title: Purification and characterisation of a plasminogen-binding protein from Haemophilus influenzae. Sequence determination reveals identity with aspartase.
abstract: 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.
authors.affiliation: Department of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden.
authors.forename: I
authors.initials: I
authors.lastname: Sjöström
authors.forename: H
authors.initials: H
authors.lastname: Gröndahl
authors.forename: G
authors.initials: G
authors.lastname: Falk
authors.forename: G
authors.initials: G
authors.lastname: Kronvall
authors.forename: M
authors.initials: M
authors.lastname: Ullberg
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C084529:plasminogen-binding protein, bacteria; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D001220:Aspartate Ammonia-Lyase