The sialate pyruvate-lyase from pig kidney: Purification, properties and genetic relationship

被引:10
作者
Sommer, U [1 ]
Traving, C [1 ]
Schauer, R [1 ]
机构
[1] Univ Kiel, Inst Biochem, D-24098 Kiel, Germany
关键词
sialate pyruvate-lyase; N-acetylneuraminate lyase; aldolase; pig kidney; purification; properties; mammalian lyases; bacterial lyases; primary structures; alignment;
D O I
10.1023/A:1007030627948
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
For further insight into the structural relationship between mammalian and microbial sialate pyruvate-lyases, the enzyme from pig kidney was purified to homogeneity from the tissue homogenate by a heat precipitation step followed by anion exchange and Hydrophobic Interaction Chromatography or native gel electrophoresis, respectively. The pure enzyme preparation exhibited an about 1000-fold increase of specific activity compared to the supernatant after the first centrifugation and revealed a single band at 34-37 kDa after SDS-PAGE, which represents the monomeric form of the protein. While the native enzyme seems to be a trimer according to the molecular weight obtained by gel filtration (108 kDa), crosslinking with dimethylpimelimidate suggests it to be a tetramer. The lyase is optimally active at about 75 degrees C and in the pH range of 7.6 to 8.0 and belongs to the class I-aldolases [1], due to its non-requirement of metal ions and the presence of lysine as the main functional residue in its catalytic centre. These data are similar to those obtained with bacterial lyases. However, peptide fragments of this enzyme show less similarity to primary lyase structures of microbia than to those derived from expressed sequence tags of mammals.
引用
收藏
页码:425 / 435
页数:11
相关论文
共 44 条
  • [1] PURIFICATION, CRYSTALLIZATION AND CHARACTERIZATION OF N-ACETYLNEURAMINATE LYASE FROM ESCHERICHIA-COLI
    AISAKA, K
    IGARASHI, A
    YAMAGUCHI, K
    UWAJIMA, T
    [J]. BIOCHEMICAL JOURNAL, 1991, 276 : 541 - 546
  • [2] BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
  • [3] BRUNETTI P, 1962, J BIOL CHEM, V237, P2447
  • [4] COMB DG, 1960, J BIOL CHEM, V235, P2529
  • [5] Corfield A.P., 1982, CELL BIOL MONOGR, V10, P195
  • [6] WHOLE-GENOME RANDOM SEQUENCING AND ASSEMBLY OF HAEMOPHILUS-INFLUENZAE RD
    FLEISCHMANN, RD
    ADAMS, MD
    WHITE, O
    CLAYTON, RA
    KIRKNESS, EF
    KERLAVAGE, AR
    BULT, CJ
    TOMB, JF
    DOUGHERTY, BA
    MERRICK, JM
    MCKENNEY, K
    SUTTON, G
    FITZHUGH, W
    FIELDS, C
    GOCAYNE, JD
    SCOTT, J
    SHIRLEY, R
    LIU, LI
    GLODEK, A
    KELLEY, JM
    WEIDMAN, JF
    PHILLIPS, CA
    SPRIGGS, T
    HEDBLOM, E
    COTTON, MD
    UTTERBACK, TR
    HANNA, MC
    NGUYEN, DT
    SAUDEK, DM
    BRANDON, RC
    FINE, LD
    FRITCHMAN, JL
    FUHRMANN, JL
    GEOGHAGEN, NSM
    GNEHM, CL
    MCDONALD, LA
    SMALL, KV
    FRASER, CM
    SMITH, HO
    VENTER, JC
    [J]. SCIENCE, 1995, 269 (5223) : 496 - 512
  • [7] Enzymatic and molecular properties of the Clostridium tertium sialidase
    Grobe, K
    Sartori, B
    Traving, C
    Schauer, R
    Roggentin, P
    [J]. JOURNAL OF BIOCHEMISTRY, 1998, 124 (06) : 1101 - 1110
  • [9] DETERMINATION OF MONO-O-ACETYLATED N-ACETYLNEURAMINIC ACIDS IN HUMAN AND RAT SERA BY FLUOROMETRIC HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
    HARA, S
    YAMAGUCHI, M
    TAKEMORI, Y
    FURUHATA, K
    OGURA, H
    NAKAMURA, M
    [J]. ANALYTICAL BIOCHEMISTRY, 1989, 179 (01) : 162 - 166
  • [10] HEUKESHOVEN J, 1986, ELEKTROPHORESEFORUM, P92