No abstract

A highly anionic peroxidase induced in suberizing cells was suggested to be the key enzyme involved in polymerization of phenolic monomers to generate the aromatic matrix of suberin. The enzyme encoded by a potato cDNA was found to be highly homologous to the anionic peroxidase induced in suberizing tomato fruit. A tomato genomic library was screened using the potato anionic peroxidase cDNA and one genomic clone was isolated that contained two tandemly oriented anionic peroxidase genes. These genes were sequenced and were 96% and 87% identical to the mRNA for potato anionic peroxidase. Both genes consist of three exons with the relative positions of their two introns being conserved between the two genes. Primer extension analysis showed that only one of the genes is expressed in the periderm of 3 day wound-healed tomato fruits. Southern blot analyses suggested that there are two copies each of the two highly homologous genes per haploid genome in both potato and tomato. Abscisic acid (ABA) induced the accumulation of the anionic peroxidase transcripts in potato and tomato callus tissues. Northern blots showed that peroxidase mRNA was detectable at 2 days and was maximal at 8 days after transfer of potato callus to solid agar media containing 10(-4) M ABA. The transcripts induced by ABA in both potato and tomato callus were identical in size to those induced in wound-healing potato tuber and tomato fruit. The anionic peroxidase peptide was detected in extracts of potato callus grown on the ABA-containing media by western blot analysis. The results support the suggestion that stimulation of suberization by ABA involves the induction of the highly anionic peroxidase.

The addition of peroxynitrite to purified cytochrome P450 2B1 resulted in a concentration-dependent loss of the NADPH- and reductase-supported or tert-butylhydroperoxide-supported 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 with IC50 values of 39 and 210 microM, respectively. After incubation of P450 2B1 with 300 microM peroxynitrite, the heme moiety was not altered, but the apoprotein was modified as shown by HPLC and spectral analysis. Western blot analysis of peroxynitrite-treated P450 2B1 demonstrated the presence of an extensive immunoreactivite band after incubating with anti-nitrotyrosine antibody. However, the immunostaining was completely abolished after coincubation of the anti-nitrotyrosine antibody with 10 mM nitrotyrosine. These results indicated that one or more of the tyrosine residues in P450 2B1 were modified to nitrotyrosines. The decrease in the enzymatic activity correlated with the increase in the extent of tyrosine nitration. Further demonstration of tyrosine nitration was confirmed by GC/MS analysis by using 13C-labeled tyrosine and nitrotyrosine as internal standards; approximately 0.97 mol of nitrotyrosine per mole of P450 2B1 was found after treatment with peroxynitrite. The peroxynitrite-treated P450 2B1 was digested with Lys C, and the resulting peptides were separated by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid sequence of the major nitrotyrosine-containing peptide corresponded to a peptide containing amino acid residues 160-225 of P450 2B1, which contains two tyrosine residues. Thus, incubation of P450 2B1 with peroxynitrite resulted in the nitration of tyrosines at either residue 190 or 203 or at both residues of P450 2B1 concomitant with a loss of 2B1-dependent activity.

The 7-ethoxycoumarin O-deethylase activity of rat liver cytochrome P450 2B1 reconstituted with NADPH-cytochrome P450 reductase and lipid was inactivated by 2-ethynylnaphthalene (2EN) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. The extrapolated KI and kinactivation were 0.08 microM and 0.83 min-1, respectively. The loss of 7-ethoxycoumarin O-deethylation activity displayed a number of characteristics consistent with mechanism-based inactivation, including irreversibility, saturability, protection by an alternate substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. The inactivation was not accompanied by a concomitant loss of spectrally detectable cytochrome P450. HPLC analysis showed that [3H]2EN was irreversibly bound to the protein moiety of cytochrome P450 and the stoichiometry of inactivation was approximately 1.3 mol of 2EN bound per mole of cytochrome P450. Liquid chromatographic and GC-MS analyses of the organic extracts from these incubations showed that the major metabolite was 2-naphthylacetic acid, and a partition ratio of 4-5 mol of acid produced per mole of cytochrome P450 2B1 inactivated was determined. A radiolabeled peptide, approximately 6.5 kDa when analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was isolated by HPLC from a tryptic digest of the [3H]2EN-inactivated cytochrome P450 and NADPH-cytochrome P450 reductase. Sequence data were obtained after cyanogen bromide cleavage of this amino-terminally blocked peptide. These results in conjunction with the results from the cleavage of the intact [3H]2EN-inactivated cytochrome P450 by cyanogen bromide and separation of the peptides either by HPLC or by Tricine-SDS-PAGE followed by transfer of the peptides to a poly(vinylidene difluoride) membrane and sequencing of the labeled peptides from both experiments, led to the identification of a 2EN-modified active-site peptide with the sequence ISLLSLFFAGTETSSTTLRYGFLLM. This corresponds to positions 290-314 in cytochrome P450 2B1. Sequence alignments of cytochrome P450 2B1 with cytochrome P450 2B1 with cytochrome P450 101 predict that this region might correspond to helix I of the bacterial protein [Poulos, T.L. (1988) Pharm. Res. 5, 67-75] that contains a highly conserved threonine residue involved in oxygen binding.

The survey method is one of the most common approaches used in the social sciences to empirically study the characteristics and interrelations of sociological and psychological variables. Its impact on research in accounting and related disciplines has been substantial. However, the method has often been criticised. This paper describes and critically assesses a survey undertaken in management information systems. Its purpose is twofold. First, it aims to counter some of the criticisms of the survey method by demonstrating how many of the potential weaknesses of surveys can be overcome. Second, it provides a prescription for rigorous survey research.