Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

Journal Article · Mon Jul 15 00:00:00 EDT 1991 · Journal of Biological Chemistry; (United States)

OSTI ID:5464015

Morris, D I; Robbins, J D; Ruoho, A E; Sutkowski, E M; Seamon, K B ^[1]

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1. Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, MD (USA)

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.

• OSTI ID: 5464015
• Journal Information: Journal of Biological Chemistry; (United States), Journal Name: Journal of Biological Chemistry; (United States) Vol. 266:20; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

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550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALDEHYDES
ANIMALS
AZIDES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY
BODY FLUIDS
BRAIN
CARBOHYDRATES
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CYCLASES
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
ERYTHROCYTES
ESTERS
GLUCOSE
HEXOSES
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
LIPIDS
LYASES
MAMMALS
MAN
MATERIALS
MEMBRANE TRANSPORT
MEMBRANES
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
MONOSACCHARIDES
NERVOUS SYSTEM
NITROGEN COMPOUNDS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANS
PHOSPHOLIPIDS
PRIMATES
RADIOISOTOPES
REACTION KINETICS
RUMINANTS
SACCHARIDES
SPECIFICITY
TRACER TECHNIQUES
VERTEBRATES