Biomedical subjects

B E Haley

Publications and source records attributed to B E Haley.

93 records · Page 6Linked to original sources

PubMedDate not supplied

Identification of adenine binding domain peptides of the ADP regulatory site within glutamate dehydrogenase.

Photoaffinity labeling with [alpha-32P]-8-azidoadenosine 5'-diphosphate (8N3ADP) and [beta-32P]-2-azidoadenosine 5'-diphosphate (2N3ADP) was used to identify overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of the regulatory ADP site of bovine liver glutamate dehydrogenase (GDH). In the absence of UV irradiation, 8N3ADP was able to activate the reverse reaction catalyzed by GDH as well as ADP. Photoinsertion of both [alpha 32P] 8N3ADP and [beta 32P]2N3ADP was reduced best by ADP in comparison to other nucleotides. Photolabeling of GDH with [alpha 32P]8N3-ADP appeared to be biphasic, with saturation occurring near 80 and 130 microM, whereas [beta 32P]2N3ADP showed saturation near 50 microM. When 60 microM [alpha 32P]8N3ADP (below the first saturation value) was used to identify peptides within the ADP binding domain, peptides corresponding to residues G156-K200 and E175-K200 (tryptic) and I158-Y183 (chymotryptic) were photolabeled. However, when 160 microM [alpha 32P]8N3ADP (above the second saturation value) was used, the peptide D403-R418 was also photolabeled. Digestion with both trypsin and chymotrypsin resulted in isolation of peptides E175-Y183 and A184-I192. [beta 32P]2N3ADP at 90 microM also photolabeled tryptic peptides G156-K200 and C270-K289. C270-K289 was shown earlier to be within the NAD+ binding site [Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2, 142-147]. These results are consistent with the residues E175-[192 being within the adenine binding domain of the ADP regulatory site.

Adenine↗

PubMedDate not supplied

Photoaffinity labeling of atrial natriuretic factor analog atriopeptin III with [gamma-32P]8N3GTP.

We have determined that atrial natriuretic factor (ANF) analog atriopeptin III (AP III) is a guanosine 5'-triphosphate (GTP) binding protein as shown by photoaffinity labeling with the GTP-analog [gamma-32P]8N3GTP (azido-GTP). The photo-insertion was saturable at 40 microM azido-GTP. Half-maximal protection from photo-insertion was observed using 40-microM concentrations of the native nucleotide GTP. The photolabeling was inhibited approximately 70% by addition of micromolar concentrations of the divalent cations MgCl2, MnCl2 or CaCl2. The related purine-containing nucleotide adenosine 5'-triphosphate (ATP) was able to elicit half-maximal protection of AP III from photo-insertion with 20 microM azido-GTP at a concentration of 120 microM. No significant protection from photoinsertion was observed with other compounds such as GDP, GMP, cGMP, UTP and pyrophosphate. Additionally, a carboxy-terminal F-R-Y deleted analog of ANF, AP I; reduced and carboxymethylated AP III; and resact, a peptide released from sea urchin eggs, were not photolabeled by azido-GTP. These results demonstrate that the AP III analog of ANF contains a functional GTP binding site that would be saturated at physiological concentrations of GTP. At present, the role of this interaction in mediating the biological effects of ANF is unknown, but we speculate that direct GTP interaction with AP III may be involved in the mechanism of action of this peptide.

Affinity Labels↗

PubMedDate not supplied

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.

Adenylyl Cyclases↗