Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed.

Sodium-dependent transport of neutral amino acids by whole cells and membrane vesicles of Streptococcus bovis, a ruminal bacterium.

Streptococcus bovis JB1 cells were able to transport serine, threonine, or alanine, but only when they were incubated in sodium buffers. If glucose-energized cells were washed in potassium phosphate and suspended in potassium phosphate buffer, there was no detectable uptake. Cells deenergized with 2-deoxyglucose and incubated in sodium phosphate buffer were still able to transport serine, and this result indicated that the chemical sodium gradient was capable of driving transport.

On the membrane topology of vertebrate cytochrome P-450 proteins.

Hydropathy profiles of 34 aligned cytochrome P-450 sequences were compared to identify potential transmembrane segments. Eleven regions with the potential to cross a membrane in at least some P-450 sequences were detected. The known sidedness of several residues and peptides was used to eliminate some of these regions from consideration.

Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production.

When mixed ruminal bacteria were inoculated into semicontinuous cultures (25% transfer every other day) containing lactate, dulcitol, pectin, or xylose and Trypticase (1 g/liter) as the sole nitrogen source, the specific activity of ammonia production increased. The greatest enrichment was observed with lactate and xylose, and in these cases the specific rate of ammonia production was eightfold higher than that of the ruminal fluid control (approximately 35 nmol of ammonia per mg of protein per min). Isolates with different morphologies were obtained from each of the enrichments, but in no case did the specific activity of any isolate exceed that of the mixed ruminal bacteria.

Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450.

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the apparent Km of the reductase for cytochrome P-450b or cytochrome P-450c using either 7-ethoxycoumarin or benzphetamine as substrates. A 20-45% decrease in the Vmax was observed except for cytochrome P-450b with 7-ethoxycoumarin as substrate, where there was a 27% increase.

Effects of additives on in vitro ruminal fermentation: a comparison of monensin and bacitracin, another gram-positive antibiotic.

Mixed ruminal bacteria (n = 4) were incubated in anaerobic media for 24 h in vitro with either hay, corn meal, protein hydrolyzate or hydrogen gas as the substrate. The ionophore monensin and the polypeptide antibiotic bacitracin were added to the incubation flasks at concentrations ranging from 0 to 10 or 40 mg/liter. As was expected, monensin decreased methane production, increased the ratio of propionate to acetate and decreased the deamination of amino acids.

Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats.

A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with beta-naphthoflavone (beta-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.

Evolution of cytochrome P-450 proteins.

Thirty-four cytochrome P-450 sequences from one bacterial and six vertebrate species have been aligned with the aid of a computer alignment algorithm. Phylogenetic trees were constructed using the unweighted-pair-group and neighbor-joining methods. The two trees differed at only a single branch point near the base of the tree.

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression.

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose.

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro.

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids.

Concentration of ammonia across cell membranes of mixed rumen bacteria.

When mixed ruminal bacteria were provided with growth rate limiting amounts of mixed carbohydrates, more than 50 mg ammonia/L were required for maximal protein synthesis. Microbial protein synthesis declined when ammonia concentration was less than 50 mg/L and unfermented carbohydrates increased. Ammonia starvation also decreased growth efficiency.

Drug metabolism in rat colon: resolution of enzymatic constituents and characterization of activity.

A direct demonstration of the basis of mixed function oxidase activity in rat colonic mucosa was achieved by resolution of microsomes into two components, cytochrome P-450 and cytochrome P-450 reductase, which on recombination with phosphatidylcholine catalyzed hydroxylation of benzo[alpha]pyrene and benzphetamine. Reconstitution of hydroxylation activity requires both the cytochrome P-450 component and the cytochrome P-450 reductase component in addition to phospholipid. Omission of either of the protein components or the phospholipid component reduces the activity almost to background levels.

Distributions and properties of cytochromes P-450 and cytochrome P-450 reductase from rat colon mucosal cells.

