Take advantage of time in your experiments: a guide to simple, informative kinetics assays

Understanding virtually any process in cellular and molecular biology depends on knowledge of the rates of the biochemical reactions, so it is regrettable that few cellular and molecular biologists take advantage of kinetics experiments in their work. Fortunately, the kinetics experiments that are most useful for understanding cellular systems are within reach for everyone whose research would benefit from this information. This essay describes simple methods to measure the valuable kinetic parameters that characterize the dynamics of life processes. These “transient-state” methods not only differ in concept from traditional approaches used to analyze enzyme reactions at steady state, but they are also applicable to learning about the dynamics of any biological process.

[1]  W. Cao,et al.  The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA. , 2008, Journal of molecular biology.

[2]  Thomas D. Pollard,et al.  A Guide to Simple and Informative Binding Assays , 2010, Molecular biology of the cell.

[3]  T. Pollard,et al.  Energetic Requirements for Processive Elongation of Actin Filaments by FH1FH2-formins* , 2009, Journal of Biological Chemistry.

[4]  J. Mitchison Cell Biology , 1964, Nature.

[5]  Carl Frieden Polymerization of actin: mechanism of the Mg2+-induced process at pH 8 and 20 degrees C. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[6]  E Michael Ostap,et al.  Kinetic and Equilibrium Analysis of the Myosin Atpase , 2022 .

[7]  K. Kovács,et al.  Properties of enzymes. IV. Non-specific superoxide dismutase activity of iron-porphyrin systems. , 1975, Enzyme.

[8]  W. Cao,et al.  Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA , 2010, Proceedings of the National Academy of Sciences.

[9]  R. Goody,et al.  The original Michaelis constant: translation of the 1913 Michaelis-Menten paper. , 2011, Biochemistry.

[10]  Huan‐Xiang Zhou,et al.  Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. , 2008, Annual review of biophysics.

[11]  T. Pollard,et al.  Transient kinetic analysis of rhodamine phalloidin binding to actin filaments. , 1994, Biochemistry.

[12]  Kenneth A. Johnson,et al.  1 Transient-State Kinetic Analysis of Enzyme Reaction Pathways , 1992 .

[13]  E. M. De La Cruz,et al.  Kinetic analysis of the guanine nucleotide exchange activity of TRAPP, a multimeric Ypt1p exchange factor. , 2009, Journal of molecular biology.

[14]  E. M. De La Cruz,et al.  Analyzing ATP utilization by DEAD-Box RNA helicases using kinetic and equilibrium methods. , 2012, Methods in enzymology.

[15]  T. Pollard,et al.  The Role of the FH1 Domain and Profilin in Formin-Mediated Actin-Filament Elongation and Nucleation , 2008, Current Biology.

[16]  E. M. De La Cruz,et al.  Vertebrate Myosin VIIb Is a High Duty Ratio Motor Adapted for Generating and Maintaining Tension* , 2005, Journal of Biological Chemistry.