Purification of the N,N'-dicyclohexylcarbodiimide-binding proteolipid of a higher plant tonoplast H+-ATPase

Journal Article · Sun Oct 25 04:00:00 EST 1987 · J. Biol. Chem.; (United States)

OSTI ID:5784410

Rea, P A; Griffith, C J; Sanders, D

The H+-ATPase of Beta vacuolar membrane (tonoplast) comprises at least three functionally distinct subunits of Mr = 67,000, 57,000, and 16,000, respectively. The hydrophobic carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the enzyme with pseudo-first order kinetics, and the concentration dependence of the reaction indicates that DCCD interacts with a single site on the enzyme to exert its inhibitory effect. The apparent pseudo-first order rate constant (k0) is reciprocally dependent on membrane protein concentration, which is expected if a large fraction of the DCCD partitions into the lipid phase. k0 has a nominal value of 1000 M-1 min-1 at a protein concentration of 250 micrograms/ml, although when phase partitioning is taken into account, the true, protein concentration-independent value of k0 is calculated to be about an order of magnitude lower. (/sup 14/C)DCCD primarily labels the Mr = 16,000 polypeptide of native tonoplast vesicles. Binding is venturicidin-insensitive and occurs at a rate similar to the rate of enzyme inactivation, implying that inhibition is a direct result of covalent modification of the Mr = 16,000 polypeptide. Labeling of the containing Mr = 8,000 subunit of mitochondrial F0F1-ATPase is, on the other hand, faster by a factor of 5 and totally abolished by venturicidin. These results confirm that the Mr = 16,000 polypeptide which copurifies with tonoplast H+-ATPase activity is a subunit of the enzyme. Most of the DCCD-reactive Mr = 16,000 subunit is extracted from acetone:ethanol-washed tonoplast vesicles by chloroform:methanol. (/sup 14/C)DCCD bound to the Mr = 16,000 polypeptide is enriched in the chloroform:methanol extract by 5-fold compared with native tonoplast and the specific activity (nmol of (/sup 14/C)DCCD/mg of protein) can be increased a further 37-fold by chromatography on DEAE-Sephadex.

• Research Organization: Univ. of York, Heslington, England
• OSTI ID: 5784410
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 262:30; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ATP-ASE
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CELL MEMBRANES
CHROMATOGRAPHY
ENZYME ACTIVITY
ENZYMES
EXTRACTION
FRACTIONATION
HYDROLASES
INHIBITION
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIPIDS
MEMBRANES
MOLECULAR WEIGHT
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PLANTS
REACTION KINETICS
SEPARATION PROCESSES
SOLUBILITY
SOLVENT EXTRACTION
TRACER TECHNIQUES