Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.
Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.
Department of Medicine, Tulane University School of Medicine, New Orleans, LA.
W J
WJ
Rossowski
T M
TM
Rossowski
S
S
Zacharia
A
A
Ertan
D H
DH
Coy
D000818:Animals; D001667:Binding, Competitive; D005260:Female; D019004:Galanin; D007583:Jejunum / Q000032:analysis*; D009130:Muscle, Smooth / Q000032:analysis*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000032:analysis* / Q000378:metabolism; D013270:Stomach / Q000032:analysis*; D013696:Temperature; D013997:Time Factors
D010446:Peptide Fragments; D010455:Peptides; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D019004:Galanin