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[Proteins of bacterial membranes. H+-adenosinetriphosphatase from Acholeplasma laidlawii cells]
Abstract:
The cytoplasmic membrane of micoplasmic cells, in particular of A. laidlawii cells, contains a proton-carrier Mg2+ -activated ATPase. A whole H+ -ATPase complex (F0-F1) was isolated from these cells and characterized. The isolation procedure included solubilization of the enzyme with Triton X-100 followed by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The enzyme was inhibited by dicyclohexylcarbodiimide (10(-4) M). The Km value for ATP hydrolysis and Ki for ADP hydrolysis were determined. The order of the constants did not differ from those measured earlier for factor F1 of the complex. The purified enzyme, similar to its hydrophylic moiety is sensitive to the action of bivalent cations. The subunit composition of the whole complex and of its water-soluble part was investigated. The complex was found to contain 11 polypeptides, five of which belong to factor F1. The molecular weights of these polypeptides were determined.
Insights
Researchers isolated and characterized a proton-carrier Mg2+-activated ATPase (H+-ATPase) complex from Mycoplasma. This enzyme, crucial for cellular energy, was purified and its properties analyzed.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Mycoplasma cytoplasmic membranes contain a Mg2+-activated ATPase involved in proton transport.
- Understanding the structure and function of this H+-ATPase complex is key to cellular energy dynamics.
Purpose of the Study:
- To isolate and characterize the complete H+-ATPase (F0-F1) complex from A. laidlawii cells.
- To investigate the enzyme's sensitivity to inhibitors and bivalent cations.
- To determine the subunit composition and molecular weights of the complex.
Main Methods:
- Enzyme isolation via Triton X-100 solubilization, DEAE-cellulose ion-exchange chromatography, and Sepharose 6B gel filtration.
- Enzyme characterization including inhibition studies with dicyclohexylcarbodiimide.
- Determination of kinetic parameters (Km for ATP hydrolysis, Ki for ADP hydrolysis).
- Analysis of subunit composition using gel electrophoresis.
Main Results:
- The whole H+-ATPase complex was successfully isolated and purified.
- The enzyme was inhibited by dicyclohexylcarbodiimide and sensitive to bivalent cations.
- Kinetic properties were consistent with previously studied F1 components.
- The complex comprises 11 polypeptides, with 5 belonging to the F1 moiety.
Conclusions:
- The study provides a detailed characterization of the A. laidlawii H+-ATPase complex.
- The findings contribute to understanding proton transport mechanisms and energy transduction in mycoplasmic cells.
- The subunit composition analysis offers insights into the complex's structure-function relationship.