Modification of adenylate cyclase by photoaffinity analogs of forskolin.
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 (see Figure 1 for structure) 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.
Ho
L T
LT
Department of Biochemistry, University of Miami School of Medicine.
Nie
Z M
ZM
Mende
T J
TJ
Richardson
S
S
Chavan
A
A
Kolaczkowska
E
E
Watt
D S
DS
Haley
B E
BE
Ho
R J
RJ
D000262:Adenylyl Cyclases / Q000032:analysis / Q000378:metabolism*; D000273:Adipose Tissue / Q000648:ultrastructure; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D005576:Colforsin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D007553:Isotope Labeling; D051381:Rats; D011919:Rats, Inbred Strains; D014466:Ultraviolet Rays
D000345:Affinity Labels; D005576:Colforsin; D000262:Adenylyl Cyclases