Partial Purification and Characterization of the Rat Parotid Gland Protein Kinase Catalyzing Phosphorylation of Matured Destrin at Ser-2

Eriko Osumi, Chihiro Kondo, Mitsumasa Mizuno, Takahiro Suzuki, Mamoru Matsubara, Kazuo Shimozato, Takao Kanamori

Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan.
Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyotogakuen University, Kyoto, Japan.
Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan.

DOI: 10.4236/aer.2014.22011                

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.

Keywords

Protein Kinase Purification, Protein Kinase Assay, Destrin Kinase, Actin-Depolymerizing Factor, Rat Parotid Gland

Conflicts of Interest

The authors declare no conflicts of interest.

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