The role of positive charges and structural segments in the presequence of rat liver aldehyde dehydrogenase in import into mitochondria.

Most mitochondrial proteins are nucleus-encoded and translated in the cytosol. They have an N-terminal presequence that allows recognition by the mitochondrial import apparatus and subsequent import into mitochondria. These presequences are rich in positive charges, mainly arginines. The role of these positive charges in the 19-amino acid presequence of rat liver aldehyde dehydrogenase was investigated by systematically replacing them with the polar but uncharged residue, glutamine. The single substitution of any of the four Arg residues in the helical segments did not affect import. Substitution of both Arg residues in the N-terminal segment (R3Q/R10Q) caused a dramatic decrease in import competence. This could be restored by using the mutant lacking the three-amino acid (RGP) linker that separates the two helical domains, determined by two-dimensional NMR (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). CD and NMR spectra of the peptide corresponding to the linker-deleted presequence showed that it was substantially more prone to helix formation than the native peptide over its entire length. A similar analysis of the peptide corresponding to the R3Q/R10Q presequence revealed that this peptide was only somewhat more helical than the native peptide and that the greater helicity did not include the residues near the N terminus. It is concluded that positively charged residues in the presequence play a vital role in the import of precursor aldehyde dehydrogenase. One of the positive charges in the N-terminal helical segment of the presequence is necessary for import competence. However, if both positive charges are removed, import competence can be retained as long as the presequence is capable of forming a relatively more stable alpha-helix near its N terminus.

Inorganic tin -- a new selective inducer of the murine coumarin 7-hydroxylase (CYP2A5).

The coumarin 7-hydroxylase of mice (Coh, CYP2A5) is known to be highly selectively inducible by both a set of heavy metals such as cobalt, indium and cerium and a variety of organic nitrogen-containing heteroaromatic compounds such as 3-amino-1,2,4-triazole, pyrazine and pyrazole. The investigations presented reveal that inorganic divalent tin has to be included in the list of selective inducers. Pretreatment of NMRI-mice with 50 mg SnCl2/kg body weight, daily for 2 days, increases the coumarin hydroxylation 40- and 20-fold in the kidney and liver, respectively. So far, the inducing potency of tin chloride is higher than that of the agents already known. The diagnostic inhibitor metyrapone strongly inhibits the coumarin model reaction. In the kidneys tin generates an almost pure fraction of a cytochrome P450 isozyme catalyzing the metabolism of coumarins, as inhibition experiments reveal.

Effect of pyrazole, cobalt and phenobarbital on mouse liver cytochrome P-450 2a-4/5 (Cyp2a-4/5) expression.

Pyrazole, cobalt and phenobarbital increase the activity of coumarin 7-hydroxylase (COH) in mouse liver. To study the mechanism of this increase, we measured the expression of the cytochrome P-450 2a-4/5 (Cyp2a-4/5) complex, which mediates testosterone 15 alpha-hydroxylase and COH activities, as a function of dose and time after the treatment of C57BL/6 (B6) and DBA/2 (D2) male mice with the inducers. COH activity and Cyp2a-4/5 steady-state mRNA levels were increased in both strains in response to the inducers. No marked effect occurred with testosterone 15 alpha-hydroxylase or activities associated with Cyp1a-1 or Cyp2e-1. A 2-7-fold increase in response to the inducers was seen in the amount of P-450Coh (cytochrome P-450 isoenzyme catalysing coumarin 7-hydroxylation) protein in Western immunoblots. PCR amplification of a 1 kb region in Cyp2a-4/5-mRNA-derived cDNA, followed by cutting at the diagnostic PstI site, showed that most of the steady-state mRNA consisted of Cyp2a-5, which is also the form most affected by pyrazole. Nuclear run-off analysis revealed no increase in the transcription rate of Cyp2a-4/5 after pyrazole or cobalt treatment, whereas a 2-3-fold increase occurred after phenobarbital pretreatment in B6 mice. Together with previous reports [Aida & Negishi (1991) Biochemistry 30, 8041-8045], the current data suggest that both pyrazole and cobalt increase COH catalytic activity by affecting Cyp2a-5 by post-transcriptional mechanisms in mice.

7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases.

A series of 7-alkoxyquinolines was synthesized and tested as substrates with hepatic microsomes prepared from male Wistar rats. Microsomal O-dealkylation rates and kinetic constants were determined for the 7-alkoxyquinolines with microsomes from control, 3-methylcholanthrene (MC)-pretreated, and phenobarbitone (PB)-pretreated rats. Structure-activity relationship studies indicated that the 7-benzyloxyquinoline was the most rapidly metabolized substrate for control microsomes and those from PB-pretreated rats, whereas the 7-ethoxy- and 7-propoxyquinolines were O-dealkylated more rapidly by microsomes of MC-pretreated animals. Differences in activities occurred in Vmax and apparent Km values; however, there does not appear to be a correlation between these two values for the different quinoline substrates. Apparent Km and Vmax values for the 7-alkoxyquinolines were: control microsomes, Km = 71-773 microM, Vmax = 0.37-8.4 nmol 7-quinolinol/min/mg protein; MC microsomes, Km = 0.5-14 microM, Vmax = 0.29-2.7 nmol 7-quinolinol/min/mg protein; PB microsomes, Km = 2.8-46 microM, Vmax = 0.9-12 nmol 7-quinolinol/min/mg protein. All of the quinoline substrates gave Type I binding spectra with control and MC microsomes. With PB microsomes, Type I. Reverse Type I, and a mixture of the two types of binding spectra were observed. Comparisons of the structure-activity relationships, levels of induction, and kinetic constants were made with 7-alkoxycoumarin and 7-alkoxyphenoxazone analogs. In addition, three new coumarin substrates (7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin) are described.

Interactions of heterocyclic Maillard products with the hepatic microsomal monooxygenase system.

1. Interactions of methyl-substituted pyrazines, and other constituents of Maillard products generated during heat treatment of food, with hepatic microsomal mixed-function oxygenases were studied in vitro. 2. Spectral interactions of N-containing heteroaromatic compounds with the cytochrome P-450 system are type I or type II depending on the state of induction, and are relatively weak. Inhibition of 7-ethoxycoumarin O-deethylation by these compounds is ten times lower than that of metyrapone, agreeing with the weak spectral interaction. Inhibition is competitive for 2,3-dimethylquinoxaline, and complex for 2,5-dimethylpyrazine and 2,3,5,6-tetramethylpyrazine. 3. Spectral and inhibitory interactions indicate biotransformation. This was studied with 2,3,5,6-tetramethylpyrazine; the metabolite formed was identified as 2-hydroxymethyl-3,5,6-trimethylpyrazine. Metabolism to the N-oxide did not occur.

Automobile seat belt practices of pregnant women.

Despite the fact that seat belts have been proved effective in reducing deaths and serious injuries, many pregnant women do not wear seat belts. A study was conducted to examine the seat belt practices of pregnant women. Of the 87 women who were interviewed, 40 reported using seat belts regularly during pregnancy. However, almost one-third of them did not adjust the belts properly for maximum protection. Older women and those with more education were more likely to use their seat belts routinely. Only 20 women recalled receiving information about seat belts during their pregnancies, but 48 women would have liked to receive more information.