Corpora allata of the larval tobacco hornworm contain a calcium/calmodulin-sensitive adenylyl cyclase

0739-4462

corpora allata ; adenylyl cyclase ; CAMP ; calcium ; calmodulin ; biogenic amines ; dopamine ; Chemistry ; Food Science, Agricultural, Medicinal and Pharmaceutical Chemistry

Wiley InterScience Backfile Collection 1832-2000

Biology

An assay was developed with which to study basic characteristics of an adenylyl cyclase in the corpora allata (CA) of the tobacco hornworm, Manduca sexta. The assay used glands collected and frozen at -80°C, to circumvent the problem of tissue availability. With this protocol for storage of tissue, less than 25% of the enzyme activity in fresh tissue was lost. Substances such as sodium fluoride (NaF) and Gpp(NH)p (a non-hydrolyzable GTP analog), which typically stimulate the adenylyl cyclases in other insect tissues, increased enzyme activity several-fold. There was a progressive decrease in the capacity of the CA adenylyl cyclase to be stimulated by NaF during the fifth stadium, suggesting a possible developmental change in the capacity of the associated G protein to be stimulated by NaF. The calcium/calmodulin (CaM) dependence of adenylyl cyclase activity was also investigated. The results demonstrated that addition of up to 10-4 M calcium to assays of enzyme activity in whole gland homogenates of both larval (day O) and prepupal (day 6) CA resulted in only a slight increase in the activity of the enzyme over basal rates in the presence of the calcium chelator EGTA. However, addition of as little as 5 m̈M CaM in the presence of 10-4 to 10-3 M calcium increased adenylyl cyclase activity three to five-fold. A similar stimulation was obtained with washed membrane preparations of day 0 and day 6 glands, but required a substantially higher concentration of CaM. Results demonstrated that the CA possess a calcium/CaM-dependent adenylyl cyclase from day 0 through day 6. A preliminary investigation of the effect of two biogenic amines on the CA adenylyl cyclase revealed that enzyme activity was not affected by octopamine, but a stage-specific effect was obtained with dopamine. Concentrations of 10-6 and 10-7 M stimulated enzyme activity in hornogenates of day 0 glands but inhibited activity in homogenates of day 6 CA. © 1995 Wiley-Liss, Inc.

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