Cytochrome P450-linked p-nitroanisole O-demethylation in the perfused lung.

Oxidative demethylation of p-nitroanisole, a cytochrome P450-linked mixed-function oxidation, was evaluated in isolated perfused rat and rabbit lungs. The product, p-nitrophenol, was monitored continuously in the lung effluent by spectrophotometric measurement. Pulmonary p-nitrophenol production in mumol/h per g dry wt was 6.2+/-0.4 by rabbits and 2.0+/-0.3 by rats (mean+/-SE). Maximal activity of the reaction required pulmonary perfusion rates in excess of 60-80 ml/min per g of dry lung. The half-maximal rate of p-nitrophenol production was observed with p-nitroanisole concentration of 13 micron. Pretreatment of rabbits with chlorpromazine increased p-nitroanisole O-demethylation activity by 63% but phenobarbital pretreatment had no effect. Ventilation with 75% carbon monoxide plus 20% O2 reversibly inhibited the reaction. Specific activity of p-nitroanisole demethylase in the microsomal fraction was 0.5 nmol/min per mg protein in rabbit lungs and 0.1 nmol/min per mg protein in rat lungs. Other rabbit lung subcellular fractions compared with microsomes had significantly lower specific activity. This study demonstrates that p-nitroanisole O-demethylation can be continuously monitored in the intact lung and describes conditions necessary for maximal activity of this pathway.

Beta-D-phosphogalactoside galactohydrolase from Streptococcus cremoris HP: purification and enzyme properties.

beta-d-phosphogalactoside galactohydrolase (beta-PGal) was isolated and purified from cell-free extracts of Streptococcus cremoris HP to apparent homogeneity to gel electrophoresis. Using the chromogenic o-nitrophenol-beta-d-galactopyranoside-6-phosphate as substrate, the purified enzyme exhibited a specific activity of 18.71 U/mg of protein and K(m) and V(max) values of 5.88 x 10(-4) M and 23.8 mumol of o-nitrophenol liberated per min per mg of protein, respectively. d-Galactose-6-phosphate was a weak competitive inhibitor of beta-PGal. Activity was relatively heat resistant and was maximal from pH 5.0 to 8.0 and over a temperature range of 45 to 52 C. Dithiothreitol, ethylenediaminetetraacetic acid, and citrate stimulated beta-PGal activity, whereas Mg(2+), Li(1+), and p-hydroxymercuribenzoate were inhibitory. Molecular weight of the enzyme was estimated at 6.76 x 10(4). Amino acid composition was similar to other beta-phosphogalactosidases previously investigated, with the exception that the S. cremoris enzyme contains a small amount of half cystine.

Methods for visualization of enzymes in polyacrylamide gels.

White bands resulting from precipitation of dodecan-1-ol liberated by hydrolysis of sodium dodecyl sulfate and decan-5-ol released by hydrolysis of decan-5-yl sulfate produced zymograms of the primary and secondary alkylsulfatases from Pseudomonas C(12)B. Gas-liquid chromatographic analyses of ether extracts of the precipitate-containing segments of the zymograms confirmed the identity of the alcohols which were not discerned in extracts of segments of the gels other than those containing precipitates. beta-Galactosidase from Escherichia coli was marked on zymograms by the liberation of o-nitrophenol from o-nitrophenyl-beta-D-galactoside, and arylsulfatase from Pseudomonas C(12)B was marked in gels by liberation of p-nitrophenol from p-nitrophenyl sulfate. Membrane-associated dissimilatory nitrate reductases from a nitrate respirer (Enterobacter aerogenes) and a denitrifier (Pseudomonas perfectomarinus) did not penetrate either 6.8 or 3% polyacrylamide gel but were demonstrable at the top of the gels. In the membrane-bound state, formate served as electron donor for nitrate reductase from E. aerogenes, and reduced nicotinamide adenine dinucleotide (NADH) served as donor for nitrate reductase from P. perfectomarinus. Both enzymes reduced nitrate at the expense of reduced benzyl viologen as well. Assimilatory nitrate reductase from E. aerogenes moved easily into the 6.8% gels (R(f) = 0.43 under the conditions of these experiments). The reduced dye served as electron donor for the assimilatory reductase, but formate and NADH did not. Incubation of the membrane-associated nitrate reductases with 2% Triton X-100 solubilized the enzymes and removed the capacity of formate and NADH to serve as electron donors. Both retained the ability to reduce nitrate at the expense of reduced benzyl viologen. The solubilized dissimilatory reductase from E. aerogenes moved further in the gels (R(f) = 0.49) than the soluble assimilatory reductase; the solubilized dissimilatory reductase from the denitrifier, P. perfectomarinus, moved further in the gels (R(f) = 0.64) than either of the enzymes from E. aerogenes.

