Brian R. Rabin

The effect of terpenoid compounds on cytochrome P-450 levels in rat liver.

We have investigated the ability of camphor, menthol, pinene, limonene and myrcene to induce in rats members of a cytochrome P-450 sub-family termed PB P-450. These proteins have recently been designated as members of the P450IIB sub-family. None of these naturally occurring terpenoids significantly changed the total content of cytochromes P-450 or cytochrome b5. Radioimmunoassay results showed that PB P-450 was induced 6-fold by camphor and to a lesser extent by menthol and pinene. The induction was confirmed by Western blotting. It was shown by nucleic acid hybridization that induction of PB P-450 by terpenoids was mediated by an increase in the amount of the corresponding mRNA. Analysis of the denaturation of mRNA-cDNA hybrids demonstrated that the mRNA induced by the terpenoids was encoded by a member of the P450IIB sub-family. None of the terpenoids had an effect on the amount of mRNA coding for P450IA2 (a cytochrome P-450 inducible by beta-naphthoflavone and isosafrole). The results indicate that cytochromes P-450 induced by a synthetic compound, phenobarbital, may have originally evolved in response to terpenoid compounds normally present in the environment.

The effects of aflatoxin B1 and steroid hormones on polysome binding to microsomal membranes as measured by the activity of an enzyme catalysing disulphide interchange

An enzyme catalysing the rearrangement of protein disulphide bonds has been found firmly attached to the endoplasmic reticulum of all tissues tested [l-4]. The biological function of the enzyme is uncertain although it is probably involved in terminal stages of the biosynthesis of secreted and lysosomal proteins, many of which possess disulphide bonds. The apparent interchange activity of microsomal membranes, measured by their ability to catalyse the reformation of native, active enzyme from “randomly-oxidised)’ ribonuclease [ 11, is a function of the number of rib@ somes bound [4] and there appears to be an equivalence between the amount of interchange enzyme and the number of polysome-binding sites. Fig. 1 shows the relationship between the apparent interchange activity (per mg microsomal protein) and the RNA: protein ratio for several sub-fractions of three different tissues, all taken from the same male, albino rats (150-220 g body weight). The plots are linear and parallel, and the parameters obtained from these plots are given in table 1. It would seem that polysome-binding sites are uniquely associated with loci of interchange enzyme activity and it is suggested that this enzyme catalyses the formation of the correct conformation of proteins “exported’ from the cell via the endoplasmic tubules. The effects of aflatoxin B1 on the relationship between the enzyme and polysome binding were investigated in vitro, since in common with most carcinogens it appears to induce “degranulation” of the endoplasmic reticulum in vivo [S] . It is also an inhibitor of protein synthesis in liver slices [6] and causes a breakdown of polysomes in HeLa cells [7].

Induction of cytochrome P-450 by phenobarbital is mediated at the level of transcription

We have previously shown that the 43-fold induction by phenobarbital of the major phenobarbital-inducible cytochrome P-450 of rat liver microsomal membranes (PB P-450) is mediated by a 20-fold increase in the amount of its mRNA in the cytoplasm. Here we demonstrate that the induction of the mRNA can be almost entirely accounted for by an increase in the rate of transcription of genes coding for PB P-450, and involves little or no change in the rates of processing, transport or degradation of the mRNA. Phenobarbital treatment resulted in no amplification or rearrangement of PB P-450 genes.

Inhibition of lactate dehydrogenase by high concentrations of pyruvate: The nature and removal of the inhibitor

The inhibition of lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1,l ,1,27) by high concentrations of the su.&rate, pyruvate, has been attributed to the formation of an abortive ternary complex of the enzyme, NAD and pyruvate [l-5] . High concentrations of pyruvate, greater than 0.5 mM, are required to observe the inhibition and demonstrate the formation of a ternary complex. The effects could be due to a contaminant or a form of the substrate present in only trace amounts. We have prepared samples of pyruvate which give no excess substrate inhibition and have obtained suggestive evidence for the chemical nature of the inhibitor present in the specimens of pyruvate normally used for investigations on lactate dehydrogenase.

The effects of ribosomes on the activity of a membrane bound enzyme catalysing thiol-disulphide interchange

An enzyme is present in the microsome fraction of rat liver, which catalyses disulphide interchange in a variety of proteins [l] . The enzyme has also been found in various other mammalian tissues [ 2,3] and in the microsome fractions of baker’s yeast, cabbage and garden peas [4]. In E. coli, the enzyme is found in the “cell-wall-membrane” fraction after sordcation [4] . To obtain maximal activity when assaying this enzyme in membrane preparations, both fl-mercaptoethanol and EDTA need to be present in the incubation mixture [5]. These studies were initiated in an attempt to elucidate the role of these activators.

