The analysis of five carbenicillin-hydrolysing enzymes by electrophoretic methods.
Five carbenicillin-hydrolysing enzymes (carbenicillinases, or CARB), PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3, CARB-4 and CARB-5, and the beta-lactamase PSE-2 were compared by analysing their isoelectric points (pI), electrophoretic mobilities (mR) and titration curves (pH gradient electrophoresis). The pI determined by isoelectric focusing were 4.3 (CARB-4), 5.3 (PSE-4/CARB-1), 5.7 (PSE-1/CARB-2), 5.75 (CARB-3), 6.1 (PSE-2) and 6.35 (CARB-5). Their mR were estimated by zone electrophoresis as congruent to 26 for PSE-1 (CARB-2), CARB-3 and CARB-5, congruent to 30 for PSE-2, congruent to 33 for PSE-4 (CARB-1) and congruent to 61 for CARB-4. Titration curve analyses indicated that (1) PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5 are closely related variants differing by a few amino acid substitutions; (2) the qualitative titration curve of CARB-4 is different from those of PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5, although their patterns are somewhat similar; and (3) PSE-2 has no structural relationship with any of the other carbenicillin-hydrolysing enzymes or carbenicillinases (CARB) studied. Electrophoretic methods, and in particular titration curve determination combined with other physicochemical and enzymatic data, allowed a rapid comparison of the molecular structures of the beta-lactamases, and hence their classification.
Vedel
G
G
Laboratoire de Bactériologie, CHU Cochin, Paris.
Picard
B
B
Paul
G
G
Philippon
A
A
Gilly
L
L
Krishnamoorthy
R
R
Névot
P
P
D000150:Acinetobacter / Q000201:enzymology*; D002228:Carbenicillin / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D001618:beta-Lactamases / Q000032:analysis*
D001618:beta-Lactamases; D002228:Carbenicillin