Antibodies directed against synthetic peptides distinguish between GTP-binding proteins in neutrophil and brain.

P Goldsmith,P Gierschik,G Milligan,C G Unson,R Vinitsky,H L Malech,A M Spiegel

doi:10.1016/s0021-9258(18)47849-0

Abstract

Antisera AS/6 and 7, raised against a synthetic peptide KENLKDCGLF corresponding to the carboxyl-terminal decapeptide of transducin-alpha, react on immunoblots with purified transducin-alpha and with proteins of 40-41 kDa in all tissues tested. The latter represent one or more forms of Gi alpha but not Go alpha, since a synthetic peptide, KNNLKDCGLF, corresponding to the carboxyl-terminal decapeptide of two forms of Gi alpha blocks AS/6 and 7 reactivity with transducin-alpha and Gi alpha on immunoblots, whereas the corresponding Go-related peptide, ANNLRGCGLY, does not. Antisera LE/2 and 3, raised against the synthetic peptide LERIAQSDYI, corresponding to an internal sequence predicted by one form of Gi alpha cDNA (Gi alpha-2) and differing by 3 residues from the sequence of another form, Gi alpha-1, react strongly with a 40-kDa protein abundant in neutrophil membranes and with the major pertussis toxin substrate purified from bovine neutrophils. LE/2 and 3 reveal a relatively faint 40-kDa band on immunoblots of crude brain membranes or of purified brain Gi/Go. LE/2 and 3 do not react with transducin-alpha or Go alpha nor with the 41-kDa form of pertussis toxin substrate in brain, Gi alpha-1. These antisera distinguish between the major pertussis toxin substrates of brain and neutrophil and tentatively identify the latter as Gi alpha-2.

Highlights

From the $Metabolic Diseases Branch, National Institute of Diabeteasnd Digestive and Kidney Diseases and **Laboratoryof Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutesof Health, Bethesda, Maryland 20892, the §Pharmacology Institute, Universityof Heidelberg, Federal Republic of Germany, the TDepartments of Biochemistry and Pharmacology, University of Glasgow, Scotland, and the IlDepartment of Biochemistry, Rockefeller University, New York, New York 10021
Gia-l.These antisera distinguish between the major At least two distinct formsof “Gin”have been identified by pertussis toxin substrates of brain and neutrophil and cDNA cloning [13]
A crude plasma membrane fractionwas prepared, and 50 munoblots of purified holotransducin.Forcomparison, we tested an immune bleed of a rabbit, AS/l, immunized with holotransducin

Summary

THEJOURNALOF BlOLOcICAL CHEMISTRY

Vol 262, No 30, Issue of October 25, pp. 14683-14688,1987 Printed in U.S.A. Antibodies Directed against Synthetic Peptides Distinguish between GTP-binding Proteins in Neutrophil andBrain*. Gia-l.These antisera distinguish between the major At least two distinct formsof “Gin”have been identified by pertussis toxin substrates of brain and neutrophil and cDNA cloning [13]. The amino acid sequence predicted by this cDNA corresponds to thsequence obtained for a 41-kDa pertussis toxin substrate purifiedfrombovine [14] and rat Afamily of guaninenucleotide-bindingproteins(G-pro- [15] brain. We refer to this form as Gin.l because it teins)’ couples diverse receptors to effectors. Of antisera directed againtswt o synthetic peptides thpartoved useful in distinguishing G-proteins in neutrophil and brain and tentativelyidentify the major pertussis toxin substrateof neutrophils as the G-proteinencoded by Gin.zcDNA. The positions of the a, p, and y subunits of transducin (TD)are indicated

RESULTS

Thesyntheticpeptide wasconjugated toKLHandthree

Coomassie Blue

DISCUSSION