Bovine pancreatic deoxyribonuclease F: isoelectric focusing, peptide mapping and primary structure.
DNAase F is a minor isoform of bovine pancreatic DNAase which can be separated from the other isoforms (DNAases A, B, C and D) present in a commercial preparation by a preparative isoelectric-focusing cell (Rotofor; Bio-Rad). The ampholytes and other contaminating proteins present in DNAase F preparations can be removed by chromatography on an affinity column (Cibacron Blue 3GA-agarose) and a hydrophobic-interaction column (phenyl-Sepharose CL-4B). The complete separation of DNAase F from the other isoforms is demonstrated on a thin-layer isoelectric-focusing gel, DNAase F being the most basic (pI 5.68). A procedure is described for tryptic peptide mapping by h.p.l.c. requiring only picomolar amounts of DNAase F protein. The DNAase F map shows two peptide peaks not present in the DNAase A map, and the DNAase F map does not have a peak at the position where a C-terminal peptide of DNAase A is normally eluted. The amino acid compositions and sequences for the two new peptides suggest that Gly240 in DNAase A is changed to Arg240 in DNAase F.
Chang
Y M
YM
Institute of Biochemistry, National Taiwan University/College of Medicine, Taipei.
Lin
S
S
Liao
T H
TH
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D007525:Isoelectric Focusing; D007527:Isoenzymes; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D015183:Restriction Mapping
D007527:Isoenzymes; D001120:Arginine; D003851:Deoxyribonucleases; D005998:Glycine