• 대한약리학회지
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• 제26권1호
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• pp.35-49
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• 1990

A highly purified $(Na^+,\;K^+)-ATPase$ from the rectal gland of Squalus acanthias and from the electric organ of Electrophorus electricus has been used to raise antibodies in rabbits. The 97,000 dalton catalytic subunit and glycoprotein derived from the rectal gland of spiny shark were also used as antigens. The two $(Na^+,\;K^+)-ATPase$ holoenzymes and the two shark subunits were antigenic. In Ouchterlony double diffusion experiments, these antibodies formed precipitation bands with their antigens. Antibodies prepared against the two subunits of shark holoenzyme also formed precipitation bands with their antigens and shark holoenzyme, but not with eel holoenzyme. These observations are in good agreement with inhibitory effect of these antibodies on the catalytic activity of $(Na^+,\;K^+)-ATPase$ both from the shark and the eel, since there is very little cross-reaction between the shark anticatalytic subunit antibodies and the eel holoenzyme. The maximum antibodies titer of the anticatalytic subunit antibodies is found to be 6 weeks after the initial single exposure to this antigen. Multiple injections of the antigen increased the antibody titer. However, the time required to produce the maximum antibody titer was approximately the same. These antibodies also inhibit catalytic activity of $(Na^+,\;K^+)-ATPase$ vesicles reconstituted by a slow dialysis of cholate after solubilization of the enzyme in a presonicated mixture of cholate and phospholipid. In these reconstituted $(Na^+,\;K^+)-ATPase$ vesicles, effects of these antibodies on the fluxes of $Na^+$, $Rb^+$, and $K^+$ were investigated. Control or preimmune serum had no effect on the influx of $^{22}Na^+$ or the efflux of $^{86}Rb^+$. Immunized sera against the shark $(Na^+,\;K^+)-ATPase$ holoenzyme, its glycoprotein or catalytic subunit did inhibit the influx of $^{22}Na^+$ and the efflux of $^{86}Rb^+$. It was also demonstrated that these antibodies inhibit the coupled counter-transport of $Na^+$ and $K^+$ as studied by means of dual labeling experiments. However, this inhibitory effect of the antibodies on transport of ions in the $(Na^+,\;K^+)-ATPase$ vesicles is manifested only on the portion of energy and temperature dependent alkali metal fluxes, not on the portion of ATP and ouabain insensitive ion movement. Simultaneous determination of effects of the antibodies on ion fluxes and vesicular catalytic activity indicates that an inhibition of active ion transport in reconstituted $(Na^+,\;K^+)-ATPase$ vesicles appears to be due to the inhibitory action of the antibodies on the enzymatic activity of $(Na^+,\;K^+)-ATPase$ molecules incorporated in the vesicles. These findings that the inhibitory effects of the antibodies specific to $(Na^+,\;K^+)-ATPase$ or to its subunits on ATP and temperature sensitive monovalent cation transport in parallel with the inhibitory effect of vesicular catalytic activity by these antibodies provide direct evidence that $(Na^+,\;K^+)-ATPase$ is the molecular machinery of active cation transport in this reconstituted $(Na^+,\;K^+)-ATPase$ vesicular system.

• Journal of Ginseng Research
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• 제45권2호
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• pp.228-235
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• 2021

Backgroud: Ginsenoside compound K (GK) is a major metabolite of protopanaxadiol-type ginsenosides and has remarkable anticancer activities in vitro and in vivo. This work used an ionic cross-linking method to entrap GK within O-carboxymethyl chitosan (OCMC) nanoparticles (Nps) to form GK-loaded OCMC Nps (GK-OCMC Nps), which enhance the aqueous solubility and stability of GK. Methods: The GK-OCMC Nps were characterized using several physicochemical techniques, including x-ray diffraction, transmission electron microscopy, zeta potential analysis, and particle size analysis via dynamic light scattering. GK was released from GK-OCMC Nps and was conducted using the dialysis bag diffusion method. The effects of GK and GK-OCMC Nps on PC3 cell viability were measured by using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Fluorescent technology based on Cy5.5-labeled probes was used to explore the cellular uptake of GK-OCMC Nps. Results: The GK-OCMC NPs had a suitable particle size and zeta potential; they were spherical with good dispersion. In vitro drug release from GK-OCMC NPs was pH dependent. Moreover, the in vitro cytotoxicity study and cellular uptake assays indicated that the GK-OCMC Nps significantly enhanced the cytotoxicity and cellular uptake of GK toward the PC3 cells. GK-OCMC Nps also significantly promoted the activities of both caspase-3 and caspase-9. Conclusion: GK-OCMC Nps are potential nanocarriers for delivering hydrophobic drugs, thereby enhancing water solubility and permeability and improving the antiproliferative effects of GK.