Method of quantitating GTP and GDP bound to a G protein and uses thereof

US5741635A · kind A · utility

Assignees

• MOUNT SINAI HOSPITAL · CA
• The Regents of the University of California · US

Inventors

• Gerry Boss · Village of La Jolla, US
• Abhijit Guha · Mississauga, CA
• Jürgen Scheele · Wildeshausen, DE
• Anthony Pawson · Toronto, CA

Key dates

• Filing date: Jan 30, 1996
• Grant date: Apr 21, 1998
• Expiry date: Jan 30, 2016

Classification

• Technology area (CPC G): Physics
• CPC primary: G01N33/6893
• WIPO field: Measurement
• WIPO sector: Instruments

Abstract

The invention features methods for measuring the total amounts of G protein-bound GDP and GTP in mammalian cells and tissue. The method of the invention is advantageous over conventional methods in that the cells need not be exposed to phosphate-free growth conditions, which can adversely affect the accuracy of the GTP determination. Moreover, the method does not require exposure of the cells to radiolabelled phosphate in the growth medium. Standardized amounts of G protein-bound GDP and GTP are determined relative to non-nuclear cellular protein content or cellular DNA content, allowing comparison of results across different cell types. The method is particularly applicable to the detection of Ras protein bound GTP and GDP. The determination of Ras-bound GTP levels by the method of the invention provides a sensitive and accurate means of evaluating the role of Ras activation in cellular processes, such as cell proliferative disease, hormonal secretion, cellular differentiation, programmed cell death, cell cycle regulation, and protein trafficking. Treatment of abnormal processes can then more accurately targeted to the alteration of G protein activation.