Halorhodopsin, a photoactivated Cl- pump of halobacteria, shows several analogies with bovine rhodopsin. As it is known that bovine rhodopsin is acylated, we have examined the possibility that fatty acids ore covalently bound to halorhodopsin too. Purified holorhodopsin was isolated from Halobacterium halobium as previously described (Duschl Aet ol, 263:17016-17022, 1988). Two purple fractions were eluted from phenylsepharose column; the absorption spectra of the two holorhodopsin fractions in the dark were identical. The two fractions showed the same 20 kDa protein bond on SDS-PAGE (Blonck A, Oesterhelt D, 6: 265-273, 2987). Lipids released by alkaline hydrolysis of the two halorhodopsin fractions were esterified with HCl/MeOH, analysed by gas chromutogruphy/mass spectrometry and quantitated using an internal standard. We have identified methyl palmitute among derivotized lipids released from both halorhodopsin fractions; the paImitate content of the first eluted fraction wus higher than the second. By udding palmitote to buffers used in the phenylsephorose chromatography, only one sharp purple bond was collected. The halorhodopsin photoreaction, studied by an optical multichunnel analyzer, was found to be dependent on the amount of polmitate associated to purified halorhodopsin.
Role of palmitic acid on isolation and properties of halorhodopsin
Corcelli Angela;Trotta Massimo
1995
Abstract
Halorhodopsin, a photoactivated Cl- pump of halobacteria, shows several analogies with bovine rhodopsin. As it is known that bovine rhodopsin is acylated, we have examined the possibility that fatty acids ore covalently bound to halorhodopsin too. Purified holorhodopsin was isolated from Halobacterium halobium as previously described (Duschl Aet ol, 263:17016-17022, 1988). Two purple fractions were eluted from phenylsepharose column; the absorption spectra of the two holorhodopsin fractions in the dark were identical. The two fractions showed the same 20 kDa protein bond on SDS-PAGE (Blonck A, Oesterhelt D, 6: 265-273, 2987). Lipids released by alkaline hydrolysis of the two halorhodopsin fractions were esterified with HCl/MeOH, analysed by gas chromutogruphy/mass spectrometry and quantitated using an internal standard. We have identified methyl palmitute among derivotized lipids released from both halorhodopsin fractions; the paImitate content of the first eluted fraction wus higher than the second. By udding palmitote to buffers used in the phenylsephorose chromatography, only one sharp purple bond was collected. The halorhodopsin photoreaction, studied by an optical multichunnel analyzer, was found to be dependent on the amount of polmitate associated to purified halorhodopsin.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
