Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of a serine residue into the Ca2+-binding site.
We purified four amphibian deoxyribonucleases I from the pancreases of one toad, two frog and one newt species, by using three different column chromatography methods in sequence. Each of the purified enzymes had a molecular mass of approx. 40 kDa and an optimal pH for activity of approx. 8.0. These values were significantly greater than those for other vertebrate DNases I. The full-length cDNA encoding each amphibian DNase I was constructed from the total RNA of the pancreas by using rapid amplification of cDNA ends. Nucleotide sequence analyses revealed two structural characteristics unique to amphibian DNases I: a stretch of approx. 70 amino acids with a high cysteine content (approx. 15%) in the C-terminal region, and the insertion of a serine residue at position 205 (in a domain containing an essential Ca2+-binding site). Expression analysis of a series of mutant constructs indicated that both of these structures are essential in generating the active form of the enzyme. 'DNase I signature sequences', which are well conserved in other vertebrate DNases I, could not be found in any of the amphibian DNases I tested, whereas a 'somatomedin B motif' was identified in the Cys-rich stretches of all four. Although DNase I has so far been considered to be a secretory glycoprotein, amphibian DNase I seems to be non-glycosylated. These structural findings indicate strongly that amphibian DNases I are situated in a unique position on the phylogenetic tree of the DNase I family.
Takeshita
H
H
Department of Legal Medicine, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan.
Yasuda
T
T
Iida
R
R
Nakajima
T
T
Mori
S
S
Mogi
K
K
Kaneko
Y
Y
Kishi
K
K
D000595:Amino Acid Sequence; D000663:Amphibians / Q000378:metabolism*; D000818:Animals; D002025:Bufonidae; D019556:COS Cells; D002522:Chlorocebus aethiops; D002852:Chromatography, Ion Exchange; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010802:Phylogeny; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012455:Salamandridae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D014162:Transfection; D014982:Xenopus laevis
D011994:Recombinant Proteins; D003850:Deoxyribonuclease I