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.
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.
Department of Legal Medicine, Shimane University School of Medicine, Izumo, Shimane 693-8501, Japan.
J
J
Fujihara
Y
Y
Hieda
Y
Y
Xue
N
N
Nakagami
K
K
Takayama
K
K
Kataoka
H
H
Takeshita
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D009928:Organ Specificity / Q000502:physiology; D010179:Pancreas / Q000201:enzymology*; D010306:Parotid Gland / Q000201:enzymology*; D046188:Protein Modification, Translational / Q000502:physiology; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013552:Swine
D003850:Deoxyribonuclease I; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase