Identification and localization of pertussis toxin-sensitive GTP-binding proteins in bovine kidney glomeruli.
The vascular tree and the mesangium in the glomerulus respond to various hormones, growth factors, and autonomic signals, leading to generation of second messengers and regulation of ion channels. Guanine nucleotide regulatory proteins (G proteins) mediate these effects in other systems. Glomerular G proteins were studied by immunoblotting and immunohistochemical techniques. Glomeruli were isolated from bovine kidney cortex by differential sieving. Glomerular proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and nitrocellulose transfers were immunoblotted with antibodies to G proteins. G alpha,common antiserum (P-960) recognized proteins with a molecular mass of 41 to 45 kDa. Antibodies against peptide sequences specific to Gi alpha and Go alpha demonstrated Gi alpha, 1/3 (molecular mass, 39 to 41 kDa), Gi alpha 2 (molecular mass, 40 kDa), and Go alpha (molecular mass, 39 kDa). Presence of these proteins was further confirmed by pertussis toxin-catalyzed ADP ribosylation of protein(s) with a molecular mass of 39 to 41 kDa in the glomeruli. Immunohistochemical staining of frozen sections from bovine kidney cortex revealed the presence of Gi alpha 2 in capillary loop distribution in glomeruli and interstitium, but Gi,1/3 or Go could not be demonstrated. The pattern of immunofluorescence with Gi alpha 2 antiserum suggested localization of Gi alpha 2 to the endothelium in glomerular and interstitial vasculature. The novel finding of Go in glomeruli requires localization of Go to specific cells and determination of its role in glomerular physiology. In conclusion, these studies demonstrate that bovine kidney glomeruli express alpha subunits of pertussis toxin-sensitive GTP-binding proteins Gi,1/3, Gi,2 and Go.(ABSTRACT TRUNCATED AT 250 WORDS)
Gupta
A
A
Renal Division, Jewish Hospital of St. Louis, MO 63110.
Bastani
B
B
Purcell
H
H
Hruska
K A
KA
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002417:Cattle; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D007678:Kidney Glomerulus / Q000378:metabolism*; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D000906:Antibodies; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins