Characterization of ATP-dependent Ca2+ uptake by canine brain microsomes with saponin

Abstract

ATP‐dependent Ca2+ uptake by brain microsomes was classified into two fractions according to the sensitivity to saponin. Properties of each fraction of Ca2+ uptake were examined and compared with those of inside‐out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum. The concentration of saponin for 50 % inhibition (IC50) of major saponin‐sensitive Ca2+ uptake was 11 μg/ml, and this uptake was enhanced by calmodulin. The minor saponin‐insensitive Ca2+ uptake fraction (IC50;90 μg/ml) was not affected by calmodulin but was enhanced by oxalate or 0.1 M KC1. The IC50 of saponin for inside‐out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum was 11.3 and 114.8 μg/ml, respectively. A characteristic ring‐like saponin‐cholesterol micellar structure was observed electron microscopically in most membrane vesicles of brain microsomes and erythrocyte membrane vesicles but not in the cardiac sarcoplasmic reticulum. These observations indicate that saponin‐sensitive and insensitive Ca2+ uptake was derived from plasma membranes and endoplasmic reticulum, respectively. Saponin proved useful for distinguishing the Ca2+ transport activity of plasma membrane from the Ca2+ uptake of other cellular organelles in the membrane preparations.

Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion

“…Therefore, the NC should not considerably increase the overall size the NC-eGFP marker, suggesting that small fusion pores formed by EnvA should be a few nanometers in diameter. This size is consistent with the diameter of a saponin pore (3–10 nm [51], [52]) which does not considerably restrict the release of viral content (Figure 6). …”

“…These highly variable rates of viral content release were not related to the differences in retention of NC-eGFP by individual viral cores. Indeed, saponin-treated viruses lost their green fluorescence within a few seconds after the onset of lysis (Figure 6A, cyan circles), consistent with the ability of saponin to form relatively large (3–10 nm) pores in membranes [51], [52]. These rather uniform rates of NC-eGFP release by saponin (Figure 6C, open bar) demonstrate that the vast differences in the release rates during fusion are due to the variable sizes of fusion pores which limit the efflux of viral content.…”

Physiological concentrations of inorganic phosphate affect MgATP-dependent Ca2+ Storage and inositol trisphosphate-induced Ca2+ efflux in microsomal vesicles from non-hepatic cells

“…The contribution of organelles other than the ER to Ca2+ accumulation in the different microsomal fractions appeared to be negligible. In fact, Pi-stimulated and -unstimulated Ca2+ accumulations were not modified in the presence of a mitochondrial inhibitor (5 mM NaN3, which was routinely included in the incubation system, or 2 #M carbonyl cyanide p-fluoromethoxyphenylhydrazone) and in the presence of digitonin at concentrations suitable to permeabilize plasma-membranederived vesicles [37]. Also, the reticular Ca2+-pump inhibitor thapsigargin (1 ,uM; [38]) suppressed any Ca2+ accumulation by hepatoma and platelet microsomes, and 70 % of Ca2+ accumulation by brain microsomes.…”

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells.

“…9 shows the relationship between the free Ca 21 concentration (pCa) and the Ca 21 content in the absence and presence of sodium azide. It is clear that the sodium azidesensitive Ca 21 store could accumulate significant amounts of Ca 21 when the free Ca 21 concentration exceeded 3 X 10 -6 M. DISCUSSION It is believed that, under appropriate conditions, saponin produces holes in the plasmalemma by forming micellar complexes with cholesterol, while not affecting the intracellular organelles such as the SR and mitochondria, which are thought to have a lower cholesterol content in skeletal and cardiac muscle (Comte et a1., 1976 ;Inamitsu and Ohtsuki, 1984;Lau et al, 1979;Ohtsuki et al, 1978). Although in smooth muscle the cholesterol content of the membranes of the SR and the sarcolemma is not known, the method of saponin skinning has been used successfully in some studies that have illuminated several important aspects of Ca" mobilization during activation of smooth muscle Itoh et al, 1981Itoh et al, , 1983Saida, 1982 ;Saida and van Breemen, 1984a, b).…”