PURIFICATION AND CHARACTERIZATION OF A PLASMINOGEN-BINDING PROTEIN FROM HAEMOPHILUS-INFLUENZAE - SEQUENCE DETERMINATION REVEALS IDENTITY WITH ASPARTASE

Plasminogen binding proteins have been described both for Gram positiv e and Gram negative bacteria. In the present work we describe the puri fication and characterization of a plasminogen binding protein from Ha emophilus influenzae (strain HI-23459). Bacteria were sonicated in ord er to solubilize plasminogen-binding proteins. The supernatant was sub jected to affinity chromatography on plasminogen kringle-4 fragment bo und to Sepharose 4B and subsequently processed by ion-exchange chromat ography on DEAE-Sepharose CL-6B. Characterization of the protein by SD S-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. Antibodi es were raised in rabbits and used in quantitative and qualitative ana lysis. However, using a FITC-conjugate we failed to demonstrate the pr esence of the purified protein on the surface of intact bacteria. The corresponding gene was isolated from a lambda EMBL3 phage library prep ared 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 a mino acid sequence of the translated gene demonstrates 97% identity wi th the recently published sequence from aspartate ammonia lyase (aspar tase) from H. influenzae. Enzymatic analysis of the purified protein r evealed a high aspartase activity.