Purification and characterisation of a plasminogen-binding protein from Haemophilus influenzae. Sequence determination reveals identity with aspartase.
Plasminogen binding proteins have been described both for Gram positive and Gram negative bacteria. In the present work we describe the purification and characterization of a plasminogen binding protein from Haemophilus influenzae (strain HI-23459). Bacteria were sonicated in order to solubilize plasminogen-binding proteins. The supernatant was subjected to affinity chromatography on plasminogen kringle-4 fragment bound to Sepharose 4B and subsequently processed by ion-exchange chromatography on DEAE-Sepharose CL-6B. Characterization of the protein by SDS-PAGE displayed a single band with a molecular mass of about 55,000, both prior to and after reduction. The purified protein stimulates tPA (tissue plasminogen activator) catalysed plasminogen activation by a factor of approximately 300, mainly due to a decrease in K(m). Antibodies were raised in rabbits and used in quantitative and qualitative analysis. However, using a FITC-conjugate we failed to demonstrate the presence of the purified protein on the surface of intact bacteria. The corresponding gene was isolated from a lambda EMBL3 phage library prepared from chromosomal DNA from the same H. influenzae strain, using an oligonucleotide probe based on the NH2-terminal amino acid sequence. An open reading frame corresponding to 472 amino acid was found. The amino acid sequence of the translated gene demonstrates 97% identity with the recently published sequence from aspartate ammonia lyase (aspartase) from H. influenzae. Enzymatic analysis of the purified protein revealed a high aspartase activity.
Sjöström
I
I
Department of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden.
Gröndahl
H
H
Falk
G
G
Kronvall
G
G
Ullberg
M
M
D000595:Amino Acid Sequence; D001220:Aspartate Ammonia-Lyase / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; C084529:plasminogen-binding protein, bacteria; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D001220:Aspartate Ammonia-Lyase