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Author Scaglione, Christine N. ♦ Xu, Qijin ♦ Ramanujan, V. Krishnan
Source United States Department of Energy Office of Scientific and Technical Information
Content type Text
Language English
Subject Keyword APPLIED LIFE SCIENCES ♦ AGING ♦ ANIMAL TISSUES ♦ ANTIOXIDANTS ♦ BIOPSY ♦ CALORIMETRY ♦ CATALASE ♦ DECISION MAKING ♦ ENZYME ACTIVITY ♦ IN VITRO ♦ LIVER ♦ MAMMARY GLANDS ♦ MICE ♦ NEOPLASMS ♦ OXIDATION ♦ OXYGEN ♦ REACTION KINETICS ♦ SUBSTRATES ♦ SURGERY
Abstract Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear demonstration of the applicability in cancer cells and aging animal tissues.
ISSN 0006291X
Educational Use Research
Learning Resource Type Article
Publisher Date 2016-01-29
Publisher Place United States
Journal Biochemical and Biophysical Research Communications
Volume Number 470
Issue Number 1


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