Abstract
Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephosphorylation of destrin. It is suggested that destrin dephosphorylation is involved in cortical F-actin disruption observed in parallel with β-agonist-induced amylase secretion. At present, the phosphorylation/dephosphorylation mechanism of destrin in parotid tissue is not known. We previously detected, in a crude rat parotid extract, a constitutively active protein kinase catalyzing phosphorylation of destrin; however, its identification has been hampered by difficulty in its enrichment. The purpose of this study was to explore a simple purification method(s) for this enzyme. To this end, we first developed a high-throughput dot-blot assay for the kinase with an anti-phosphodestrin antibody and then studied its purification by column chromatography on several media. We found that the kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite columns. In each chromatography, however, the kinase could be eluted, at the cost of resolution, only by sharp increases in the elution power of the eluent; gradual increases in the elution power resulted in unacceptably poor recovery. We confirmed that enzymatic properties of the kinase were not basically altered during the purification. Further purification of the kinase was achieved by native polyacrylamide gel electrophoresis (PAGE), which resolved the kinase activity into two bands and separated the activity from most proteins (the kinase activity after PAGE was detected with destrin-coated polyvinylidene difluoride membranes and the anti-phosphodestrin antibody). The two bands seem to constitute the major destrin-phosphorylating activity in the resting rat parotid gland. We here report its partial purification and characterization together with the detection methods.
Highlights
Introduction βAdrenergic stimulation of the rat parotid gland induces potent α-amylase secretion through cAMP-dependent protein kinase activation [1] [2]
Because the cortical actin layer disappears upon β-adrenergic stimulation [6], it seems possible that β-adrenergic stimulation-induced dephosphorylation of destrin and/or cofilin is involved in depolymerization of cortical F-actin and thereby in α-amylase secretion [3]
To detect destrin phosphorylated during incubation, we employed western blotting, which was time-consuming and occasionally provided poor results derived from uneven transfer of proteins
Summary
Adrenergic stimulation of the rat parotid gland induces potent α-amylase secretion through cAMP-dependent protein kinase activation [1] [2]. During the search for endogenous substrates of the kinase, we unexpectedly observed that two endogenous proteins are dephosphorylated in response to β-adrenergic stimulation of this tissue and identified them as destrin and cofilin [3] [4]. The two proteins are structurally related actin-binding proteins [5], and their dephosphorylated forms are active in binding to F- and G-actin and in depolymerizing and severing F-actin [5]. Destrin is called actin-depolymerizing factor (ADF) [5]. Because the cortical actin layer disappears upon β-adrenergic stimulation [6], it seems possible that β-adrenergic stimulation-induced dephosphorylation of destrin and/or cofilin is involved in depolymerization of cortical F-actin and thereby in α-amylase secretion [3]
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