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One-Step Purification of Mammalian Deoxyribonucleases I and Differences among Pancreas, Parotid, and Pancreas-Parotid (Mixed) Types Based on Species- and Organ-Specific N-Linked Glycosylation

J. Fujihara, Y. Hieda, Y. Xue, N. Nakagami, K. Takayama, K. Kataoka, and H. Takeshita*

Department of Legal Medicine, Shimane University School of Medicine, 89-1 Enya, Izumo, Shimane 693-8501, Japan; fax: +81-853-20-2155; E-mail: htakeshi@med.shimane-u.ac.jp

* To whom correspondence should be addressed.

Received June 16, 2005; Revision received July 14, 2005
Mammalian deoxyribonucleases I (DNase I) are classified into three types, namely, pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations. In this study, DNase I purification by concanavalin A-wheat germ agglutinin mixture-agarose column from rat (parotid type), rabbit (mixed type), and pig (pancreas type) is described. This method permits a relatively easy one-step purification of DNase I from rat and rabbit parotid glands, the rat submaxillary gland, and porcine pancreas. To elucidate differences among the three types, these DNases I were subjected to enzymatic deglycosylation either by peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). Following deglycosylation, digests were separated on DNA-casting polyacrylamide gel electrophoresis. PNGase F produced a single lower mobility product in all samples. Endo H produced a double band in rat and rabbit parotid glands and porcine pancreas, and a single band in the rabbit pancreas corresponding with the PNGase F product. DNase I activity of the porcine pancreas was completely extinguished by deglycosylation, while that of the parotid glands and rabbit pancreas was unaffected. Our results suggest that the distinct properties of DNase I exhibited by the three types may be attributed to differences in the extent of post-translational N-linked glycosylation of the enzyme.
KEY WORDS: Con A, DNase I, glycosylation, pancreas, parotid gland, submaxillary gland, WGA

DOI: 10.1134/S0006297906130116