Properties of adenotin reconstituted into phospholipid vesicles.
Adenotin is a low affinity adenosine binding protein that has amino terminal homology with mammalian and avian stress proteins. Human placental adenotin was solubilized and reconstituted into phospholipid vesicles with an overall yield of 30%. The properties of adenotin in vesicles were similar to the native membranes as follows: association has a Kobs of 0.61 +/- 0.03 minute-1; equilibrium is reached in approximately 15 minutes; and the first order dissociation constant is 5.0 +/- 0.3 minute-1. Displacement analysis reveals an agonist potency order and Ki values as follows: N-ethylcarboxamidoadenosine, 0.35 microM; 2-chloroadenosine, 1.5 microM; R-phenylisopropyladenosine, greater than 1000 microM. The addition of 100 microM 5'-guanylylimidodiphosphate did not decrease binding of 5'-N-ethylcarboxamidoadenosine (NECA) at 37 degrees C or 4 degrees C but did decrease the IC50 for PC12 and JAR cell membrane agonist binding from 9.9 to 3.3 microM and increase the binding to 150-211% of the control value at 37 degrees C. The latter studies at 37 degrees C showed high variability. Using binding sites reconstituted into vesicles and gel filtration chromatography and agonist related guanine nucleotide release, the authors investigated whether these changes were related to an interaction between adenotin and a guanine nucleotide regulatory protein. No evidence for such an interaction was found. These data suggest that adenotin retains its binding properties when reconstituted into phospholipid vesicles. The function of this low affinity adenosine binding site remains to be discovered. However, the reconstitution of adenotin into phospholipid vesicles provides a method to study its function.
Hutchison
K A
KA
Department of Internal Medicine, University Hospital, Ann Arbor, Michigan 48109-0108.
Prasad
M
M
Work
C
C
Fox
I H
IH
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D019830:Adenosine-5'-(N-ethylcarboxamide); D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane; D002784:Cholesterol; D002850:Chromatography, Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D008861:Microsomes; D008970:Molecular Weight; D010713:Phosphatidylcholines; D015398:Signal Transduction
D002352:Carrier Proteins; D010713:Phosphatidylcholines; C064982:adenotin; D006165:Guanylyl Imidodiphosphate; D019830:Adenosine-5'-(N-ethylcarboxamide); D002784:Cholesterol; D019204:GTP-Binding Proteins; D000241:Adenosine