Studies on the plasma membrane H sup + -ATPase of oat roots: Preparation and assay, cytological localization, and sulfhydryl chemistry

Miscellaneous · Sun Jan 01 00:00:00 EST 1989

OSTI ID:5613872

Katz, D B

Biochemical and cytological studies were performed on the plasma membrane proton pump (H{sup +}-ATPase) of oat roots (Avena sativa cv. Stout). H{sup +}-ATPase activity in oat root plasma membranes is inhibited by N-ethylmaleimide (NEM), a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced in the presence of ADP or MgADP. An M{sub r} = 100,000 plasma membrane polypeptide showed reduced labelling by ({sup 3}H)NEM in the presence of ADP. When tryptic peptides from ({sup 3}H)NEM-labeled M{sub r} = 100,000 polypeptide were separated by reverse-phase high-pressure liquid chromatography (HPLC), only one radioactive peak consistently showed labeling in the presence of ADP. In order to determine the location and identity of the NEM-reactive residue, the radioactive peptide in this peak was further purified by HPLC. The amino acid sequence(s) in the resulting sample were then determined by Edman degradation on an automated gas-phase sequenator. The PTH-amino acids released at each cycle of the degradation were separated by HPLC. Analysis of the chromatograms suggested that the radio-labeled residue was located in a peptide of sequence V-E-N-Q-D-A-I-D-A-C{sup *}-M-V-G-M-L-A-D-P-K. The NEM-reactive residue was cysteine, based on the retention time of the radioactivity released. The ATP-hydrolyzing activity observed in electron micrographs by lead-precipitation of enzymically released inorganic phosphate was compared with that observed in in vitro assays of the soluble and plasma membrane fractions of oat root homogenates. Although an ATP-hydrolyzing activity was observed on the plasma membrane in the electron micrographs, its substrate specificity and inhibitor sensitivity was identical to that observed for phosphatase activity.

• Research Organization: Wisconsin Univ., Madison, WI (USA)
• OSTI ID: 5613872
• Resource Relation: Other Information: Thesis (Ph. D.)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ATP-ASE
ENZYME ACTIVITY
NEM
BIOLOGICAL EFFECTS
POLYPEPTIDES
AMINO ACID SEQUENCE
ADP
BIOCHEMISTRY
FRACTIONATION
LIQUID COLUMN CHROMATOGRAPHY
MEMBRANE PROTEINS
OATS
PROTONS
ROOTS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ACID ANHYDRASES
ANTIMITOTIC DRUGS
BARYONS
CEREALS
CHEMISTRY
CHROMATOGRAPHY
DRUGS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
GRAMINEAE
HADRONS
HYDROGEN COMPOUNDS
HYDROLASES
IMIDES
ISOTOPE APPLICATIONS
LILIOPSIDA
MAGNOLIOPHYTA
MOLECULAR STRUCTURE
NUCLEONS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDES
PHOSPHOHYDROLASES
PLANTS
PROTEINS
RADIOSENSITIZERS
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques
560300 - Chemicals Metabolism & Toxicology