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Phosphorylation increases the catalytic activity of rainbow trout gill cytosolic carbonic anhydrase

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Abstract

Cytoplasmic carbonic anhydrase (CAc) in the gill of teleost fish contributes to ionic regulation and acid–base balance by catalyzing the reversible reaction of CO2 and water, CO2 + H2O ↔ H+ + HCO3 −. Regulation of CAc abundance and activity therefore is expected to fine-tune responses to ionic or acid–base challenges. The present study investigated the potential for gill CAc of rainbow trout, Oncorhynchus mykiss (tCAc), to undergo reversible phosphorylation. The activity of tCAc was approximately doubled by phosphorylation achieved through in vitro stimulation of endogenous protein kinases; kinase stimulation doubled phospho-threonine content from that observed in tCAc isolated under conditions where both kinases and protein phosphatases were inhibited. In vitro incubation to preferentially stimulate specific kinases implicated protein kinase G (PKG) in mediating the increase in tCAc activity. The kinetic parameters of turnover number (k cat) and substrate affinity (K m) were similarly affected by stimulation of either kinase or phosphatase action. However, phosphorylation via kinase stimulation significantly increased the efficiency of tCAc (V max /K m), and this factor may have contributed to the elevation of tCAc activity. In addition, phosphorylation of tCAc by kinase stimulation significantly increased the inhibition constant (K i) for acetazolamide. These results demonstrate that tCAc is subject to reversible phosphorylation; future work should focus on identifying the physiological situation(s) in which phosphorylation of trout branchial CAc occurs.

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Acknowledgments

This work was supported by NSERC of Canada Discovery and Research Tools and Instruments grants to KMG. DC was supported by an NSERC postgraduate scholarship and an Ontario graduate scholarship. Thanks are extended to Bill Fletcher and Christine Archer for their excellent care of the trout in the aquatic facility.

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  1. Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON, K1N 6N5, Canada

    Daniel Carrie & Kathleen M. Gilmour

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  1. Daniel Carrie
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Correspondence to Kathleen M. Gilmour.

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Communicated by H.V. Carey.

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Carrie, D., Gilmour, K.M. Phosphorylation increases the catalytic activity of rainbow trout gill cytosolic carbonic anhydrase. J Comp Physiol B 186, 111–122 (2016). https://doi.org/10.1007/s00360-015-0942-4

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  • DOI: https://doi.org/10.1007/s00360-015-0942-4

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