Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50μl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev. Sci. Instrum. 47, 1383–1393]. This equipment can apply pressure changes of ±20MPa (maximally) in time periods as short as 80μs and follow the resulting change in fluorescence signals. The system is relatively simple to use with fast (approx. 1min) exchange of samples. In the present study, we show that this system can perturb the binding of 2′(3′)-O-(N-methylanthraniloyl)-ADP to myosin subfragment-1(S1) from skeletal and smooth muscles. The kinetic data are consistent with previous work, and in addition show that, although 2′(3′)-O-(N-methylanthraniloyl)-ADP binds with a similar affinity to both proteins, the increase in molar volume for the skeletal-muscle S1 binding to ADP is half of that for the smooth-muscle protein. This high-volume change for smooth-muscle S1 may be related to the ability of ADP to induce a 23° tilt in the tail of S1 bound to actin.
Skip Nav Destination
Article navigation
September 2002
- PDF Icon PDF LinkFront Matter
Research Article|
September 01 2002
A novel pressure-jump apparatus for the microvolume analysis of protein–ligand and protein–protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1
David S. PEARSON;
David S. PEARSON
∗Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
Search for other works by this author on:
Georg HOLTERMANN;
Georg HOLTERMANN
†Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany,
Search for other works by this author on:
Patricia ELLISON;
Patricia ELLISON
‡Department of Biochemistry, University of Nevada, Reno, NV, U.S.A.
Search for other works by this author on:
Christine CREMO;
Christine CREMO
‡Department of Biochemistry, University of Nevada, Reno, NV, U.S.A.
Search for other works by this author on:
Michael A. GEEVES
Michael A. GEEVES
1
∗Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
1To whom correspondence should be addressed (e-mail M.A.Geeves@ukc.ac.uk).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
March 25 2002
Revision Received:
May 09 2002
Accepted:
May 15 2002
Accepted Manuscript online:
May 15 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (2): 643–651.
Article history
Received:
March 25 2002
Revision Received:
May 09 2002
Accepted:
May 15 2002
Accepted Manuscript online:
May 15 2002
Citation
David S. PEARSON, Georg HOLTERMANN, Patricia ELLISON, Christine CREMO, Michael A. GEEVES; A novel pressure-jump apparatus for the microvolume analysis of protein–ligand and protein–protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1. Biochem J 1 September 2002; 366 (2): 643–651. doi: https://doi.org/10.1042/bj20020462
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.