Target inactivation analysis applied to determination of molecular weights of rat liver proteins in the purified state and in microsomal membranes.

In principle, target inactivation analysis provides a means of determining the molecular weights (Mr) and states of aggregation of proteins in native environments where they are functionally active. We applied this irradiation technique to the rat liver microsomal membrane proteins: cytochrome b5, epoxide hydrolase, flavin-containing monooxygenase, NADH-ferricyanide reductase, NADPH-cytochrome P-450 reductase, and seven different forms of cytochrome P-450. Catalytic activities, spectral analysis of prosthetic groups, and sodium dodecyl sulfate-polyacrylamide electrophoresis/peroxidase-coupled immunoblotting were used to estimate apparent Mr values in rat liver microsomal membranes. Except in one case (cytochrome P-450PCN-E), the estimated Mr corresponded most closely to that of a monomer. Purified cytochrome P-450PB-B, NADPH-cytochrome P-450 reductase and epoxide hydrolase were also subjected to target inactivation analysis, and the results also suggested monomeric structures for all three proteins under these conditions. However, previous hydrodynamic and gel-exclusion results clearly indicate that all three of these proteins are oligomeric under these conditions. The discrepancy between target inactivation Mr estimates and hydrodynamic results is attributed to a lack of energy transfer between monomeric units. Thus, while P-450PCN-E may be oligomeric in microsomal membranes, target inactivation analysis does not appear to give conclusive results regarding the states of aggregation of these microsomal proteins.

Topological studies of cytochromes P-450scc and P-45011 beta in bovine adrenocortical inner mitochondrial membranes. Effects of controlled tryptic digestion.

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria were studied by using controlled digestion with trypsin of purified inner mitochondrial membranes. Inhibition of steroid hydroxylase activity by trypsin was only observed in inner mitochondrial membranes which had been disrupted by various techniques. The steroid hydroxylase activity of intact inner membranes was not inhibited by trypsin. The effect of tryptic digestion was monitored by measuring 11 beta-hydroxylase and cholesterol side chain cleavage activities, as well as cytochrome P-450 reduction. The effect of trypsin on the steroid-induced difference spectra using pregnenolone, 20 alpha-hydroxycholesterol, and deoxycorticosterone was also measured. The results were similar regardless of which procedure was utilized and strongly suggest that both cytochrome P-45011 beta and cytochrome P-450scc are located on the matrix side of the mitochondrial inner membrane.

Isolation and characterization of a Mycobacterium species capable of degrading three- and four-ring aromatic and aliphatic hydrocarbons.

Mycobacterium sp. strain CH1 was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated freshwater sediments and identified by analysis of 16S rDNA sequences. Strain CH1 was capable of mineralizing three- and four-ring PAHs including phenanthrene, pyrene, and fluoranthene. In addition, strain CH1 could utilize phenanthrene or pyrene as a sole carbon and energy source. A lag phase of at least 3 days was observed during pyrene mineralization. This lag phase decreased to less than 1 day when strain CH1 was grown in the presence of phenanthrene or fluoranthene. Strain CH1 also was capable of using a wide range of alkanes as sole carbon and energy sources. No DNA hybridization was detected with the nahAc gene probe, indicating that enzymes involved in PAH metabolism are not related to the well-characterized naphthalene dioxygenase gene. DNA hybridization was not detected when the alkB gene from Pseudomonas oleovorans was used under high-stringency conditions. However, there was slight but detectable hybridization under low-stringency conditions. This suggests a distant relationship between genes involved in alkane oxidation.

Osmoregulatory Responses of Fungi Inhabiting Standing Litter of the Freshwater Emergent Macrophyte Juncus effusus.

Standing litter of emergent macrophytes often forms a major portion of the detrital mass in wetland habitats. Microbial assemblages inhabiting this detritus must adapt physiologically to daily fluctuations in temperature and water availability. We examined the effects of various environmental conditions on the concentrations of osmoregulatory solutes (polyols and trehalose) and the respiratory activities of fungal assemblages inhabiting standing litter of the freshwater emergent macrophyte Juncus effusus. Under field conditions, the concentrations of osmolytes (polyols plus trehalose) in fungal decomposers were negatively correlated with plant litter water potentials (r = -0.75, P < 0.001) and rates of microbial respiration (r = -0.66, P < 0.001). The highest concentration of osmolytes (polyols plus trehalose) occurred in standing litter exposed to desiccating conditions (range from wet to dry, 0.06 to 0.68 mumol . mg of fungal biomass). Similar fluctuations in polyol and trehalose concentrations were observed in standing litter wetted and dried under laboratory conditions and for four predominant fungal decomposers of J. effusus grown individually on sterilized Juncus leaves. These studies suggest that fungal inhabitants associated with standing litter of emergent macrophytes can adjust their intracellular solute concentrations in response to daily fluctuations in water availability.