Selective autocytotoxicity in a model system of Escherichia coli K-12 recombinants.

Autocytotoxicity was shown by Lac(+) recombinant strains of Escherichia coli K-12 when their growth was inhibited in media containing o-nitrophenyl-beta-d-galactopyranoside. Lac(-) strains without lactose permease or beta-galactosidase activity grew well. Selective autocytotoxicity was shown by simultaneous inhibition of Lac(+) cells and multiplication of Lac(-) cells grown together in this medium.

Isolation and characterization of acid phosphatase mutants in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strain H-42 seems to have two kinds of acid phosphatase: one which is constitutive and one which is repressible by inorganic phosphate. The constitutive enzyme was significantly unstable to heat inactivation, and its K(m) of 9.1 x 10(-4)m for p-nitrophenylphosphate was higher than that of the repressible enzyme (2.4 x 10(-4)m). The constitutive and the repressible acid phosphatases are specified by the phoC gene and by the phoB, phoD, or phoE gene, respectively. Results of tetrad analysis suggested that the phoC and phoE genes are linked to the lys2 locus on chromosome II. Since both repressible acid and alkaline phosphatases were affected simultaneously in the phoR, phoD, and phoS mutants, it was concluded that these enzymes were under the same regulatory mechanism or that they shared a common polypeptide. The phoR mutant produced acid phosphatase constitutively, and the phoR mutant allele was recessive to its wild-type counterpart. The phoS mutant showed a phenotype similar to that of a mutant defective in one of the phoB, phoD, or phoE genes. However, the results of genetic analysis of the phoS mutant clearly indicated that the phoS gene is not a structural gene for either of the repressible acid and alkaline phosphatases, but is a kind of regulatory gene. According to the proposed model, the phoS gene controls the expression of the phoR gene, and inorganic phosphate would act primarily as an inducer for the formation of the phoR product which represses phosphatase synthesis.

Biochemical characterization of the ctr mutants of Escherichia coli.

A test procedure based on complementation in mixed extracts is described for the assay of heat-stable protein and enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. The test was used to assay a collection of pleiotropic carbohydrate mutants of Escherichia coli (ctr mutants) and revertants of these mutants. All mutants were found to lack enzyme I of the phosphoenolpyruvate-dependent transferase system. Revertants of these mutants to complete wild phenotype regained enzyme I-forming ability. Reversion to partial wild type was not accompanied by restoration of enzyme I-forming ability.

Salmonella screening procedure with tests for beta-galactosidase and flagellar antigens.

Differentiation of Salmonella from other gram-negative bacilli requires several biochemical and serological tests. A simplified 24-hr screening procedure has been devised which allows discarding of large numbers of isolates (picked from selective plating media) before they are subjected to this extensive testing. Cultures of gram-negative organisms isolated to triple sugar-iron slants during routine examination of products for Salmonella were tested for the presence of beta-galactosidase and Salmonella flagellar antigens. beta-Galactosidase-positive cultures which did not agglutinate in polyvalent flagellar antiserum were considered to be nonsalmonellae. Of 1,103 Salmonella cultures tested, none of the 61 different serotypes was missed by this procedure, whereas 673 (82.3%) of 818 nonsalmonellae were excluded from further testing. This screening procedure eliminates most nonsalmonellae and augments the proportion of cultures undergoing further biochemical and serological testing which will be confirmed as Salmonella.