Olfactory adenylyl cyclase

Partially purified adenylyl cyclase preparations of high specific activity (60 +/- 10 mumol cAMP/(mg.min)) were obtained from rat brain synaptosomes (Orlando, C., d'Alayer, J., Baillat, G., Castets, F., Jeannequin, O., Mazié, J. C., & Monneron, A. (1992) Biochemistry 31, 3215-3222). Adenylyl cyclase activity was stimulated 4-fold by Ca2+/calmodulin and 2-fold by forskolin or by Mn2+. These preparations contained two major proteins of 140 and 110 kDa. The 140-kDa protein was identified as the neural cell adhesion molecule. The 110-kDa protein was specifically recognized by affinity-purified antibodies directed against a peptide corresponding to sequence 976-1013 of adenylyl cyclase type I. It was photolabeled by [alpha-32P]8- and 2-N3ATP in a light-dependent manner and was by far the most heavily labeled component of FC fractions. Saturation was obtained with 30 microM [32P]8-N3ATP. Photoinsertion of N3ATP into the protein was largely prevented by ATP or adenylyl imidodiphosphate but not by ADP, AMP, or adenosine. A modest incorporation of N3cAMP and photoinsertion of [alpha-32P]N3GTP into the 110-kDa protein were observed. Although some of the properties of the synaptosomal 110-kDa protein described here would match those expected from adenylyl cyclase type I, it appears that its specific activity is on the order of 1 mmol cAMP/(mg.min), about 200-fold that measured for brain adenylyl cyclases type I.

Section: Discussionmentioning

confidence: 99%

A Brain Synaptosomal Adenylyl Cyclase of High Specific Activity Is Photolabeled with Azido-ATP

Castets¹,

Baillat²,

Mirzoeva³

et al. 1994

“…1994 American Chemical Society by calcium/calmodulin and Gsa subunit association (Brostrom et al, 1977;Westcott et al, 1979;Coussen et al, 1986;Rosenberg & Storm, 1987; Mollner & Pfeuffer, 1988;Pfeuffer et al, 1989). Calmodulin-sensitive adenylyl cyclase was purified, and cDNA clones that encode this protein (type I)…”

mentioning

confidence: 99%

Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs.alpha.

Sutkowski

Tang²,

Broome³

et al. 1994

146

Several forms of adenylyl cyclase (types I, II, V, and VI) have been expressed using the recombinant baculovirus expression system in Sf9 cells. The activation of type I adenylyl cyclase by forskolin and Gs alpha was not greater than additive. In contrast, there was synergistic activation of type II, V, and VI adenylyl cyclases by Gs alpha and forskolin. Gs alpha potentiated the effect of forskolin on type II adenylyl cyclase to the greatest extent. Type I and II adenylyl cyclases were photolabeled specifically by an iodinated photoaffinity derivative of forskolin ([125I]-6-AIPP-Fsk). Type I adenylyl cyclase was photolabeled efficiently in the absence of Gs alpha, and the addition of Gs alpha only slightly increased the labeling efficiency. In contrast, type II adenylyl cyclase was not photolabeled efficiently in the absence of Gs alpha, and the addition of Gs alpha greatly enhanced the labeling efficiency. Photolabeling of type V and VI adenylyl cyclases was detected only in the presence of Gs alpha. Neither calcium/calmodulin nor G protein beta gamma subunits modulated the photolabeling of type I or II adenylyl cyclases. Another iodinated derivative of forskolin, [125I]-6-IHPP-fsk, bound to Sf9 cell membranes expressing type I adenylyl cyclase with high affinity in a filtration binding assay, and the specific binding was not enhanced by the addition of Gs alpha. In contrast, specific binding of [125I]-6-IHPP-Fsk to membranes expressing type II adenylyl cyclase was detected only in the presence of Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

“…Different odorants activate distinct subpopulations of olfactory receptor cells, thus generating distinct patterns of neuronal activity that encode odor quality and concentration (Duchamp et al, 1974;Sicard & Holley, 1984).Current evidence supports the notion that odorant recognition and olfactory transduction are initiated at the mucociliary surface. Olfactory cilia are enriched in a tissue-specific form of adenylate cyclase (Pfeuffer et al, 1989;Bakalyar & Reed, 1990), which is regulated by a distinct GTP-binding protein that is closely related to Gs (Pace & Lancet, 1986;Anholt et al, 1987) and has been designated G0if (Jones & Reed, 1989…”

mentioning

confidence: 99%

Olfactomedin: purification, characterization, and localization of a novel olfactory glycoprotein

Snyder¹,

Rivers²,

Yokoe³

et al. 1991

119

111

We have identified a novel glycoprotein expressed exclusively in frog olfactory neuroepithelium, which we have named "olfactomedin". Olfactomedin is a 57-kDa glycoprotein recognized by seven monoclonal antibodies, previously shown to react solely with proteins of olfactory cilia preparations. It undergoes posttranslational modifications, including dimerization via intermolecular disulfides and attachment of complex carbohydrate moieties that contain N-acetylglucosamine and beta-D-galactoside sugars. Olfactomedin strongly binds to Ricinus communis agglutinin I and has been purified to homogeneity by lectin affinity chromatography. Polyclonal rabbit antiserum raised against purified olfactomedin confirmed that it is expressed only in olfactory tissue. Immunohistochemical studies at the light microscopic and electron microscopic level show that olfactomedin is localized in secretory granules of sustentacular cells, in acinar cells of olfactory glands, and at the mucociliary surface. The massive production of olfactomedin and its striking deposition at the chemosensory surface of the olfactory neuroepithelium suggest a role for this protein in chemoreception.