The reaction of iodoacetate and iodoacetamide with proteins as determined with a silver/silver iodide electrode.

Abstract An iodide electrode suitable for measuring the iodide produced in the reaction of iodocompounds with low concentrations of thiols is described. It has been used to determine the number of SH groups in proteins and other thiol compounds. The presence of two reactive thiol groups in creatine phosphokinase is confirmed and it is shown that their rate of alkylation parallels loss of enzyme activity. Urea-denatured ovalbumin is shown to react rapidly with iodoacetate to produce two equivalents of iodide per mole, after which a slow reaction, in which a further 9.8 equivalents are liberated, is observed. Native ovalbumin is shown to undergo a slow rection with iodoacetamide.

Induction by phenobarbital of the mRNA for a specific variant of rat liver microsomal cytochrome P-450

The treatment of rats for 4 days with phenobarbital causes an apparent 3-fold increase in the amount of total liver cytochrome P-450. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, metyrapone binding and immunoprecipitation, this increase was found to be due to a much larger increase in a restricted number of specific cytochrome P-450 variants. A radioimmunoassay technique demonstrated that the major phenobarbital-inducible variant, of molecular weight 52 000, is induced 24-fold by phenobarbital. Immunoprecipitation analysis of products of translation in vitro with an antibody specific to the 52 000-mol.wt. cytochrome P-450 showed that phenobarbital induces the mRNA in polyribosomes for this variant 20-fold. Evidence is presented for the action of phenobarbital at the transcriptional and translational levels.

The binding of polysomes to smooth membranes of rat liver promoted by steroid hormones and extracts from either rough endoplasmic reticulum or from polysomes of the opposite sex

The attached polysomes can be removed from the membranes of rough endoplasmic reticulum of rat liver by treatment with EDTA and the resultant ‘degranulated’ membranes will rebind polysomes in the presence of magnesium ions at physiological pH values [ 1,2] . In sharp contrast, smooth membranes will not interact with polysomes under these conditions. It has recently been demonstrated [ 1,3,4] that the attachment of polysomes to smooth membranes can be achieved by adding a steroid hormone. The hormone requirements are totally sex specific, with oestradiol and testosterone as the most effective substances yet discovered for promoting polysome attachment to smooth membranes from male and female rat liver respectively. We report in this paper the steroid hormone requirements for the interaction of membranes with polysomes from the opposite sex. We show that ‘activating substances’ can be obtained from polysomes by extraction with ethyl acetate which have remarkable sex specific effects. In the membrane-polysome binding systems the extract from female polysomes will replace oestradiol but not testosterone, and the extract from male polysomes, testosterones but not oestradiol. An extract from male rough endoplasmic reticulum wilI replace both hormones. The implications of these fmdings are discussed.

The chemical reactivity of the thiol group in the active centre of ficin

1. 1. The irreversible inhibition of ficin (EC 3.4.4.12) by chloroacetamide or iodoacetamide is due to the alkylation of the thiol group of one particular cysteine residue in the enzyme. 2. 2. The variation of the rate of alkylation of ficin with pH shows that the reactive thiol group of ficin behaves essenitally as a freely ionising roup, pKa′ = 8.55 (25 °; I, 0.1). Hence it is concluded that this group is unlikely to exist in a hydrogen-bonded form in the free enzyme. 3. 3. The rates of reaction of ficin with chloroacetamide or iodacetamide were accelerated in the presence of substrates or products of catalytic activity without the stoichiometry of the overall reaction being affected. The rate of addition of the essential thiol of ficin to N-ethylmaleimide was unaffected by substrate or substrate analogues. 4. 4. The nucleophilicity of the essential thiol group of ficin in SN2 reactions was found to be greater than that of simple thiol compounds whereas in the addition reaction to N-ethylmaleimide the converse was found. 5. 5. A possible reason for the high nucleophilicyt of ficin in SN2 reactions and the acceleration caused by the presence of substrate or substrate analogues is given.

Differential effect of phenobarbital and β‐naphthoflavone on the mRNAs coding for cytochrome P450 and NADPH cytochrome P450 reductase

The induction in rat liver of a specific variant(s) of cytochrome P450 (PB‐P450) by phenobarbital and its repression by β‐naphthoflavone occur through corresponding changes in the levels of mRNA coding for the protein(s). The level of translatable mRNA coding for NADPH‐cytochrome P450 reductase in rat liver increases on treatment with phenobarbital but not β‐naphthoflavone.