| Effects of Channel Cytoplasmic Regions on the Activation Mechanisms of Cardiac versus Skeletal Muscle Na Channels Biophysical Journal, Volume 77, Issue 6, 1 December 1999, Pages 2999-3009 Eric S. Bennett Abstract Functional comparison of skeletal muscle (rSkM1) and cardiac (hH1) voltage-gated sodium channel isoforms expressed in Chinese hamster ovary cells showed rSkM1 half-activation () and inactivation () voltages 7 and 10mV more depolarized than hH1 and , respectively. Internal papain perfusion removed fast inactivation from each isoform and caused a 20-mV hyperpolarizing shift in hH1 , with an insignificant change in rSkM1 . Activation voltage of the inactivation-deficient hH1 mutant, hH1Q3, was nearly identical to wild-type hH1 , both before and after papain treatment, with hH1Q3 also shifted by nearly 20mV after internal papain perfusion. These data indicate that while papain removes both hH1 and rSkM1 inactivation, it has a second effect only on hH1 that causes a shift in activation voltage. Internal treatment with an antibody directed against the III-IV linker essentially mimicked papain treatment by removing some inactivation from each isoform and causing a 12-mV shift in hH1 , while rSkM1 remained constant. This suggests that some channel segment within, near, or interacting with the III-IV linker is involved in establishing hH1 activation voltage. Together the data show that rSkM1 and hH1 activation mechanisms are different and are the first to suggest a role for a cytoplasmic structure in the voltage-dependent activation of cardiac sodium channels. Abstract | Full Text | PDF (206 kb) |
| Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy Biophysical Journal, Volume 60, Issue 3, 1 September 1991, Pages 623-628 Z.A. Ping and D.A. Butterfiel Abstract A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme. Abstract | PDF (589 kb) |
| Design, Synthesis, and Evaluation of In Vivo Potency and Selectivity of Epoxysuccinyl-Based Inhibitors of Papain-Family Cysteine Proteases Chemistry & Biology, Volume 14, Issue 5, 29 May 2007, Pages 499-511 Amir Masoud Sadaghiani, Steven H.L. Verhelst, Vasilena Gocheva, Kimberly Hill, Eva Majerova, Sherman Stinson, Johanna A. Joyce and Matthew Bogyo Summary The papain-family cathepsins are cysteine proteases that are emerging as promising therapeutic targets for a number of human disease conditions ranging from osteoporosis to cancer. Relatively few selective inhibitors for this family exist, and the in vivo selectivity of most existing compounds is unclear. We present here the synthesis of focused libraries of epoxysuccinyl-based inhibitors and their screening in crude tissue extracts. We identified a number of potent inhibitors that display selectivity for endogenous cathepsin targets both in vitro and in vivo. Importantly, the selectivity patterns observed in crude extracts were generally retained in vivo, as assessed by active-site labeling of tissues from treated animals. Overall, this study identifies several important compound classes and highlights the use of activity-based probes to assess pharmacodynamic properties of small-molecule inhibitors in vivo. Summary | Full Text | PDF (1484 kb) |
Copyright © 1980 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 30, Issue 1, 99-118, 1 April 1980
doi:10.1016/S0006-3495(80)85079-X
Research Article
T.R. Chay
Based on Eyring's multibarrier activation process, a mathematical model and equation is developed to account for proton diffusion through an immobilized protein and enzyme membrane perfused with an electrolyte, substrate, and a buffer. With this model we find that, in the presence of a buffer, our solution approaches the continuum case very rapidly. We apply our model to membranes composed of papain and bovine serum albumin and find that our theory closely stimulates the experimental observations on the effect of salt and buffer on proton diffusion. Our theory shows that the pH oscillations observed in the diffusion controlled papain-benzoyl-L-arginine ethyl ester (BAEE) reaction may be the result of CO2 dissolved in the bath at high pH. In our theory, under certain conditions and in agreement with experimental observation, the buffer penetration depth oscillates near the boundary of a papain membrane in a solution containing BAEE and borate. We also find that at low ionic strength small ions as well as a buffer are seen to oscillate if a membrane is highly charged.