Eva Kärgel

Candida maltosa NADPH‐cytochrome P450 reductase: Cloning of a full‐length cDNA, Heterologous expression in Saccharomyces cerevisiae and function of the N‐terminal region for membrane anchoring and proliferation of the endoplasmic reticulum

A full‐length cDNA for NADPH‐cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes.

Inducible Membranes in Yeast: Relation to the Unfolded‐Protein‐Response Pathway

Overproduction of an endoplasmic reticulum (ER)‐resident membrane protein (cytochrome P450 52A3) and of a secretory protein (invertase) was used to study the regulation of the luminal ER protein Kar2p under conditions that lead to ER proliferation and secretory overload, respectively. In both cases we found (i) a significant increase of Kar2 protein and mRNA levels, (ii) a transcriptional regulation based on the function of the 22 bp unfolded‐protein‐response element of the KAR2 promoter and (iii) an essential role of the transmembrane kinase Ire1p for upregulation of KAR2 gene expression. These results show that the same mechanism operates when KAR2 induction is triggered by overproduction of cytochrome P450 or invertase and that this mechanism shares the known features of the unfolded‐protein‐response pathway. Disruption of the IRE1 gene resulted in a marked decrease of the invertase protein levels produced. In contrast, a functional IRE1 gene was not required to reach high‐level production of the integral membrane protein cytochrome P450 52A3. Moreover, IRE1 gene disruption did not prevent P450‐induced ER proliferation. We suggest that Ire1p‐mediated KAR2 induction is, in the case of cytochrome P450 52A3 overproduction, a process which follows on ER proliferation, thereby monitoring the increase of ER size and adjusting the level of Kar2p accordingly.

Cytochrome P450–Dependent Renal Arachidonic Acid Metabolism in Desoxycorticosterone Acetate–Salt Hypertensive Mice

Cytochrome P450 (P450)-dependent arachidonic acid metabolites may act as mediators in the regulation of vascular tone and renal function. We studied arachidonic acid hydroxylase activities in renal microsomes from normotensive NMRI mice, desoxycorticosterone acetate (DOCA)-salt hypertensive mice, and DOCA-salt mice treated with either lovastatin or bezafibrate, both of which improve hemodynamics in this model. Control renal microsomes had arachidonic acid hydroxylase activities of 175±12 pmol · min−1 · mg−1. The metabolites formed were 20- and 19-hydroxyarachidonic acid, representing ≈80% and ≈20% of the total hydroxylation. Treatment with DOCA-salt resulted in significantly decreased hydroxylase activities (to 84±4 pmol · min−1 · mg−1) of the total microsomal P450 content and a decrease in immunodetectable Cyp4a proteins. Lovastatin had no effect on these variables, whereas bezafibrate increased arachidonic acid hydroxylase activities to 163±12 pmol · min−1 · mg−1. In situ hybridization with probes for Cyp4a-10, 12, and 14 revealed that Cyp4a-14 was the P450 isoform most strongly induced by bezafibrate. The expression was concentrated in the cortical medullary junction and was localized predominantly in the proximal tubules. In conclusion, these results suggest that the capacity to produce 20-hydroxyarachidonic acid is impaired in the kidneys of DOCA-salt hypertensive mice. Furthermore, bezafibrate may ameliorate hemodynamics in this model by restoring P450-dependent arachidonic acid hydroxylase activities. Lovastatin, on the other hand, exerts its effects via P450-independent mechanisms.

Electrochemical investigations of the intermolecular electron transfer between cytochrome c and NADPH-cytochrome P450-reductase

Abstract The electron exchange reaction of cytochrome c with NADPH-cytochrome P450 reductase was investigated electrochemically. For this purpose the electrochemical behavior of cytochrome c in solution, adsorbed or covalently immobilized on a modified gold electrode surface was studied. In the case when cytochrome c was in solution or only electrostatically adsorbed on the electrode surface, fast electron transfer was observed with NADPH-cytochrome P450 reductase. When cytochrome c was covalently bound to the electrode surface, in spite of quasi-reversible electron exchange with the electrode, no electron transfer was observed with NADPH-cytochrome P450 reductase. These results suggest that electrostatically adsorbed cytochme c on the modified electrode surface has some mobility that allows re-orientation as required to interact both with the electrode and with the NADPH-cytochrome P450 reductase. In contrast, covalent binding of cytochrome c leads to permanent orientation towards the electrode surface and thus blocks the cytochrome c electron accepting site from the reductase.

N-alkanes induce the synthesis of cytochrome P-450 mRNA in Candida maltosa.

In the yeast Candida maltosa the level of cytochrome P-450 was 100-300-fold higher in alkane-grown cells than in glucose-grown ones (detected by a radioimmunoassay). It was shown immunochemically (1) that this was not the result of an assembly of preexisting apoenzyme with the prosthetic heme group. By cell-free translation of total poly(A)RNA in a wheat germ system and subsequent immunoprecipitation it was shown that the amount of mRNA coding for cytochrome P-450 paralleled its concentration in the cell.