Biomedical subjects

E Nekrasova

Publications and source records attributed to E Nekrasova.

3 recordsLinked to original sources

PubMed1997

The regulation of the cGMP-binding cGMP phosphodiesterase by proteins that are immunologically related to gamma subunit of the photoreceptor cGMP phosphodiesterase.

The cGMP phosphodiesterase from retinal rods (PDE-6) is an alphabetagamma2 heterotetramer. The alpha and beta subunits contain catalytic sites for cGMP hydrolysis, whereas the gamma subunits serve as a protein inhibitor of the enzyme. Visual excitation of photoreceptors enables the activated GTP-bound form of the G-protein transducin to remove the inhibitory action of the gamma subunit, thereby triggering PDE-6 activation. The type 5 phosphodiesterase (PDE-5) isoform shares a number of similar characteristics with PDE-6, including binding of cGMP to noncatalytic sites, the cyclic nucleotide specificity, and inhibitor sensitivities. Although the functional role of PDE-5 remains unclear, it has been shown to be activated by protein kinase A (PKA) (Burns, F., Rodger, I. W. & Pyne, N. J. (1992) Biochem. J. 283, 487-491). Here we report that both the recombinant gamma subunit and a peptide corresponding to amino acids 24-46 in this protein inhibited the activation of PDE-5 by PKA. Furthermore, immunoblotting airway smooth muscle membranes with a specific antibody against amino acids 24-46 of the PDE-6 gamma subunit identified two major immunoreactive small molecular mass proteins of 14 and 18 kDa (p14 and p18). These appear to form a complex with PDE-5, because PDE activity was immunoprecipitated using antibody against the PDE-6 gamma subunit. p14 and p18 were also substrates for phosphorylation by a unidentified kinase that was stimulated by a pertussis toxin-sensitive G-protein. Phosphorylation of p14/p18 in membranes treated with guanine nucleotides correlated with a concurrent reduction in the activation of PDE-5 by PKA. We suggest that p14 and p18 share an epitope common to PDE-6 gamma and that this region may interact with PDE-5 to prevent its activation by PKA.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1996

Overexpression, solubilization and purification of rat and human olfactory receptors.

The superfamily of olfactory receptor genes, whose products are thought to be activated by odorant ligands, is critical for odor recognition. Two olfactory receptors, olp4 from rat and OR17-4 from human, were overexpressed in Sf9 insect cells. The presence of the proteins in cell membranes was monitored by immunoblotting with peptide-specific polyclonal antibodies directed against the C-terminal sequences of these receptors and with a mAb against an N-terminal octapeptide epitope tag. A DNA sequence that codes for a His6 tag, which binds tightly to a Ni2+-chelate-affinity column, was incorporated into the N-termini of both genes. The expressed olfactory receptors were found mainly in the cell-membrane fraction. The proteins were difficult to solubilize by many detergents and only lysophosphatidylcholine was found to be both suitable for efficient solubilization of the overexpressed olfactory receptors and compatible with the purification system used. After solubilization, the olfactory receptors were purified to near homogeneity by affinity chromatography on nickel nitrilotriacetic acid resin and by cation-exchange chromatography. Electrophoresis of the purified proteins and visualization with Coomassie Blue staining or by immunoblotting with specific antibodies, revealed bands of 32, 69 and 94 kDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins. The oligomeric forms were resistant to reduction and alkylation, and are therefore thought to be held together by non-covalent hydrophobic interactions that are resistant to SDS. This finding is similar to previous observations for other guanine-nucleotide-binding-regulatory-protein-coupled receptors. Reconstitution in phospholipid vesicles showed that the purified olfactory receptors insert specifically into the lipid bilayer. This provides a means to study functional reconstitution with putative transduction components such as olfactory guanine-nucleotide-binding-regulatory protein.

Amino Acid Sequence↗

PubMed1994

Olfactory receptor proteins. Expression, characterization and partial purification.

A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation was a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a specific polyclonal antibody against a C-terminal peptide of olp4. Translation in vitro, followed by immunoprecipitation, showed a major olp4-specific band of 27-29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag-olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag-olp4 product on the cell surface of the infected host Sf9 cells, with the N-terminus and C-terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.

Amino Acid Sequence↗