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S Danko

Publications and source records attributed to S Danko.

5 recordsLinked to original sources
PubMed2001

Organization of cytoplasmic domains of sarcoplasmic reticulum Ca(2+)-ATPase in E(1)P and E(1)ATP states: a limited proteolysis study.

In order to characterize the domain organization of sarcoplasmic reticulum Ca(2+)-ATPase in different physiological states, limited proteolysis using three proteases (proteinase K (prtK), V8 and trypsin) was conducted systematically and quantitatively. The differences between E(2) and E(2)P were examined in our previous study and E(2)P was characterized by the complete resistance to all three proteases (except for trypsin attack at the very top of the molecule (T1 site)). The same strategies were employed in this study for E(1)ATP, E(1)PADP and E(1)P states. Because of the transient nature of these states, they were either stabilized by non-hydrolyzable analogues or made predominant by adjusting buffer conditions. Aluminum fluoride (without ADP) was found to stabilize E(1)P. All these states were characterized by strong (E(1)ATP) to complete (E(1)PADP and E(1)P) resistance to prtK and to V8 but only weak resistance to trypsin at the T2 site. Because prtK and V8 primarily attack the loops connecting the A domain to the transmembrane helices whereas the trypsin T2 site (Arg(198)) is located on the outermost loop in the A domain, these results lead us to propose that the A domain undergoes a large amount of rotation between E(1)P and E(2)P. Combined with previous results, we demonstrated that four states can be clearly distinguished by the susceptibility to three proteases, which will be very useful for establishing the conditions for structural studies.

Adenosine Monophosphate↗
PubMed2001

ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca(2+)-ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin.

Sarcoplasmic reticulum Ca(2+)-ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca(2+). Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with P(i) and phosphorylated state analogues produced by the binding of F(-) or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg(505)). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues.

Adenosine Diphosphate↗
PubMed1986

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↗
PubMed1985

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↗