Howard M. Steinman

Construction of an Escherichia coli K-12 strain deleted for manganese and iron superoxide dismutase genes and its use in cloning the iron superoxide dismutase gene of Legionella pneumophila

SummaryAn Escherichia coli K-12 strain deleted for sodA and sodB (manganese and iron superoxide dismulases) was constructed and characterized by Southern blotting, enzyme assays, and physiological analyses. The sod deletion strain was used to clone the iron superoxide dismutase gene of Legionella pneumophila by complementation to paraquat resistance.

γ-Glutamyl transpeptidase from WI-38 fibroblasts: Purification and active site modification studies

γ-Glutamyl transpeptidase has been purified to homogeneity from WI-38 human fetal lung fibroblasts, following extraction with Triton X-100 in the absence of added proteases. The specific activity of the purified enzyme is 16 units/mg protein at the optimum of pH 8.0. Although this activity value is low, the WI-38 enzyme is very similar to previously described γ-glutamyl transpeptidases in its molecular properties. The native molecule (apparent molecular weight of 82,000) is composed of one light and one heavy subunit (apparent molecular weights of 20,000 and 62,000, respectively). Papain digestion reduces the native molecular weight to an apparent value of 73,000 by proteolysis of the heavy chain. The known active site modifying agent and glutamine analog 6-diazo-5-oxo-l-nor-leucine, completely inactivates the enzyme, coincident with its stoichiometric incorporation into the light subunit. This inactivation is accelerated by maleate and prevented by S-methylglutathione. The WI-38 γ-glutamyl transpeptidase is also inactivated by the fluorescent alkylating agent, 5-iodoacetamidofluorescein. Selective reaction of this reagent with an active site residue is suggested by prevention of the inactivation by S-methylglutathione, the stoichiometric incorporation of the fluorescein moiety, and the loss of one methionine residue per molecule of protein accompanying inactivation.

Bacterial superoxide dismutases.

Superoxide dismutase (SOD) has played a major role in establishing the biological relevance of oxyradicals. To many oxyradical researchers, “superoxide dismutase” connotes a copper-zinc SOD (CuZnSOD), the first dismutase to be purified and the form of prime interest clinically. However, superoxide dismutases containing manganese or iron as cofactor are also known and are widespread among bacteria. The manganese and iron SODs (MnSOD and FeSOD) are homologous in primary and three-dimensional structure and have clearly evolved from a common ancestor.2,3 Although structurally homologous, the Mn- and FeSODs are functionally distinct. Only in rare instances can the endogeneous Mn (or Fe) be replaced by Fe (or Mn) with retention of catalytic activity. The Mn- and FeSODs are further distinguished in their distribution among bacterial species. Strict anaerobes contain one SOD, an FeSOD. Bacterial aerobes usually contain an MnSOD or both Mn- and FeSODs. (The MnSOD is also widely found in eukaryotes. The FeSOD is also found in primitive eukaryotes and some green plants. Distributions of the Mn- and FeSODs among eukaryotes have been discussed elsewhere.1,4)

Purification and characterization of γ-Glutamyltransferase from rat pancreas

γ-Glutamyltransferase ((5-giutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68 000, composed of one heavy and one light subunit, with respective molecular weights 43 000 and 25 000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with l-γ-glutamyl-pnitroanilide as substrate (Km = 0.9 mM) and 20 mM glycylgiycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-l-norleucine, through a specific and stoichiometric reaction with the light subunit (Ki = 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney γ-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.

Catalase-peroxidase activity is decreased in a Caulobacter crescentus rho mutant.

A Caulobacter crescentus rho::Tn5 mutant strain presenting a partially functional transcription termination factor Rho is highly sensitive to hydrogen peroxide in both exponential and stationary phases. The mutant was shown to be permanently under oxidative stress, based on fluorophore oxidation, and also to be sensitive to tert-butyl hydroperoxide and paraquat. However, the results showed that the activities of superoxide dismutases CuZnSOD and FeSOD and the alkylhydroperoxide reductase ahpC mRNA levels in the rho mutant were comparable to the wild-type control in the exponential and stationary phases. In contrast, the KatG catalase activity of the rho mutant strain was drastically decreased and did not show the expected increase in the stationary phase compared with the exponential phase. Transcription of the katG gene was increased in the rho mutant and the levels of the immunoreactive KatG protein do not differ considerably compared with the wild type in the stationary phase, suggesting that KatG activity is affected in a translational or a post-translational step.