Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1999-09-02
2000-12-05
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 4, 435 75, 435968, C12Q 126, C12Q 100, G01N 3353
Patent
active
061565296
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for the specific labeling of a protein containing selenocyst(e)ine and/or cyst(e)ine groups.
The examination of receptors, such as the G protein coupled receptors, the polypeptide chain of which folds into a structure with seven membrane-spanning helices is of great importance in terms of both science and economy. These so-called 7-transmembrane receptors are a large group of receptors which are very old phylogenetically and the amino acid sequence of which contains characteristic cysteines. The corresponding ligands belong to a wide variety of chemical classes of substances and molecular sizes, from lipids, metal ions and monoamines to peptides and proteins. However, to date, little is known about possible structural changes of such receptors which represent the link between ligand binding and signal transduction.
It is believed that ligand-induced conformational changes of the 7-transmembrane receptors are involved in the initial step of the dissociation of the heterotrimeric G protein complex with exchange of GDP/GTP. Schwartz and Rosenkilde (TiPS, Vol. 17, 1996) present a model in which the capability of stabilizing an active receptor conformation is mentioned as the only requirement for a new agonist, resulting in the conclusion that a common "lock" for all the "keys" in the superfamily of 7-transmembrane receptors does not exist.
Generally, in the examination of proteins, but also, in particular, in the investigation of the signal cascade, cysteine-specific reagents are used in order to shed light upon the functional and structural properties of the cysteines (Creighton, "Proteins", Freeman & Co., 1984). Korner et al. (The Journal of Biological Chemistry, Vol. 257, No. 7, pp. 3389-3396, 1982) describe a method for examining the .beta..sub.2 -adrenergic receptor using N-ethylmaleinimide (NEM). In order to react thiol groups which are already exposed prior to the actual experiment, the membrane samples were pretreated with NEM in the presence of the antagonist propranolol in a first process step. In a further process step, they were incubated with [.sup.3 H]isoproterenol with addition of NEM. In particular, the authors come to the conclusion that NEM interacts, not with the receptor itself, but with an associated component, the G protein. According to Korner et al., specific thiol groups of the G protein are being exposed when the G protein interacts with the hormone-activated receptor. Accordingly, the method of Korner et al. is not suitable for labeling the actual receptor.
Andre et al. (Biochemical Pharmacology, Vol. 31, No. 22, 3657-3662, 1982) describe the effect of NEM on agonist-induced conformational changes of the .beta.-adrenergic receptor. The process performed on membrane preparations is characterized by three incubation steps: incubation with NEM, followed by incubation with NEM in the presence of the agonist (-)-isoproterenol, followed by incubation with the labeled antagonist (-)-[.sup.3 H]DHA. Andre et al. observed that the effects brought about by NEM can also be induced by GTP in their experiments, so that evidently G proteins are critically involved.
Lipson et al. (Biochemistry 1986, 25, 5678-5685) describe a method for examining the glucagon receptor N protein complex using N-ethylmaleinimide and other reagents which alkylate thiols. It could be demonstrated, for example, that the presence of NEM prior to, during or after the association reaction of .sup.125 I-glucagon with partially purified, protein-containing liver membranes promotes the release of bound hormone in a subsequent dissociation reaction.
In a survey article, in which the use of the alkylating agent N-ethylmaleinimide (NEM) in the investigation of G protein coupled receptors was last mentioned (Lefkowitz et al., Ann. Rev. Biochem. 52, 159-86, 1983), the authors note that it is uncertain whether the critical sulfhydryl groups are localized on the receptor, on the G protein, or even on both components.
Today, the particular importance of the cysteines in the .alpha..beta.-hetero
Henco Karsten
Hunt Nicholas
Obermann-Pless Heike
Willey Kevan
EVOTEC BioSystems AG
IHF Institut fur Hormon-und Fortpfianzungsforschung GmbH
Leary Louise N.
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