A study of the yeast plasma membrane proton-translocating ATPase
Four proteases have been used to assess the topology of the H{sup +} ATPase from Saccharomyces cerevisiae reconstituted into phospholipid vesicles. Limited proteolysis by trypsin and {alpha}-chymotrypsin inactivates the enzyme and produces stable, membrane-bound fragments. Sequence analyses of these peptides have located the peptide bonds hydrolyzed. The labile bonds are on opposite sides of a central hydrophilic domain containing consensus sequences for the site of phosphorylation and fluorescein isothiocyanate binding of several related ATPases. Limited proteolysis of the ATPase by elastase cuts approximately 50 amino acids from the C-terminus, leaving the remaining membrane-bound fragments active. Proteolysis by carboxy-peptidase Y suggests that the C-terminus is on the inside of the vesicle. A model for the transmembrane arrangement of the polypeptide is proposed. In this model, the C-terminus is on the inside of the vesicle, the ATP binding region is on the outside, and the polypeptide passes through the membrane a minimum of five times. Photoaffinity labeling of the active site of the enzyme has been studied with 2-azido-AMP and 2-azido-ATP. The incorporation of ({alpha}{sup {minus}32}P)-2-azido-AMP-into the enzyme and the in inhibition of ATPase activity have comparable time courses. ATP protects the ATPase from incorporation of and photoinactivation by 2-azido-ATP or 2-azido-AMP. In the dark, 2-azido-ATP inhibits the ATPase at concentrations comparable to the apparent Michaelis constant for MgATP. Purification and sequence analysis of labeled peptides indicates that both nucleotide analogues modify a highly conserved region of the ATPase including aspartate 560 through lysine 566. These residues conform to a consensus sequence for ATP binding derived from phosphofructokinase. The hydrophilic domain of the ATPase has been cloned and expressed in E. coli.
- Research Organization:
- Cornell Univ., Ithaca, NY (USA)
- OSTI ID:
- 6322540
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP-ASE
CHEMICAL COMPOSITION
AMINO ACID SEQUENCE
AMP
ATP
CELL MEMBRANES
LABELLING
MATHEMATICAL MODELS
PHOSPHORUS 32
SACCHAROMYCES CEREVISIAE
TRACER TECHNIQUES
ACID ANHYDRASES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
ENZYMES
EUMYCOTA
FUNGI
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MEMBRANES
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PHOSPHORUS ISOTOPES
PLANTS
RADIOISOTOPES
SACCHAROMYCES
YEASTS
550201* - Biochemistry- Tracer Techniques