skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Modification of adenylate cyclase by photoaffinity analogs of forskolin

Abstract

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 activitymore » 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.« less

Authors:
; ; ; ; ; ; ; ;  [1]
  1. (Univ. of Miami School of Medicine, FL (USA))
Publication Date:
OSTI Identifier:
6855244
Alternate Identifier(s):
OSTI ID: 6855244
Resource Type:
Journal Article
Journal Name:
Second Messengers Phosphoproteins; (USA)
Additional Journal Information:
Journal Volume: 12:5-6; Journal ID: ISSN 0895-7479
Country of Publication:
United States
Language:
English
Subject:
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

Citation Formats

Ho, L.T., Nie, Z.M., Mende, T.J., Richardson, S., Chavan, A., Kolaczkowska, E., Watt, D.S., Haley, B.E., and Ho, R.J. Modification of adenylate cyclase by photoaffinity analogs of forskolin. United States: N. p., 1989. Web.
Ho, L.T., Nie, Z.M., Mende, T.J., Richardson, S., Chavan, A., Kolaczkowska, E., Watt, D.S., Haley, B.E., & Ho, R.J. Modification of adenylate cyclase by photoaffinity analogs of forskolin. United States.
Ho, L.T., Nie, Z.M., Mende, T.J., Richardson, S., Chavan, A., Kolaczkowska, E., Watt, D.S., Haley, B.E., and Ho, R.J. Sun . "Modification of adenylate cyclase by photoaffinity analogs of forskolin". United States.
@article{osti_6855244,
title = {Modification of adenylate cyclase by photoaffinity analogs of forskolin},
author = {Ho, L.T. and Nie, Z.M. and Mende, T.J. and Richardson, S. and Chavan, A. and Kolaczkowska, E. and Watt, D.S. and Haley, B.E. and Ho, R.J.},
abstractNote = {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.},
doi = {},
journal = {Second Messengers Phosphoproteins; (USA)},
issn = {0895-7479},
number = ,
volume = 12:5-6,
place = {United States},
year = {1989},
month = {1}
}