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Purification and enzymatic characterization of pp60c-src from human platelets
1Department of Microbiology and Immunology, University of California Medical Center, San Francisco 94143.
The Journal of Biological Chemistry
|May 15, 1990
Summary
Researchers purified pp60c-src protein from human platelets, overcoming previous challenges. This breakthrough enables detailed study of its enzymatic properties, advancing our understanding of this crucial protein.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- pp60c-src protein purification is challenging due to low cellular levels and proteolysis.
- Platelets offer a high-abundance source for pp60c-src, enabling large-scale purification.
Purpose of the Study:
- To develop a method for purifying intact pp60c-src from human platelets.
- To characterize the enzymatic properties of purified pp60c-src in vitro.
Main Methods:
- A two-step protocol involving immunoaffinity chromatography and phenyl-Sepharose was employed.
- Purification was performed on isolated platelet membranes for high yield.
Main Results:
- Near-homogenous pp60c-src was obtained with a specific activity of 25 nmol P/min/mg using enolase as a substrate.
- Kinetic analysis revealed distinct Km values for enolase phosphorylation and autophosphorylation.
- Enzyme activity was dependent on Mn2+ or Mg2+, and both ATP and GTP served as phosphate donors.
Conclusions:
- A robust method for purifying pp60c-src from human platelets was established.
- The study provides insights into the enzymatic characteristics and autophosphorylation mechanism of pp60c-src.