Two types of 1-lactamase were found in the cell-free extract from Pseudomonas maltophilia GN12873. One was an inducible penicillin 3-lactamase, and the other was an inducible cephalosporin 3-lactamase. The purified penicillin 1B-lactamase gave a single protein band on polyacrylamide gel electrophoresis. The isoelectric point was 6.9, and the approximate molecular weight was 118,000 by gel filtration and 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this enzyme consisted offour subunits. For the hydrolysis of penicillin G, the optimal pH was 8.0 and the optimal temperature was 35°C. The enzyme activity was inhibited by cephamycin derivatives, carpetimycins A and B, iodine, and HgCl2, but not by clavulanic acid. Furthermore, ,B-lactamase activity was almost completely inhibited by EDTA but was recovered by the addition of zinc ion. The enzyme showed a unique substrate profile, hydrolyzing N-formimidoyl thienamycin at a significant rate.

An inducible cephalosporinase was purified from Pseudomonas maltophilia GN12873. The pI was 8.4, and the molecular weight was ca. 56,000 by gel filtration or 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this enzyme had two subunits. The optimal pH and optimal temperature were 7.5 and 45°C, respectively. Enzyme activity was inhibited by clavulanic acid, sulbactam, cephamycin derivatives, carbapenem antibiotics, iodine, HgCI2, and p-chloromercuribenzoate. The enzyme showed a broad substrate profile, hydrolyzing cephaloridine, cefazolin, cefsulodin, penicillin G, ceftizoxime, and ampicillin at a high rate.There have not been many investigations of the P-lactamases of Pseudomonas maltophilia, even though most clinical isolates of this organism are resistant to many P-lactam antibiotics, including imipenem (N-formimidoyl thienamycin) and aztreonam (6,8,9,14).Recently we reported that seven strains of P. maltophilia produced two types of ,B-lactamase, designated as L-1 and L-2 (17, 18). The L-1 enzyme purified from the representative strain GN12873 was found to be a new type of penicillinase from its unusual enzymological properties. The L-2 enzyme appeared to be a cephalosporinase and seemed to play an important role in the resistance mechanisms of P. maltophilia to various cephalosporins.The present paper compares the physicochemical and enzymological properties of the L-2 enzyme produced by P. maltophilia GN12873 with other P-lactamases.MATERIALS AND METHODS Bacterial strain. Strain GN12873 is a clinical isolate of P. maltophilia. It was stored at -80°C in skim milk containing 5% glucose (pH 7.4).

An inhibitor of blood coagulation, a new protein with an apparent molecular weight of 34,000 and an isoelectric point of 4.9, was purified from human placental tissue by EDTA extraction. Five cDNA clones were isolated from the human placental lambda gt11 cDNA library using the mouse monoclonal antibody raised against the coagulation inhibitor as the probe. The longest insert consists of 1,566 nucleotides, and contains 960 nucleotides entirely encoding the 320 amino acids of the inhibitor, and a poly A tail. The deduced amino acid sequence was corroborated by chemical analyses of the protein. The entire amino acid sequence shows homology to those of lipocortin I, lipocortin II, and endonexin-related proteins. The cDNA for the inhibitor was expressed in Escherichia coli under the regulation of the trc promotor of the plasmid pKK233-2. The resulting recombinant protein manifested inhibitory activities against both blood coagulation and phospholipase A2 activity, as did the coagulation inhibitor isolated from human placenta.

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Renal dipeptidase purified from swine kidney hydrolyzed N -formimidoyl thienamycin, carpetimycins A and B, and Sch29482, but not azthreonam, penicillin G, or cephaloridine.