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 halorhodopsin 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 halorhodopsin 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 HCI/MeOH, analysed by gas chromatography/mass spectrometry and quantitated using an internal standard. We have identified methyl palmitate among derivatized lipids released from both halorhodopsin fractions; the paImitate content of the f i r s t eluted fraction was higher than the second. By adding palmitate to buffers used in the phenylsephorose chromatography, only one sharp purple bond was collected. The halorhodopsin photoreaction, studied by an optical multichannel analyzer, was found to be dependent on the amount of palmitate associated to purified halorhodopsin.
ROLE OF PALMITiC ACID ON ISOLATION AND PROPERTIES OF HALORHODOPSIN
GUERRIERI, Antonio;
1994-01-01
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 halorhodopsin 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 halorhodopsin 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 HCI/MeOH, analysed by gas chromatography/mass spectrometry and quantitated using an internal standard. We have identified methyl palmitate among derivatized lipids released from both halorhodopsin fractions; the paImitate content of the f i r s t eluted fraction was higher than the second. By adding palmitate to buffers used in the phenylsephorose chromatography, only one sharp purple bond was collected. The halorhodopsin photoreaction, studied by an optical multichannel analyzer, was found to be dependent on the amount of palmitate associated to purified halorhodopsin.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
