Relationship between hydrocarbon structure and induction of P450: effect on RNA levels.

1. Exposure to simple aromatic hydrocarbons has been shown to induce P450-dependent activities and the expression of particular P450 isozymes in a manner related to the molecular structure of the inducing hydrocarbon. In an attempt to identify the structural relationship controlling P450 induction, the effect of hydrocarbon treatment on the RNA levels for specific P450 isozymes was examined. 2. Rats were treated with daily injections of hydrocarbons (benzene, toluene, ethylbenzene, n-propylbenzene, m- and p-xylene) for 3 days, and the effects on specific RNA levels were examined by Northern blot hybridization. 3. Although P4502B1 mRNA was not elevated after hydrocarbon treatment, a significant elevation in 2B2 mRNA was observed after exposure to the larger aromatic hydrocarbons, ethylbenzene and m-xylene. It is interesting to note that despite the substantial elevation of P4502B protein levels, only a small elevation of P4502B1 and 2B2 RNA was observed. 4. P4502C11 mRNA was only suppressed by ethylbenzene administration, despite the depression of 2C11 protein levels by several hydrocarbons. 5. P4501A1 mRNA was not detectable and 2E1 mRNA was not changed by any aromatic hydrocarbon treatment investigated in this study. 6. The data indicate that the levels of mRNA species for a number of P450 isozymes are differentially regulated by exposure to hydrocarbons, and that small changes in hydrocarbon size or isomeric structure can influence the levels of these mRNA species.

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction.

Metabolism of therapeutic drugs in the body by the mixed function oxidase system is an important consideration in the analysis of a drug's effectiveness. P450-dependent metabolism within the brain of a neuro-specific drug may affect the drug's course of action. To determine whether cytochrome P450 was expressed in brain, RNA was isolated from the whole brains of rats treated with a variety of known hepatic P450 inducers, including amitriptyline, imipramine, isosafrole, phenobarbital, and beta-naphthoflavone. The RNA was analyzed for the presence of P450 isozymes by the PCR technique. Differential expression of P450IA1, P450IIB1, P450IIB2, P450IID, and P450IIE1 was detected in the brain samples, depending on the treatment. Cytochrome P450 reductase expression was also detected in the brain samples, giving strong evidence that the brain contains a competent mixed function oxidase system under all conditions studied.

The Saccharomyces cerevisiae SRK1 gene, a suppressor of bcy1 and ins1, may be involved in protein phosphatase function.

The Saccharomyces cerevisiae SRK1 gene, when expressed on a low-copy shuttle vector, partially suppresses the phenotype associated with elevated levels of cyclic AMP-dependent protein kinase activity and suppresses the temperature-sensitive cell cycle arrest of the ins1 mutant. SRK1 is located on chromosome IV, 3 centimorgans from gcn2. A mutant carrying a deletion mutation in srk1 is viable. SRK1 encodes a 140-kDa protein with homology to the dis3+ protein from Schizosaccharomyces pombe. The ability of SRK1 to alleviate partially the defects caused by high levels of cyclic AMP-dependent protein kinase and the similarity of its encoded protein to dis3+ suggest that SRK1 may have a role in protein phosphatase function.

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line.

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IA1 or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P450IA1 and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P450IA1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.

Replacement of a conserved proline and the alkaline conformational change in iso-2-cytochrome c.

Although point mutations usually lead to minor localized changes in protein structure, replacement of conserved Pro-76 with Gly in iso-2-cytochrome c induces a major conformational change. The change in structure results from mutation-induced depression of the pK for transition to an alkaline conformation with altered heme ligation. To assess the importance of position 76 in stabilizing the native versus the alkaline structure, the equilibrium and kinetic properties of the pH-induced conformational change have been compared for normal and mutant iso-2-cytochrome c. The pKapp for the conformational change is reduced from 8.45 (normal iso-2) to 6.71 in the mutant protein (Gly-76 iso-2), suggesting that conservation of Pro-76 may be required to stabilize the native conformation at physiological pH. The kinetics of the conformational change for both the normal and mutant proteins are well-described by a single kinetic phase throughout most of the pH-induced transition zone. Over this pH range, a minimal mechanism proposed for horse cytochrome c [Davis, L. A., Schejter, A., & Hess, G. P. (1974) J. Biol. Chem. 249, 2624-2632] is consistent with the data for normal and mutant yeast iso-2-cytochromes c: NH KH----N + H+ kcf in equilibrium kcb A NH and N are native forms of cytochrome c with a 695-nm absorbance band, A is an alkaline form that lacks the 695-nm band, KH is a proton dissociation constant, and kcf and kcb are microscopic rate constants for the conformational change. The Gly-76 mutation increases kcf by almost 70-fold, but kcb and KH are unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c.

