ISSN:
1432-1424
Schlagwort(e):
Protein kinase A
;
Na+/HCO 3 − cotransporter
;
Trypsin digestion
;
Regulatory protein
;
Protein phosphorylation
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
,
Chemie und Pharmazie
Notizen:
Abstract The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na+/ HCO 3 − cotransporter. We investigated whether the activity of Na+/HCO 3 − cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased 22Na uptake in the presence of HCO3 but not in the presence of gluconate, indicating that trypsin does not alter diffusive 22Na uptake but directly stimulates the Na+/HCO 3 − cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na+/HCO 3 − cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P 〈 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na+/HCO 3 − cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na+/HCO 3 − cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na+/HCO 3 − cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S−). With the addition of fraction S−, the CSU of cAMP-PKA failed to inhibit the Na+/HCO 3 − cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P 〈 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na+/HCO 3 − cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na+/HCO 3 − cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF00233307
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