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A novel adenylylation process in liver plasma membrane-bound proteins
E San José1, A Benguría, A Villalobo
1Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
The Journal of Biological Chemistry
|November 25, 1990
Summary
Rat liver plasma membranes contain five glycoproteins that bind adenosine monophosphate (AMP). One acts as a phosphoesterase, while others may have regulatory roles, suggesting complex signaling in the cell membrane.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Rat liver plasma membranes possess multiple polypeptides that interact with adenosine triphosphate (ATP).
- Understanding the function and interactions of these membrane proteins is crucial for cellular signaling research.
Purpose of the Study:
- To identify and characterize polypeptides in rat liver plasma membranes that bind adenosine triphosphate (ATP).
- To elucidate the roles of these polypeptides, distinguishing between catalytic and regulatory functions.
Main Methods:
- Incubation of plasma membranes with radiolabeled ATP ([alpha-32P]ATP and [gamma-32P]ATP).
- Identification of covalently bound adenosine monophosphate (AMP) using nonhydrolyzable ATP analogues.
- Chase experiments with nonradiolabeled phosphocompounds and inhibitor sensitivity assays.
- Electrophoretic analysis, including experiments without beta-mercaptoethanol to assess disulfide bonds.
- Triton X-100 solubilization and dilution experiments with nonhydrolyzable ATP analogues.
Main Results:
- Five distinct polypeptides (130, 120, 110, 100, and 86 kDa) were labeled with [alpha-32P]ATP and [gamma-32P]ATP.
- The 86-kDa polypeptide was identified as a phosphoesterase forming a catalytic intermediate.
- Higher molecular mass polypeptides (130, 120, 110, 100 kDa) showed slow turnover, suggesting a regulatory role for bound AMP.
- The 130-kDa polypeptide was identified as a potential target of an adenylylating system.
- Disulfide bonds were implicated in a dimeric structure (240 kDa) involving the 120 kDa and/or 110 kDa polypeptides.
- All five labeled polypeptides are glycoproteins.
Conclusions:
- Rat liver plasma membranes contain at least five adenylylated glycoproteins.
- The 86-kDa polypeptide functions catalytically as a phosphoesterase.
- The higher molecular mass adenylylated polypeptides likely play regulatory roles in cellular processes.
- Complex structural interactions, including disulfide bonds, exist among these membrane proteins.