Hisaaki Taniguchi

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.

Regulation of cytochrome P-450 CYPIA1 gene expression and protooncogene expression by growth factors in primary hepatocytes

The effect of growth factors on the cytochrome P‐450 (CYPIA1) gene expression was studied in primary mouse hepatocytes. Of the three growth factors used, i.e. epidermal growth factor (EGF), transforming growth factor α (TGFα) and insulin, only EGF or TGFα completely blocked CYPIA1 expression in the presence of the CYPIA1 inducer 3‐methylcholanthrene (3‐MC). This repression was not linked to cell cycle progression of the hepatocyte because insulin was active to induce ‘early immediate genes’ and DNA replication as well as EGF/TGFα but failed to suppress CYPIA1 expression. A specific EGF/TGFα receptor‐mediated function may repress CYPIA1 gene expression and contribute to the acquisition of a xenobiotic drug resistance phenotype.

Glutathione S-transferase is an in vitro substrate of Ca++-phospholipid-dependent protein kinase (protein kinase C).

Glutathione S-transferase was found to be a good substrate of Ca++-phospholipid-dependent protein kinase in vitro. Of 6 isozymes of glutathione transferase purified from rat liver cytosol (1-1, 1-2, 2-2, 3-3, 3-4, 4-4), only isozymes 1-1, 1-2 and 2-2 were significantly phosphorylated by the kinase purified from rabbit brain. Phosphorylation was more pronounced in subunit 1 than in subunit 2, and the degree of the phosphorylation was similar in all three homo- and heterodimers, where 1 mol of phosphoryl group per mol subunit was transferred to the subunit 1. The phosphorylated transferase 1-1 showed decreased affinity for bilirubin, suggesting that the phosphorylation affects the function of glutathione S-transferase in an isozyme-specific manner.

Phosphorylation of microsome-bound cytochrome P-450 LM2

The phosphorylation of a microsomal protein of rabbit liver by catalytic subunit of cyclic AMP-dependent protein kinase was shown, and the protein was identified as cytochrome P-450 LM2 on basis of comparative peptide-mapping. Acid hydrolysis of microsome-bound phosphorylated cytochrome P-450 revealed that phosphorylation occurred exclusively on serine residues. This serine residue was identified as the same residue phosphorylated in purified, soluble P-450, that is, serine in position 128.

Fast liquid chromatographic assay of androgen aromatase activity

A rapid, sensitive nonradiometric assay method for the enzymatic aromatization of androgens has been developed using reversed-phase fast liquid chromatography. The use of a 3-microns-particle, 5-cm-long column allowed analysis of androstenedione, estrone, testosterone, and estradiol within 1 min. The reliability of the method was demonstrated by measuring the aromatase activity of human placental microsomes and that of the purified reconstituted aromatase system using both substrates. The rapid analysis enabled the processing of a large number of samples in a short time, which makes the present method especially suitable for the analysis of chromatographic fractions obtained during enzyme purifications and for enzyme kinetic studies.

Phospholipid bilayer membranes play decisive roles in the cytochrome P-450-dependent monooxygenase system

SummaryHepatic microsomal monooxygenase was reconstituted by incorporating cytochrome P-450 and NADPH-cytochrome P-450 reductase, which had been purified from phenobarbital-pretreated rabbit liver microsomes, into phospholipid liposomal membranes. The NADPH-dependent monooxygenase activity of the reconstituted system was found to be dependent on the phospholipid-to-protein ratio, i.e., the two-dimensional concentration of the two proteins on the plane of the membranes. A similar concentration dependence was also observed in the cytochromeb5 and NADH-cytochromeb5 system, which had been incorporated into liposomal membranes. The diffusion process of the proteins in the membrane, therefore, plays an important role in the monooxygenase system. When the fluidity of the membrane was changed by utilizing a synthetic dimyristoylphosphatidylcholine, which shows a well-defined gel to liquid crystalline phase transition, the activation energy of the monooxygenase reaction was changed at around the phase transition temperature, suggesting a conformational change of cytochrome P-450 caused by the fluidity change of the membrane. The incorporation of P-450 into liposomes was also found to affect the binding of substrates to cytochrome P-450. The decrease in the apparent dissociation constant of substrates upon incorporation into membranes suggests that the lipid membrane acts as a pool for hydrophobic substrates, which are concentrated in the lipid phase, and that cytochrome P-450 takes substrates directly from the membrane phase. Phospholipid membranes, therefore, play very important roles in various phases of the reaction of cytochrome P-450-dependent monooxygenase.