As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine [Wood, L. C., Muthukrishnan, K., White, T. B., Ramdas, L., & Nall, B. T. (1988) Biochemistry (preceding paper in this issue)]. For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 degrees C (delta G degrees u = 3.8 kcal/mol for normal Pro-76 iso-2 versus 2.6 kcal/mol for Gly-76 iso-2). The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing. The rates for fast and slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2. A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfolding of the mutant protein. The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species. The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manual mixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and characterization of mutant iso-2-cytochromes c with replacement of conserved prolines.

Oligonucleotide-directed mutagenesis has been used to construct two mutant forms of iso-2-cytochrome c. In one, Pro-30 is replaced by threonine; in the other, Pro-76 is replaced by glycine. Both prolines are fully conserved among mitochondrial cytochromes c and play important structural and functional roles. Yeast with either the Pro-30 or the Gly-76 mutation has appreciable levels of mutant protein in vivo and grows on media containing nonfermentable carbon sources. Thus, neither mutation blocks protein targeting to mitochondria, uptake by mitochondria, covalent attachment of heme, or in vivo function. As judged by ultraviolet-visible spectrophotometry and proton nuclear magnetic resonance spectroscopy, the nativelike conformation of purified Gly-76 iso-2 at pH 6 is almost indistinguishable from that of the normal protein at pH 6. Ultraviolet second-derivative spectrophotometry, however, suggests an increase in the average number of exposed tyrosine side chains, with 2.25 out of 5 residues exposed for the mutant compared to 1.95 for normal iso-2. Above neutral pH, the protein folds to a mutant conformation possibly related to alkaline cytochrome c. Nuclear Overhauser difference spectroscopy of the reduced nativelike conformation allows assignment of several proton resonances and comparison of side-chain conformations of the heme ligand Met-80 in the mutant and the normal proteins. The proton chemical shifts for the assigned resonances are the same within errors for Gly-76 iso-2 and normal iso-2 at pD 6, 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in conformation and slow refolding kinetics in mutant iso-2-cytochrome c with replacement of a conserved proline residue.

Using oligonucleotide-directed mutagenesis, we have produced a mutant form of iso-2-cytochrome c of yeast in which threonine (Thr-71) replaces a conserved proline residue (Pro-71) located between two short alpha-helical segments in the native protein. Optical spectroscopy indicates that, at pH 7.2, Thr-71 iso-2-cytochrome c folds to a nonnative conformation possibly related to the alkaline form of the native protein. On titration to pH 5.2, Thr-71 iso-2-cytochrome c regains many of the optical properties of the normal protein. We have shown that the proline residue at position 71 has no effect on the kinetics of fluorescence-detected slow refolding. However, between pH 5 and pH 7.2 the amplitude for absorbance-detected slow folding is strongly pH dependent in the mutant protein but is largely independent of pH in the normal protein. We believe this to be due to the folding of Thr-71 iso-2-cytochrome c to a nonnative conformation at pH 7.2 that does not require the slow, absorbance-detected conformational changes observed in folding to the more native-like state at pH 5-6.

Transformation of a Bacillus subtilis L-form with bacteriophage deoxyribonucleic acid.

A stable L-form, sal-1, of Bacillus subtilis was transformed with deoxyribonucleic acid (DNA) from bacteriophages phi 25 and phi 29 to determine whether exogenous DNA can be introduced into this organism. The viral transformation (transfection) was successful with the use of polyethylene glycol. In the presence of the fusogen, bacteriophage phi 25 DNA initiated a single cycle of infection. When compared with transfection of competent cells of Bacillus subtilis, the appearance of viral particles was delayed and their production occurred over a longer time period. L-form cells were best able to support intracellular replication of phi 25 viral particles when in balanced growth in a rich medium. The addition of polyethylene glycol also induced infection of sal-1 with whole bacteriophage phi 25 particles which could not otherwise infect the L-form and enhanced infection by intact phi 29 particles. Primary recombination was shown to be required for polyethylene glycol-mediated phi 25 transfection, but not phi 29 transfection or for whole bacteriophage phi 25 infection mediated by polyethylene glycol. Successful transfection of sal-1 suggests that the L-form may be amenable to genetic modification with exogenous DNA.