Changes in bacterial species composition in enrichment cultures with various dilutions of inoculum as monitored by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis revealed changes in the bacterial species obtained from enrichment cultures with different inoculum dilutions. This inoculum dilution enrichment approach may facilitate the detection and isolation of a greater number of bacterial species than traditional enrichment techniques.

Biological and molecular characterization of cellular differentiation in Tetrahymena vorax: a potential biocontrol protozoan.

Tetrahymena vorax (T. vorax) is an indigenous fresh water protozoan with the natural biological potential to maintain a specific aquatic microbial flora by ingesting and eliminating specific microorganism. To investigate the molecular mechanisms controlling Tetrahymena vorax (T. vorax) cellular differentiation from a small-mouth vegetative cell to a voracious large-mouth carnivore capable of ingesting prey ciliates and bacteria from aquatic environments, we use DNA subtraction and gene discovery techniques to identify and isolate T. vorax differentiation-specific genes. The physiological necessity for one newly discovered gene, SUBII-TG, was determined in vivo using an antisense oligonucleotide directed against the 5' SUBII-TG DNA sequence. The barriers to delivering antisense oligonucleotides to the cytoplasm of T. vorax were circumvented by employing a new but simple procedure of processing the oligonucleotide with the differentiation stimulus, stomatin. In these studies, the antisense oligonucleotide down-regulated SUBII-TG mRNA expression, and blocked differentiation and ingestion of prey ciliates. The ability to down-regulate SUBII-TG expression with the antisense oligonucleotide suggests that the molecular mechanisms controlling the natural biological activities of T. vorax can be manipulated to further study its cellular differentiation and potential as a biocontrol microorganism.

Effect of substrate and cell surface hydrophobicity on phosphate utilization in bacteria.

We measured the rates of utilization of hydrophobic and hydrophilic phosphate compounds in gram-negative bacteria with different surface hydrophobicities, isolated from wetland habitats. Three hydrophobic and two hydrophilic bacterial species were selected for study by measuring cell adherence to hydrocarbons. The bacteria were grown under phosphorus-limited conditions with P(infi), hydrophilic (beta)-glycerophosphate, or hydrophobic phosphatidic acid as the phosphate source. Hydrophilic bacteria grew most rapidly on P(infi), followed by (beta)-glycerophosphate. Phosphatidic acid did not support growth or did so at a much later time (40 h) than did the other phosphate treatments. Although all hydrophobic species grew well on these substrates, the rate of growth of two Acinetobacter baumannii isolates on phosphatidic acid exceeded the rate of growth on phosphate or (beta)-glycerophosphate. A membrane phospholipid and lipopolysaccharide were used as a source of phosphorus by hydrophobic species, whereas hydrophilic species could not use the membrane phospholipids and used lipopolysaccharide to a lesser extent. Besides hydrophobic interaction between cells and substrate, phosphatase activity, which was cell bound in hydrophilic species but 30 to 50% unbound in hydrophobic species, affected cell growth. Dialyzed culture supernatant containing phosphatase from hydrophobic species increased the phosphate availability to hydrophilic species. Additionally, cellular extracts from a hydrophilic species, when added to hydrophilic cells, permitted growth on hydrophobic phosphate sources. Naturally occurring amphiphilic humic acids affected the utilization of P(infi) and (beta)-glycerophosphate in bacteria with hydrophilic surfaces but did not affect hydrophobic bacteria. Our results indicate that hydrophobic phosphate sources can be used by bacteria isolated from aquatic environments as the sole phosphorus source for growth. This utilization, in part, appears to be related to cell surface hydrophobicity and extracellular enzyme production.

Topological studies of the steroid hydroxylase complexes in bovine adrenocortical mitochondria.

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria was studied by using nonpenetrating artificial electron acceptors and the impermeable protein reagent diazobenzenesulfonate. Inhibition of steroid hydroxylase activity by ferricyanide and dichlorophenolindophenol sulfonate was only observed in mitochondria which had been damaged by various techniques. Intact mitochondria were not inhibited by these reagents. The reaction was monitored by oxygen uptake due to hydroxylation of deoxycorticosterone, as well as P-450 reduction and corticosterone formation. The results obtained were similar regardless of how the activity was measured. Labeling of the mitochondria with the nonpenetrating protein reagent diazobenzenesulfonate also inhibited P-450 reduction and corticosterone formation in mitochondria which had been damaged prior to addition of this reagent. Intact mitochondria which were labeled with this reagent showed very little inhibition of both activities. These results strongly suggest that all protein components of the steroid 11beta-hydroxylase system are located on the matrix side of the mitochondrial inner membrane. The inability of ferricyanide, dichlorophenolindophenol sulfonate, and diazobenzenesulfonate to inhibit the malate-dependent reduction of P-450 in intact mitochondria implies that all the P-450-dependent mitochondrial steroid hydroxylase systems are located on the matrix side of the inner mitochondrial membrane.

Inhibition of mitochondrial respiration by cationic rhodamines as a possible teratogenicity mechanism.

