Structure and function engineered Pseudomonas mendocina lipase.
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Amino Acid Substitution↗
Biomedical subjects
S Danko
Publications and source records attributed to S Danko.
Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum.
Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.
Adenosine Triphosphate↗
Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. II. The effects of dantrolene on Ca2+ release induced by caffeine, Ca2+ and depolarization.
The effects of dantrolene, which is a known muscle relaxant, on Ca2+ release from the isolated sarcoplasmic reticulum induced by several different methods [1) addition of caffeine, (2) Ca2+ jump, and (3) membrane-depolarization produced by choline chloride replacement of potassium gluconate) were investigated. Dantrolene inhibited caffeine-induced Ca2+ release with C1/2 = 2.5 microM, whereas there was no effect on Ca2+ release induced by a Ca2+ jump. The amount of Ca2+ released by depolarization was reduced if Ca2+ release was triggered in an earlier phase of the steady state of Ca2+ uptake (time elapsed between the addition of ATP and the triggering of Ca2+ release, tATP less than 4 min); while, if triggered in a latter phase (tATP greater than 4 min) dantrolene enhanced depolarization-induced Ca2+ release. C1/2 for the inhibition and that for enhancement of depolarization-induced Ca2+ release were 1.0 and 0.3 microM, respectively. These results suggest that dantrolene affects several different steps of the mechanism by which Ca2+ release is triggered. The sarcoplasmic reticulum and T-tubule membrane fractions had 7.9 nmol dantrolene-binding sites/mg (Kassoc = 1.0 X 10(5) M-1) and 21.0 nmol/mg (Kassoc = 1.1 X 10(5) M-1), respectively. The time-course of dantrolene binding to sarcoplasmic reticulum was monophasic, while that to T-tubules was biphasic.
Animals↗