Purification, analysis, and enzymatic activity of recombinant human synovial fluid phospholipase A2 and N-terminal variants.
Recombinant human synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized. The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges. Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded. Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.
Di Marco
S
S
Biotechnology Department, Ciba-Geigy, Ltd., Basel, Switzerland.
Märki
F
F
Hofstetter
H
H
Schmitz
A
A
van Oostrum
J
J
Grütter
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013582:Synovial Fluid / Q000201:enzymology
D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2