activity was localized on linear density gradients. On sucrose gradients, the activity cosedimented with the marker for endoplasmic reticulum at 1.11 g/cc. Sucrose gradients prepared in the presence of MgCl/sub 2/ were used to shift the ER marker to a heavier density, away from proton pumping activity. Linear dextran gradients also resulted in a clear separation of ATP-stimulated methylamine, thiocyanate, and /sup 36/Cl/sup -/ uptake, from markers for ER, Golgi, mitochondria, and plasma membranes. The tonoplast ATPase was solubilized with octylglucoside and partially purified on linear sucrose gradients. The specific activity of the KNO/sub 3/-sensitive ATPase increased 30-fold during purification.
- Research Organization:
- California Univ., Santa Cruz (USA)
- OSTI ID:
- 6105301
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ATP-ASE
ENZYME ACTIVITY
PURIFICATION
CHLORINE COMPOUNDS
MEMBRANE TRANSPORT
DIAZO COMPOUNDS
BIOLOGICAL EFFECTS
IMIDES
MAGNESIUM
POTASSIUM COMPOUNDS
STILBESTROL
VANADIUM
CARBON 14 COMPOUNDS
CHLORINE 36
COLEOPTILE
MAIZE
MICROSOMES
PROTONS
TRACER TECHNIQUES
ACID ANHYDRASES
ALKALI METAL COMPOUNDS
ALKALINE EARTH METALS
AROMATICS
BARYONS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
CEREALS
CHLORINE ISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELEMENTARY PARTICLES
ELEMENTS
ENZYMES
FERMIONS
GRASS
HADRONS
HALOGEN COMPOUNDS
HYDROLASES
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
METALS
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PHENOLS
PHOSPHOHYDROLASES
PLANTS
POLYPHENOLS
RADIOISOTOPES
TRANSITION ELEMENTS
YEARS LIVING RADIOISOTOPES
560301* - Chemicals Metabolism & Toxicology- Cells- (-1987)
550201 - Biochemistry- Tracer Techniques