RESUMO
In an effort to determine the functionality of palmitoylation in rhodopsin, a number of physicochemical properties of depalmitoylated rhodopsin were monitored. Approximately 70% of the rhodopsin was depalmitoylated in rod outer segments by a mild hydroxylamine treatment that resulted in minimal bleaching of rhodopsin. Subsequent purification by affinity chromatography could be used to remove hydroxylamine-bleached rhodopsin. Parallel physical studies were performed on both purified, detergent-solubilized rhodopsin and rhodopsin in rod outer segments. No effect was seen on the rate of metarhodopsin II formation for depalmitoylated rhodopsin. A small effect was seen in the biphasic behavior of the rate of retinal regeneration. The circular dichroism spectrum of depalmitoylated, purified rhodopsin was virtually identical to that of the native protein. These results suggest that depalmitoylation does not greatly affect the conformational structure of rhodopsin. Circular dichroism at 222 nm was used to monitor the thermal denaturation of depalmitoylated and native rhodopsin. A small but significant decrease in the in rod outer segments. In both cases, the van't Hoff parameters showed an increase in positive enthalpy for denaturation relative to the native state. This is largely counterbalanced by an increase in positive entropy relative to the native states. The circular dichroism of the "denatured" state showed a high alpha-helix content. Depalmitoylated rhodopsin had a lower helix content than native protein in this high-temperature state. The changes in the thermodynamics upon depalmitoylation were attributed to structural changes in the denatured state.
Assuntos
Ácidos Palmíticos/metabolismo , Rodopsina/química , Animais , Bovinos , Fenômenos Químicos , Físico-Química , Dicroísmo Circular , Temperatura Alta , Cinética , Ácido Palmítico , Desnaturação Proteica , Retina/metabolismo , Rodopsina/análogos & derivados , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/química , TermodinâmicaRESUMO
Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer containing four copies each of four distinct subunits: alpha, beta, gamma, and delta. By intramolecular zero-length crosslinking with transglutaminase, we have previously demonstrated that the regulatory alpha and beta subunits abut one another in the holoenzyme [Nadeau, O. W., and Carlson, G. M. (1994) J. Biol. Chem. 269, 29670-29676]. Selective partial proteolysis of the 138 kDa alpha subunit in holoenzyme that had been crosslinked by transglutaminase has revealed a high molecular weight conjugate corresponding to full-length beta subunit crosslinked to a 60 kDa N-terminal fragment of alpha (determined by SDS-PAGE, Western blotting and N-terminal sequencing). This conjugate was also observed when the enzyme was first activated by partial proteolysis of alpha and then crosslinked by transglutaminase. Both forms of the kinase, generated by either sequential crosslinking and proteolysis or the reverse, coeluted with non-crosslinked hexadecameric control enzyme in size exclusion chromatography, indicating that the crosslinking was intramolecular, i.e., within hexadecamers. This is the first demonstration of any intersubunit interaction involving the N-terminal domain of the alpha subunit and the first region of any subunit shown to interact with the beta subunit. The results are consistent with the predicted path of the polypeptide backbone of the alpha subunits within the holoenzyme and with the proposed location of the beta subunits.
Assuntos
Reagentes de Ligações Cruzadas , Fosforilase Quinase/química , Fosforilase Quinase/metabolismo , Transglutaminases/metabolismo , Animais , Quimotripsina , Eletroforese em Gel de Poliacrilamida , Holoenzimas/química , Holoenzimas/metabolismo , Substâncias Macromoleculares , Peso Molecular , Fibras Musculares de Contração Rápida/enzimologia , Músculo Esquelético/enzimologia , Fragmentos de Peptídeos/isolamento & purificação , Fosforilase Quinase/isolamento & purificação , CoelhosRESUMO
Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.
Assuntos
Calmodulina/química , Calmodulina/ultraestrutura , Fosforilase Quinase/química , Fosforilase Quinase/ultraestrutura , Animais , Calmodulina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Ouro , Holoenzimas/química , Holoenzimas/ultraestrutura , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão e Varredura , Modelos Moleculares , Músculo Esquelético/enzimologia , Fosforilase Quinase/isolamento & purificação , Estrutura Quaternária de Proteína , Subunidades Proteicas , Coelhos , Radioisótopos de EnxofreRESUMO
Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer consisting of four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha, beta, and delta, the last being endogenous calmodulin). The enzyme is activated by a variety of effectors acting through its regulatory subunits. To probe the quaternary structure of nonactivated and activated forms of the kinase, we used the heterobifunctional, photoreactive cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Mono-derivatization of the holoenzyme with the succinimidyl group, followed by photoactivation of the covalently attached azido group, resulted in intramolecular cross-linking to form two distinct heterodimers: a major (alphagamma) and a minor (betadelta) conjugate. Formation of both conjugates was significantly altered in activated conformations of the enzyme induced by phosphorylation, alkaline pH, and several allosteric activators (ADP, exogenous calmodulin/Ca2+, and Ca2+ alone). Of these activating mechanisms, all increased formation of alphagamma, except Ca2+ alone, which inhibited its formation. When cross-linking was carried out at alkaline pH or in the presence of ADP or exogenous calmodulin/Ca2+, the cross-linked enzyme remained activated following removal of the activators; however, cross-linking in the presence of Ca2+ resulted in sustained inhibition. The results indicate that perturbations in the subunit cross-linking forming the alphagamma dimer reflect the subsequent extent of sustained activation of the holoenzyme that is measured. The region cross-linked to the catalytic gamma subunit was confined to the C-terminal 1/6th of the alpha subunit, which contains known regulatory regions. These results suggest that activators of the phosphorylase kinase holoenzyme perturb interactions between the C-terminal region of the inhibitory alpha subunit and the catalytic gamma subunit, ultimately leading to activation of the latter.