Nucleotide sequence of the bla(RTG-2) (CARB-5) gene and phylogeny of a new group of carbenicillinases.

We determined the nucleotide sequence of the bla gene for the Acinetobacter calcoaceticus beta-lactamase previously described as CARB-5. Alignment of the deduced amino acid sequence with those of known beta-lactamases revealed that CARB-5 possesses an RTG triad in box VII, as described for the Proteus mirabilis GN79 enzyme, instead of the RSG consensus characteristic of the other carbenicillinases. Phylogenetic studies showed that these RTG enzymes constitute a new, separate group, possibly ancestors of the carbenicillinase family.

Characterization and nucleotide sequence of CARB-6, a new carbenicillin-hydrolyzing beta-lactamase from Vibrio cholerae.

A clinical strain of Vibrio cholerae non-O1 non-O139 isolated in France produced a new beta-lactamase with a pI of 5.35. The purified enzyme, with a molecular mass of 33,000 Da, was characterized. Its kinetic constants show it to be a carbenicillin-hydrolyzing enzyme comparable to the five previously reported CARB beta-lactamases and to SAR-1, another carbenicillin-hydrolyzing beta-lactamase that has a pI of 4.9 and that is produced by a V. cholerae strain from Tanzania. This beta-lactamase is designated CARB-6, and the gene for CARB-6 could not be transferred to Escherichia coli K-12 by conjugation. The nucleotide sequence of the structural gene was determined by direct sequencing of PCR-generated fragments from plasmid DNA with four pairs of primers covering the whole sequence of the reference CARB-3 gene. The gene encodes a 288-amino-acid protein that shares 94% homology with the CARB-1, CARB-2, and CARB-3 enzymes, 93% homology with the Proteus mirabilis N29 enzyme, and 86.5% homology with the CARB-4 enzyme. The sequence of CARB-6 differs from those of CARB-3, CARB-2, CARB-1, N29, and CARB-4 at 15, 16, 17, 19, and 37 amino acid positions, respectively. All these mutations are located in the C-terminal region of the sequence and at the surface of the molecule, according to the crystal structure of the Staphylococcus aureus PC-1 beta-lactamase.

Endogenous proteolysis of membrane-bound red cell cytochrome-b5 reductase in adults and newborns: its possible relevance to the generation of the soluble "methemoglobin reductase".

The problem of the low activity of so-called methemoglobin reductase in red cells from newborns was reinvestigated in view of our current knowledge of this enzyme, i.e., (1) its being cytochrome-b5 reductase and (2) its presence in two forms: soluble and membrane-bound. We found that red cells from cord blood and newborns exhibited a 50% decrease of soluble cytochrome-b5 reductase activity, whereas membrane-bound activity was in the adult range. Ghosts from these cells possessed diminished ability to solubilize membrane-bound cytochrome-b5 reductase in the course of in vitro auto-incubation. This autosolubilizing ability increased with age and reached adult level concomitantly with soluble cytochrome-b5 reductase activity at 6 mo. We conclude that the relative deficiency of soluble cytochrome-b5 reductase observed at birth is due to diminished post-translational processing of the membrane-bound enzyme during erythropoiesis of fetal cells. This processing is calcium-dependent related to calmodulin.

3',5' Cyclic nucleotide phosphodiesterase from human platelets: effect of heat upon the multiple forms and their interconversion.

A 33,000 g supernatant from human platelets showed a biphasic heat inactivation curve at 45, 50 and 55 degrees C of the cAMP and cGMP phosphodiesterase. This could suggest the presence of two differently heat sensitive phosphodiesterases. However, a preparation heated for 30 min at 55 degrees C, where only the apparently thermostable form of the enzyme remained, still displayed the same characteristics as the starting material, i.e. two apparent Km values for cAMP, a cAMP specific activity lower at low protein concentration (less than 50 micrograms/ml) than at high protein concentration(greater than 100 micrograms/ml), and three peaks of activity upon linear sucrose density gradient. Moreover, a biphasic inactivation curve was again observed after a second heat treatment. These results demonstrated that the heat effect is not a simple protein denaturation of one of two independent species. A study at different temperatures of the profile of the cAMP phosphodiesterase upon sucrose gradient demonstrated that the dissociated form was predominant at high temperature whereas lower temperature favored the associated form. During heat treatment, the dissociated form is at first denatured and this leads to a shift in the equilibrium between the associated and dissociated forms of the phosphodiesterase in favor of the dissociated form. From the overall results, one can draw a model for phosphodiesterase regulation by dissociation-reassociation.

Membrane-bound cytochrome b5 reductase (methemoglobin reductase) in human erythrocytes. Study in normal and methemoglobinemic subjects.

In this study we present evidence that in human erythrocytes NADH-cytochrome b5 reductase (methemoglobin reductase) is not only soluble but also tightly bound to the membrane. The membrane methemoglobin reductase-like activity is unmasked by Triton X-100 treatment, and represents about half of the total activity in the erythrocytes. Like the amphiphilic microsomal-bound cytochrome b5 reductase, the erythrocyte membrane-bound enzyme is solubilized by cathepsin D. Because this treatment is effective on unsealed ghosts but not on resealed (inside-in) ghosts, it is concluded that the enzyme is strongly bound to the inner face of the membrane. The erythrocyte membrane enzyme is antigenically similar to the soluble enzyme. The two forms of enzyme are specified by the same gene, in that both were found defective in six patients with recessive congenital methemoglobinemia. We suggest that the cytochrome b5 reductase of the erythrocyte membrane is the primary gene product. A posttranslational partial proteolysis probably gives rise to the soluble form of the enzyme, which serves as a methemoglobin reductase.

Diaphorase P: a new fetal isozyme identified in human placenta.

Human placenta contains a thermostable, cytosolic NADH-diaphorase which is different from the other diaphorases and which we designate as diaphorase P. It is specific for NADH and reduces artificial substrates such as dichlorophenol and tetrazolium derivatives, but not natural substrates such as methemoglobin, cytochrome b5 or lipoate. It is antigenically distinct from the ubiquitous red-cell type NADH-diaphorase (soluble cytochrome b5 reductase) specified by the DIA1 locus. Using electrophoretic and immunologic methods, it was possible to detect diaphorase P in various fetal tissues (brain, liver, kidney, muscle), whereas was not found in adult tissues with the exception of the brain. This enzyme, the physiological role of which remains unknown, appears to belong, therefore, to the category of fetal proteins. Its resurgance in primary liver cancer was demonstrated in three cases.