Characterization of heparinase from an oral bacterium Prevotella heparinolytica.
Heparinase was purified to homogeneity from the cell extract of an oral bacterium, Prevotella heparinolytica, by a combination of anion exchange chromatography, gel filtration chromatography, and hydroxyapatite chromatography. Properties of the purified P. heparinolytica heparinase (P. heparinase) were investigated. The enzyme exhibited a maximum activity in 50 mM Tris-HCl buffer, pH 7.5-8.0, containing 75 mM sodium acetate, 0.1 M NaCl, and 1 mM CaCl2. Optimum conditions for the maximum activity of P. heparinase were similar to those of the heparinase from Flavobacterium heparinum (F. heparinase). The two enzymes also yielded similar digestion profiles of various glycosaminoglycans and heparin tetrasaccharides, suggesting that they have a similar substrate specificity. Kinetic study of the P. heparinase reaction using porcine intestinal heparin as substrate gave a Km value of 3.8 x 10(-5) M and a Vmax value of 11.4 micromol/min x mg protein. The Michaelis constant of P. heparinase was slightly larger than but not significantly different from that of F. heparinase. The amino acid composition of P. heparinase was also similar to that of F. heparinase, but its N-terminal sequence of 20 amino acid residues was different and hitherto unreported. These results together indicate that these heparinases are different proteins with closely similar enzymatic properties. Since F. heparinum produces not only heparinase but also heparitinase II, which has a broad substrate specificity, F. heparinase may be contaminated with this enzyme. In contrast, P. heparinolytica does not produce heparitinase II, and P. heparinase should prove a useful tool for degrading heparin without the risk of contamination with heparitinase II.
Watanabe
M
M
Department of Biochemistry, Kobe Pharmaceutical University.
Tsuda
H
H
Yamada
S
S
Shibata
Y
Y
Nakamura
T
T
Sugahara
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D005417:Flavobacterium / Q000201:enzymology; D019763:Heparin Lyase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009055:Mouth / Q000382:microbiology*; D010446:Peptide Fragments / Q000032:analysis; D018720:Prevotella / Q000201:enzymology*; D017421:Sequence Analysis; D013379:Substrate Specificity
D000596:Amino Acids; D010446:Peptide Fragments; D019763:Heparin Lyase