Azumolene inhibits a component of store-operated calcium entry coupled to the skeletal muscle ryanodine receptor.

Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca(2+) entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.

Critical segment of apocytochrome c for its insertion into membrane.

Apocytochrome c has a potent ability to insert spontaneously into membrane. To identify which sequences were critical for this insertion activity, a series of peptides N19, C8, C15 and C21, corresponding to sequences 1-19, 81-88, 74-88 and 68-88 of apocytochrome c, respectively, were synthesized and purified. Insertion ability into phospholipid monolayer, intrinsic fluorescence emission spectra, and the accessibility of peptide C21 to fluorescence quenchers: KI, acrylamide and HB showed that only segment 68-88 could insert into membrane, while other segments did not. CD spectra demonstrated that its interaction with liposomes containing negatively charged phospholipid could induce a partial alpha-helical conformation in peptide C21. It is interesting to note that a cooperation exists between segment 68-88 and 1-19 in the insertion of apocytochrome c and consequently translocation across membrane.

Peptide 68-88 of apocytochrome c plays a crucial role in its insertion into membrane and binding to mitochondria.

Apocytochrome c (Apocyt. c) is the precursor of cytochrome c. It is synthesized in the cytosol and posttranslationally imported into mitochondria. In order to determine the crucial sequence in apocyt. c translocation, deleted mutant and chemically synthesized peptides with different length were used. Obtained results showed that sequence 68-88 of apocyt. c plays a critical role in its insertion into membrane and binding to mitochondria.

Change of apocytochrome c translocation across membrane in consequence of hydrophobic segment deletion.

Wild-type apocytochrome c and its hydrophobic segment deleted mutants, named delta28-39, delta72-86 and delta28-29/72-86 were constructed, expressed and highly purified respectively. Insertion ability into phospholipid monolayer, inducing leakage of entrapped fluorescent dye fluorescein sulfonate (FS) from liposomes, and translocation across model membrane system showed that the wild-type apoprotein and delta28-39 almost exhibited the same characteristics, while mutants with segment 72-86 deletion did not. Furthermore, CD spectra, intrinsic fluorescence emission spectra, and the accessibility of the protein to the fluorescence quenchers: KI, acrylamide and HB demonstrated that the segment 72-86 deletion has a significant effect on the conformational changes of apocytochrome c following its interaction with phospholipid. On the basis of these results it is postulated that the C-terminal hydrophobic segment 72-86 plays an important role in the translocation of apocytochrome c across membrane.

Studies on the Conformation of Apocytochrome c in Different Folding States Inserted into the Membranes.

Correlation between the folding states of the chicken heart apocytochrome c and its membrane insertion ability was studied by the monolayer experiment. The penetration ability of apocytochrome c decreased with its folding. Intrinsic fluorescence emissions of apocytochrome c interacted with soybean phospholipids liposomes suggested that the conformations of apocytochrome c with different folding states in aqueous solution were also different in the membranes. The conformational differences were further characterized by CD spectra of apocytochrome c interacted with DMPC and DMPG liposomes. The results showed that apocytochrome c, which were randomly coiled in aqueous solution, adopted alpha-helical conformations after interaction with DMPG liposome. The apocytochrome c with a folded state in aqueous solution also adopted alpha-helical conformations, but the alpha-helical contents were less than the former. When DMPC liposomes were used, no distinct conformational change was observed.

The Self-association of Melittin at Low Concentration in Aqueous Solution.

The intrinsic fluorescence emission, polarization and Trp-19 quenching of melittin in the aqueous solution showed that even with concentration as low as 2 &mgr;M, the self-association of melittin still occurred. The transition preferred to occur under acidic condition and the stability of the associated melittin was dependent on the pH value of the solution.