LIPOSOME-INDUCED RELEASE OF CELL-MEMBRANE PROTEINS FROM INTACT TISSUEEPITHELIUM

During extraction and purification, membrane proteins very often under go denaturation and deactivation. To overcome this problem, the author s have tried to establish a better methodology to make the study at in vivo tissue level, not at the isolated cellular level, possible and e asier. This is in vivo direct exposure of animal tissue to the liposom e that contains an artificial boundary lipid (D14DPC, 1,2-dimyristamid o-1,2, deoxyphosphatidylcholine). Bullfrog and rat tongues were used. To confirm the reasonableness of this methodology, several different t echniques were adopted; the nerve response study, gel electrophoretic analysis, quartz crystal microbalance (QCM) measurement and the affini ty gelchromatography. When the tongue was exposed to the D14DPC-contai ning DMPC liposome, a significant amount of membrane protein was found in the recovered liposome (this was the production of proteoliposome) . The nerve response in the neurophysiological measurement to several taste stimuli, such as L-alanine, L-leucine, sucrose and quinine hydro chloride significantly decreased when the tongue was exposed to the sa me liposome. These phenomena were common to both bullfrog and rat tong ues. The nerve response to the stimulation with L-alanine was the most remarkably affected in the liposomal treatment. Therefore, the L-alan ine-binding protein was focused upon to confirm the reasonableness of the QCM measurement and the affinity gelchromatography. The D14DPC-con taining proteoliposome always showed significant binding to both the L -alanine affinity gel and the L-alanine-conjugated QCM. The results re vealed that membrane proteins can be directly and effectively released , even from intact animal tissue epithelium, using the artificial boun dary lipid-containing liposome.