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), Vol. 266:20; ISSN 0021-9258
• Country of Publication: United States
• Language: English

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