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

Abstract

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 bymore » 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.« less

Authors:
Publication Date:
Research Org.:
Wisconsin Univ., Madison, WI (USA)
OSTI Identifier:
5613872
Resource Type:
Miscellaneous
Resource Relation:
Other Information: Thesis (Ph. D.)
Country of Publication:
United States
Language:
English
Subject:
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

Citation Formats

Katz, D B. Studies on the plasma membrane H sup + -ATPase of oat roots: Preparation and assay, cytological localization, and sulfhydryl chemistry. United States: N. p., 1989. Web.
Katz, D B. Studies on the plasma membrane H sup + -ATPase of oat roots: Preparation and assay, cytological localization, and sulfhydryl chemistry. United States.
Katz, D B. Sun . "Studies on the plasma membrane H sup + -ATPase of oat roots: Preparation and assay, cytological localization, and sulfhydryl chemistry". United States.
@article{osti_5613872,
title = {Studies on the plasma membrane H sup + -ATPase of oat roots: Preparation and assay, cytological localization, and sulfhydryl chemistry},
author = {Katz, D B},
abstractNote = {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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1989},
month = {1}
}

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