Exposure of mice to cationic rhodamines, Rh 123 and Rh 6G, has been found to be associated with developmental toxicity, while neutral rhodamines (e.g., Rh B) had no such effect. When mouse embryos from dams given ip injections of Rh 123, Rh 6G (15 mg/kg), or Rh B (30 mg/kg) on gestation Day (GD) 10 were examined, Rh 123, Rh 6G were present in embryonic tissue in fluorescent bodies within the average dimensions of mitochondria. Rh B was evenly distributed in the cytoplasm. With in vitro exposure of isolated mitochondria to rhodamines on GD 12, 3-4 times more Rh 123 was associated with mitochondria under energized conditions than under nonenergized conditions; the amount of Rh 6G associated with mitochondria was much less under either condition. Treatment of pregnant mice (ip) with Rh 123 (15 mg/kg/day) or Rh 6G (0.5 mg/kg/day) on GD 7-10 resulted in inhibition of state 3 respiration of embryonic mitochondria isolated on GD 12. When isolated embryonic mitochondria were exposed to the cationic rhodamines, inhibition of state 3 respiration was dose dependent. With 5 micrograms of Rh 123/mg mitochondrial protein, state 3 respiration decreased by 31%, while Rh 6G (1 microgram/mg) decreased state 3 respiration by 27%. In vivo exposure of maternal liver mitochondria to cationic rhodamines did not result in inhibition of respiration 2 days later, whereas in vitro results were similar to those for embryonic mitochondria. In vivo or in vitro exposure to Rh B had no effects on mitochondrial respiration. These results indicate that interference with embryonic energy metabolism is a possible mechanism by which cationic rhodamines exert adverse effects on embryogenesis.

Purification and characterization of a (R)-3-hydroxybutyrate dehydrogenase deletion mutant. Evidence for C-terminal involvement in enzyme activation by lecithin.

(R)-3-Hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) is a lipid-requiring enzyme with a specific requirement of phosphatidylcholine for optimal function. The purified enzyme, devoid of lipid, can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. In order to obtain insight into the mechanism of lipid activation, a C-terminal deletion mutant was constructed which contained 18 amino acids less than BDH. The purified deletion mutant had low, but detectable catalytic activity in the absence of phospholipid. However, the addition of either soluble lecithin or phospholipid vesicles containing lecithin had no effect on enzymatic function. Further experiments were conducted to determine if the deletion mutant had also lost its ability to bind to phospholipid vesicles and natural membranes. Our findings indicate that the mutant enzyme binds to both liposomes and rat liver microsomes. These results suggest that the binding of BDH to the phosphatidylcholine head group is independent of its interaction with the apolar core of the phospholipid bilayer.

A simple, efficient method for the separation of humic substances and DNA from environmental samples.

Three different gels (Sepharose 4B, Sephadex G-200, and Sephadex G-50) were evaluated as a means of removing humic contaminants from DNA extracts of environmental samples. Sepharose 4B gave superior separation of DNA from humics, and DNA purified in this way showed consistently greater amplification than DNA purified by the other materials.

Characterization of a rat liver cytochrome P-450UT-H cDNA clone and comparison of mRNA levels with catalytic activity.

A rat liver cDNA library was prepared using the expression vector bacteriophage lambda gt11 and plaques were screened using polyclonal antibodies raised to purified rat liver cytochrome P-450UT-H, the major enzyme involved in debrisoquine 4-hydroxylation, bufuralol 1'-hydroxylation, and sparteine delta 5-oxidation. A clone was selected which contained a 1.3-kb insert. The Escherichia coli beta-galactosidase fusion protein had a molecular weight greater than that of native beta-galactosidase (and reacted with anti-P-450UT-H after electrophoresis) and was also shown to compete with microsomal P-450UT-H for anti-P-450UT-H, partially relieving catalytic inhibition by anti-P-450UT-H in rat liver microsomes. Hybrid selection experiments with the cloned cDNA also support the view that the insert is related to P-450UT-H. mRNA electrophoresis/hybridization experiments indicated that the 1.3-kb cDNA probe recognized primarily only a single size class of mRNA (2.0 kb) in rat liver. mRNA blotting and in vitro translation/immunoprecipitation experiments both indicated that levels of P-450UT-H mRNA are similar in male and female Sprague-Dawley rats. Dark Agouti strain rats of both sexes contained significantly less P-450UT-H mRNA than did Sprague-Dawley rats and the females had approximately one-half the level of the males. These results are consonant with sex and strain differences in measured levels of P-450UT-H and bufuralol 1'-hydroxylase and sparteine delta 5-oxidase activities. Analysis of genomic DNA indicated that several DNA restriction fragments hybridized to this partial length cDNA; no differences were found between the rat strains and sexes. The results suggest that the basis for the variation in P-450UT-H and its activities among rat strains and sexes is at the level of mRNA concentrations.

Polymorphism of human cytochrome P-450.

The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS)