Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L. ) storage tissue

Journal Article · Fri May 01 00:00:00 EDT 1987 · Arch. Biochem. Biophys.; (United States)

DOI:https://doi.org/10.1016/0003-9861(87)90145-7· OSTI ID:6351278

Giannini, J L; Gildensoph, L H; Briskin, D P

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.

• Research Organization: Univ. of Illinois, Urbana
• OSTI ID: 6351278
• Journal Information: Arch. Biochem. Biophys.; (United States), Vol. 2
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
59 BASIC BIOLOGICAL SCIENCES
BEETS
MEMBRANE TRANSPORT
NITRATES
BIOLOGICAL EFFECTS
VANADIUM
ATP
CELL MEMBRANES
ION CHANNELING
MICROSOMES
PLANTS
POTASSIUM CHLORIDES
POTASSIUM IODIDES
PROTONS
SODIUM IODIDES
VEGETABLES
ALKALI METAL COMPOUNDS
BARYONS
CELL CONSTITUENTS
CHANNELING
CHLORIDES
CHLORINE COMPOUNDS
ELEMENTARY PARTICLES
ELEMENTS
FERMIONS
FOOD
HADRONS
HALIDES
HALOGEN COMPOUNDS
INORGANIC PHOSPHORS
IODIDES
IODINE COMPOUNDS
MEMBRANES
METALS
NITROGEN COMPOUNDS
NUCLEONS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANOIDS
OXYGEN COMPOUNDS
PHOSPHORS
POTASSIUM COMPOUNDS
SODIUM COMPOUNDS
TRANSITION ELEMENTS
560300* - Chemicals Metabolism & Toxicology
550200 - Biochemistry