Posttranslational modifications of the cytochrome P-450 monooxygenase system.

SummaryTwo forms of enzymatic posttranslational modifications of the monooxygenase system are described: modification by phosphatase and modification by protein kinase. Phosphatase treatment of microsomes isolated from phenobarbital-pretreated rabbits and rats caused a marked decrease of monooxygenase activity which was paralleled by a comparable decrease of NADPH-cytochrome P-450 reductase activity while the second essential component of the system, cytochrome P-450, remained unaltered. Thus phosphatase attacks monooxygenase via reductase. Protein kinases showed the opposite preference; while cytochrome P-450 was phosphorylated, NADPH-cytochrome P-450 reductase was not. Thus the kinase affects monooxygenase via cytochrome P-450. The phosphorylation of cytochrome P-450 turned out to be a specific reaction observed only with certain cytochrome P-450 isoenzymes and certain protein kinases.

Catalytic subunit of cAMP-dependent protein kinase from bovine heart: several isoforms demonstrated by high resolution focusing in immobilized pH gradient.

The catalytic subunit (C) of cAMP-dependent protein kinase holoenzyme type II from bovine cardiac muscle was separated by isoelectric focusing in Immobiline polyacrylamide gels into 9 protein forms. The major forms (i) appeared at pH 7.1, 7.4, 7.5, and 7.7, (ii) exhibited protein kinase activity and were inhibited by heat and acid stable inhibitor, (iii) represented approx. 30%, 4%, 64%, and 1% of the protein respectively, (iv) refocused in the same position from which they had been eluted from the first gel. Antibodies against C detected additional proteins at approx. pH 7.55, 7.75, and 7.8. Two more bands became detectable at approx. pH 7.3 and 7.45 by application of antibody against C beta (Uhler, M.D. & McKnight G.S. 1987, J.Biol.Chem. 262, 15202-15207). The relation of the different forms of C to the fractions CA and CB (Kinzel V. et al. 1987 Arch. Biochem. Biophys. 253, 341-349) is demonstrated.

Posttranslational Modification by Phosphorylation: Control of Cytochrome P450 and Associated Enzymes

Regulation of proteins by posttranslational modification is of crucial importance for eucaryotic cells in particular because of the relatively long half-lives of their mRNAs and the resulting lack of rapid protein regulation at the nucleic acid level. The phosphorylation of proteins (and their dephosphorylation) has emerged as the most widespread and important posttranslational regulatory device. It is now known to occur in all types of cells and in virtually all cellular compartments and organelles (1.2).

Phosphorylation and Dephosphorylation of the Cytochrome P-450-Dependent Monooxygenase System: Regulatory Devices by Posttranslational Modification

Cyclic AMP-dependent protein kinase and unspecific phosphatase are described as posttranslational modifiers of the hepatic microsomal cytochrome P-450-dependent monooxygenase system. Kinase and phosphatase affect the system not by a protein phosphorylation-dephosphorylation mechanism but rather independently of each other at different sites of the monooxygenase system. The protein kinase introduces a phosphoryl group into cytochrome P-450, the terminal monooxygenase itself, at a critical serine residue causing a conformational change which converts P-450, into its inactive form P-420. Since only certain P-450 isoenzymes carry the kinase recognition sequence— Arg-Arg-X-Ser-, the phosphorylation may act as an isoenzyme-specific form of regulation of the monooxygenase system through the control of the degradation of P-450. In contrast, the phosphatase digests FMN, one of the two prosthetic flavins of the NADPH-cytochrome P-450 reductase, thereby interrupting the electron transport chain which leads to an unspecific decrease of the overall monooxygenase activity. Neither kinase nor phosphatase affect significantly the rest of the protein components of the monooxygenase system.