Acetyl adenylate preparation

SOLUTION Acetate reacts with ATP to form acetyl adenylate, which then reacts with CoASH to form acetyl-CoA (Section 17.20). Because malonyl-CoA is prepared from acetyl-CoA, the thioester carbonyl carbon of malonyl-CoA will also be labeled. Examining each step of the mechanism for the biosynthesis of isopentenyl pyrophosphate from acetyl-CoA and malonyl-CoA allows you to determine the locations of the radioactively labeled carbons in isopentenyl pyrophosphate. Similarly, the locations of the radioactively labeled carbons in geranyl pyrophosphate can be determined from the mechanism for its biosynthesis from isopentenyl pyrophosphate. And the locations of the radioactively labeled carbons in farnesyl pyrophosphate can be determined from the mechanism for its biosynthesis from geranyl pyrophosphate. Knowing that squalene is obtained from a tail-to-tail linkage of two farnesyl pyrophosphates tells you which carbons in squalene will be labeled. 

This possibility was confirmed by injecting 5 yl N-acetyl OA (26 nmoles) into the cockroach haemocoel and monitoring levels of the derivative at various times following injection. If it is assumed that the haemolymph volume is 175 yl (24), the concentration of N-acetyl OA immediately following injection is 148.57 yM and, as indicated by the results in Table III, this value is reduced by 44% within 30 min. N-Acetyl OA is inactive against neuromuscular preparations from decapod crustaceans (25) and studies from this laboratory demonstrate that the derivative has no effect on adenylate cyclase activity in the insect nervous system (26). The relatively low rate of removal of N-acetyl OA compared with that of OA (see Table II) suppports the contention that the N-acetylation pathway produces a neurally non-active product. 

Fig. 11. Response of inactivation of acetyl-CoA carboxylase to the adenylate energy charge. Acetyl-CoA carboxylase-protein kinase preparation was preincubated as described in Fig. 1 at 37°C for 30 minutes except without AMP. Following this preincubation, inactivation and phosphorylation of the carboxylase were measured by determining the carboxylase activity remaining after incubation for 4 minutes in the presence of different adenylate energy charge systems containing ATP and AMP, or ATP alone as indicated. Carboxylase was then assayed in the presence of a final concentration of 10...

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