Modification of adenylate cyclase by photoaffinity analogs of forskolin

Journal Article · Sun Jan 01 00:00:00 EST 1989 · Second Messengers Phosphoproteins; (USA)

OSTI ID:6855244

Ho, L T; Nie, Z M; Mende, T J; Richardson, S; Chavan, A; Kolaczkowska, E; Watt, D S; Haley, B E; Ho, R J ^[1]

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1. Univ. of Miami School of Medicine, FL (USA)

Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.

• OSTI ID: 6855244
• Journal Information: Second Messengers Phosphoproteins; (USA), Vol. 12:5-6; ISSN 0895-7479
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CYCLASES
ENZYME ACTIVITY
PHOSPHOLIPIDS
BIOCHEMICAL REACTION KINETICS
ADIPOSE TISSUE
AFFINITY
AUTORADIOGRAPHY
CELL MEMBRANES
DOSE-RESPONSE RELATIONSHIPS
FAT CELLS
IODINE 125
LABELLING
RATS
ANIMAL CELLS
ANIMAL TISSUES
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CELL CONSTITUENTS
CONNECTIVE TISSUE
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
ESTERS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
KINETICS
LIPIDS
LYASES
MAMMALS
MEMBRANES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
RADIOISOTOPES
REACTION KINETICS
RODENTS
SOMATIC CELLS
TISSUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques