Binding of the pheromonal steroid, 5alpha-androst-16-en-3-one, to membrane-enriched fractions of boar and rat olfactory epithelium; preliminary evidence for binding protein being a glycoprotein.
A high degree of binding of 5alpha-[3H]-androstenone was recorded in membrane-enriched fractions of porcine olfactory tissue. The specific (i.e. high affinity, low capacity) binding had a mean Ka approximately 2x10(8)M(-1). A Hill plot of the data showed a Hill coefficient of approximately 2, possibly suggesting co-operativity of binding, with binding constants increasing from 8x10(7) to 1.6x10(9)M(-1) with increasing substrate concentration. The level of specific binding of 5alpha-[3H]-androstenone was nearly 10-fold higher than in corresponding respiratory tissue preparations and was markedly reduced in the presence of excess (approximately 1 microM) unlabelled 5alpha-androstenone. Corresponding fractions derived from rat olfactory tissue showed only 25% of the binding recorded for the pig. After incubation of 5alpha-[3H]-androstenone with solubilised olfactory cilial tissue (porcine), gel filtration and chromatography on a typical "glycoprotein" column (Concanavalin A-Sepharose B) were performed. Specific binding was recorded only in fractions corresponding to glycoproteins with Mr of approximately 70-90 kDa. In a third series of experiments, fractions containing high concentrations of cilia, some still attached to the dendritic endings (as shown by electron microscopy) were obtained by a novel method involving stripping them off the nasal epithelium. The basal adenylate cyclase (AC) activity was very significantly (P<0.01) higher in olfactory, compared with respiratory, cilia; storage at -70 degrees C for 3 weeks greatly reduced AC activity. When fresh male and female porcine olfactory cilia preparations were incubated with 5alpha-androstenone plus GTP, AC activity was increased fourfold (P<0.01). However, responses of porcine respiratory cilia were not significant statistically, neither were changes in basal levels of AC activities in rat olfactory cilia.
Division of Biochemistry and Molecular Biology, United Medical and Dental Schools, Guy's Hospital, London SE1 9RT, UK.
M A
MA
Kraevskaya
M J P
MJ
Higgins
M T
MT
Wilson
D B
DB
Gower
D000262:Adenylyl Cyclases / Q000378:metabolism; D000736:Androstenes / Q000378:metabolism*; D000818:Animals; D002845:Chromatography; D002923:Cilia / Q000201:enzymology / Q000378:metabolism; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D008970:Molecular Weight; D009831:Olfactory Mucosa / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D051381:Rats; D013552:Swine; D014316:Tritium
D000736:Androstenes; D006023:Glycoproteins; D014316:Tritium; C000214:androst-16-en-3-one; D000262:Adenylyl Cyclases