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A study of the adenyl cyclase activity in testis of maturing chinook salmon (Oncorhynchus tshawytscha)

University of British Columbia

Some properties of the adenyl cyclase activity in the maturing testis of Oncorhynchus tshawytscha (chinook salmon) were characterized. The enzymic reaction was linear at 30° and at 15° for at least 60 min. The divalent cation requirement of the salmon testis enzyme was reexamined (33). The optimal concentration of Mg was about 10 mM and of Mn2+ was 5 mM; Mn2+ concentrations above 15 mM caused a marked decrease in enzyme activity. A higher maximal activity was achieved in the presence of Mn2+ than in the presence of Mg2+. Stimulation of the enzyme with the optimal concentration of F-, 12 mM, resulted in a 7-fold increase in the reaction rate over the basal activity. In efforts to solubilize the enzyme, it was found that Lubrol PX and Triton X-100 destroyed enzymic activity but Nonidet P40 and Tween 80 did not. The adenyl cyclase activity in salmon testis homog-enates was stable for at least 6 hours at 0° to 4° but was very unstable at 24°; storage of the homogenate for 24 hours at either 0° to 4° or 24° resulted in a total loss of activity. Differential centrifugation of salmon testis homog-enates which were prepared in isotonic medium revealed tnat all subcellular fractions contained some adenyl cyclase activity. About 55% of the activity sedimented at 600g while only 10% of the activity was recovered in the 105,000g supernatant. The 6300g sediment had a very high specific activity compared with the specific activity of the other fractions. The ATP analogue, adenylyl imidodipho3phate (AMP-PNP), tritium labeled in adenosine, was synthesized from tri-butylammonium imidodiphosphate and adenosine-5’ phosphor-imidazolate. Salmon testis adenyl cyclase catalyzed the conversion of AMP-PNP to cyclic AMP.

Text

2010-01-19

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http://hdl.handle.net/2429/18684

Biochemistry and Molecular Biology

University of British Columbia

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