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.

[Kinetic observation and analysis of the radioactivity distribution of 125I-labelled F(ab')2 fragments of monoclonal antibody against human lung cancer cells in normal mice].

Radioactivity distribution of 125I-labelled F(ab')2 fragments in 14 organs and tissues of normal mice for 11 time-phases from 0.5 hour to 7 days after injection was observed. The radioactivity-time curves of these organs and tissues were divided into three types according to their characteristics: excreting type (blood and liver), absorbing type (thyroid), absorbing-excreting type (the other organs and tissues). The organs fully perfused with blood had shorter peak-time and higher peak-value, such as heart, lung, kidney and liver. All of the peak-times were equal to or less than 2 days after injection, therefore the image taking should be kept away from this period. The excreting rate constants of slow metabolic component were similar for various organs and tissues except thyroid and brain. The brain had a low peak in the curve and small excreting rate constant. It was demonstrated that F(ab')2 fragments were able to penetrate the blood-brain barrier but absorption and excretion were slower compared with the other organs and tissues. The radioiodine was obviously concentrated in the subcutaneous tissue, which may have been the main cause of the subcutaneous tissue tumor induced by radioiodine. The radioactivity-time curve in which the peak value and inflect point existed simultaneously shows that radioactivity metabolism after injection of 125I-F(ab')2 follows three compartment models. The biological meanings corresponding to three sections of the blood radioactivity-time curve are pure distribution, distribution-recycle and catabolic-excretory phases, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)