Cytochrome P-450 reductase and cytochrome P-450 fractions have been separated and partially purified from colonic mucosal microsomes of rat pretreated with phenobarbital or beta-naphthoflavone. Colonic cytochrome P-450 reductase has a molecular weight of 76,000. The Km values of colonic cytochrome P-450 reductase for the artificial electron acceptors cytochrome c, ferricyanide, and dichlorophenolindophenol and the electron donor NADPH are 6, 50, 11 and 11 microM, respectively.

Cytochrome P-450-mediated redox cycling of estrogens.

The cytochrome P-450-mediated reactions of the synthetic stilbene estrogen (E)-diethylstilbestrol (DES) and of 2-hydroxyestradiol have been investigated in vitro. Depending on the cofactor used, microsomal enzymes catalyzed reductions and/or oxidations of the estrogens: Phenobarbital-induced rat liver microsomes catalyzed the oxidation of DES to 4',4"-diethylstilbestrol quinone (DES quinone) with cumene hydroperoxide as cofactor. The quinone was unstable and spontaneously rearranged to (Z,Z)-dienestrol.

Effect of pH and energy spilling on bacterial protein synthesis by carbohydrate-limited cultures of mixed rumen bacteria.

Rumen contents were obtained from a cow fed 2.5 kg concentrates and 2.5 kg timothy hay twice daily, and the mixed rumen bacterial inoculum (pH 6.

Cytochrome P-450 mediated reductive dehalogenation of the perhalogenated aromatic compound hexachlorobenzene.

Hexachlorobenzene (HCB) elicits concentration-dependent and saturable type 1 binding spectra when added to oxidized (Fe3+) cytochrome P-450 (CYT P-450) in control, phenobarbital- (PB) induced, and beta-naphthoflavone- (BNF) induced male Sprague-Dawley rat liver microsomes. The spectral binding constants (Ks) for HCB in control and PB-induced microsomes are 180 microM and 83 microM, respectively, and correlate inversely with the specific content of CYT P-450 (0.9 and 2.

Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase.

NADPH-cytochrome P-450 reductase releases FAD upon dilution into slightly acidic potassium bromide. Chromatography on high performance hydroxylapatite resolved the FAD-dependent reductase from holoreductase. The FAD dependence was matched by a low FAD content, with the ratio of FAD to FMN as low as 0.

Aging modifies the expression of hepatic microsomal cytochromes P-450 after pretreatment of rats with beta-naphthoflavone or phenobarbital.

The various forms of hepatic cytochrome P-450 respond differentially to aging and induction. We examined the levels of six forms of cytochrome P-450, designated as Forms 1 through 5 and Form b, as a function of age and induction. Radial immunodiffusion analysis of rat liver microsomes indicate that cytochrome P-450 Forms 1 and 2 respond to induction by beta-naphthoflavone or phenobarbital less well in aging rats than in young rats.

Aging affects the drug metabolism systems of rat liver, kidney, colon and lung in a differential fashion.

Microsomes prepared from the liver, lungs, colon and kidney cortex of Sprague Dawley rats of ages 2, 4, 10, 24 and 78 weeks were assessed for hydroxylation activity with the substrate benzo[alpha]pyrene. Liver microsomal activity declined after reaching a peak of activity at 10 weeks. The hydroxylation of benzo[alpha]pyrene by colon, kidney and lung microsomes, however, either remained the same or decreased only slightly.

Metabolism of the carcinogen 1,2-dimethylhydrazine by isolated human colon microsomes and human colon tumor cells in culture.

Human colon microsomes catalyze the metabolism of the model colon carcinogen 1,2-dimethylhydrazine. Activity appears to be distributed in a gradient towards the lower end of the colon. Highest activities were observed for microsomes prepared from the descending segment of the colon with the transverse segment exhibiting lower activities, while the ascending segment showed the lowest rate of metabolism.

Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat hepatoma.

NADPH-cytochrome P-450 reductase was purified to homogeneity from hepatoma 5123t.c.(H) microsomes from phenobarbital and hydrocortisone-treated rats by detergent solubilization and affinity chromatography with an overall 8% recovery.