Isotopic and spectrophotometric assays show that micromolar concentrations of heavy metal ions (particularly Ag⁺ and Hg²⁺) stimulate ATPase activity but inhibit Ca²⁺ uptake in isolated SR vesicles. Both effects are caused by increased Ca²⁺ permeability of the membrane, apparently the result of activation of the Ca²⁺ release system of the vesicles due to heavy metal binding to a critical sulfhydryl group associated with the Ca²⁺ release channels. CU²⁺catalyzed co-oxidation of this sulfhydryl with exogenous cysteine to form a mixed disulfide also results in activation of the Ca²⁺ release system. The rate and extent of Cu²⁺/cysteine-induced release is maximal at physiological pH and is inhibited by local anaesthetics and Mg²⁺, suggesting that the redox state of this sulfhydryl may play a role in activation of the Ca²⁺ release system of intact muscle. Modification of the SR with the proteolytic enzyme trypsin also increases the Ca²⁺ permeability of the SR, and enhances the rate of Ca²⁺ release activated by cAMP, doxorubicin, Hg²⁺, and Cu²⁺/cysteine. The rates of release activated by all reagents are stimulated by a factor of five after five minutes exposure to trypsin. Hg²⁺- and Cu²⁺/cysteine-activated release are not stimulated further, while cAMP- and doxorubicin-activated release continue to increase up to maximum of 20-fold stimulation after 15 minutes exposure to trypsin. Inhibitors of the Ca²⁺ release system such as Mg²⁺ and ruthenium red still inhibit release from proteolytically modified SR, and the binding affinities of activators and inhibitors to their sites are not significantly altered by proteolysis; only the rates of Ca²⁺ transport are affected. The most probable mechanisms of tryptic stimulation of Ca²⁺ release are (1) removal of a regulatory protein or subunit of the Ca²⁺ release system, making more channels available for transport; (2) increasing the single channel unitary conductance; (3) increasing the open time of activated channels. The biphasic character of proteolytic stimulation of cAMP- and doxorubicin-activated release (as opposed to monophasic stimulation of Hg²⁺- and Cu²⁺/cysteine-activated release} suggests that more than one of the above parameters are involved in tryptic stimulation of the Ca²⁺ release system.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-2347 |
| Date | 01 January 1988 |
| Creators | Trimm, Jonathan Lee |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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