No abstract

To study the molecular mechanisms of circadian gene expression, we have sought to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. Here, we report that the Cyp2a4 gene, encoding the cytochrome P450 steroid 15alpha-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver was significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5 genes in the mouse liver.

enzymes are known to be controlled by many different stimThe influence of cell density and epidermal growth uli, including physiopathological, environmental, and genetic factor (EGF) on the expression and inducibility of cytofactors, so that changes in the battery of CYP enzymes are chrome P450 (CYP) genes of the CYP3A and CYP1A famiexpected to occur not only from one individual to another, lies in adult human hepatocytes in primary culture has but also in the same individual as a function of time, for been evaluated. Only when cultured at subconfluence example. These changes may have critical consequences in and in the presence of EGF did hepatocytes exhibit a pharmacology and toxicology, especially in those individuals proliferative response, assessed by measuring DNA synwho are significantly exposed to xenobiotics. thesis and cyclin A accumulation. In the absence of EGF,The CYP3A and CYP1A families have been investigated the accumulation of CYP3A4 and CYP1A2 messenger extensively because they play a critical part both in pharmaRNAs (mRNAs) in response to their respective inducers (rifampicin and dioxin) was dramatically decreased in cology and toxicology. 3,4 In humans, the CYP3A family has subconfluent culture with respect to confluent cultures. three functional genes, including CYP3A4, CYP3A5, and The presence of EGF only slightly decreased the accu-CYP3A7. 1,2,5 CYP3A4 is the major CYP protein expressed in mulation of these mRNAs in both confluent and subcon-the adult liver, where it may represent up to 60% of the fluent cultures. The accumulation of CYP2D6 and total amount of CYP proteins. 6 This CYP is involved in the CYP2E1 proteins, which are constitutively expressed in oxidative metabolism of the majority of drugs in current use, confluent cultures, and the production of fibrinogen and as well as in the activation of many procarcinogens, including apolipoprotein (Apo) B100 exhibited similar behavior, polycyclic aromatic hydrocarbons and aflatoxins. 5,7 CYP3A4 while nicotinamide adenine dinucleotide phosphate cy-is inducible by many structurally unrelated compounds such tochrome c reductase activity was affected neither by as rifampicin (RIF), glucocorticoids, imidazole-derivatives, cell density nor by EGF. In contrast, the accumulation and phenobarbital. 8-10 CYP3A7 is the major form of CYP exof CYP1A1 mRNA in response to dioxin was similar in pressed in the human fetal liver. 11,12 Shortly before birth, confluent and subconfluent cultures, irrespective of the however, the accumulation of CYP3A7 messenger RNA presence of EGF. Interestingly, CYP3A7, a gene that is (mRNA) and protein decreases to a low or undetectable level preferentially expressed in the fetal liver, was expressed in the newborn and adult liver. In contrast, the level of constitutively neither in confluent nor in subconfluent CYP3A4 mRNA and protein rapidly increases during the cultures, irrespective of the presence of EGF. It is con-perinatal period. 13 CYP3A5, which is expressed in a polymorcluded that the loss of cell-cell contacts ...

The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mM GTP and 3.4 M glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction.Microtubules are highly dynamic structures that switch between growing and shrinking phases both in vivo and in vitro. These cytoskeleton polymers are necessary for many functions within the cell including intracellular transport, motility, morphogenesis, and cell division. The intrinsic dynamic instability of microtubules is further modified in the cell by numerous protein factors that favor alternatively elongation, shortening, or anchoring of these polymers. Because the mitotic spindle plays a crucial role in cell division, it has been used for decades as an important target in cancer chemotherapy. Many tubulin poisons have been identified, some of them, taxanes and vinca alkaloids, have demonstrated therapeutic value. However, all tubulin poisons are not of clinical utility. This has led to extensive efforts to explore other targets that could affect spindle integrity. A promising approach is to identify the protein regulators that modulate tubulin polymerization and to investigate their mechanism of action.The dynamic instability of microtubules is controlled in vivo by several classes of cellular factors including depolymerizing kinesins (MCAK/XKCM1) (1, 2), stathmins (3), and microtubule-associated proteins (MAPs).2 This last group is composed of structural MAPs (MAP2, tau) that were first identified in brain tissue and of a group of XMAP215-related proteins whose generic member was first characterized in Xenopus eggs (4). TOGp (HUGO gene CKAP5), which is highly expressed in tumors and brain (5), is the human homolog of XMAP215. TOGp promotes microtubule assembly both in solution and from n...

The spin state of camphor-bound cytochrome P-450 is shown to depend largely on medium and temperature in aqueous as well as in mixed organic buffer. At sub-zero temperatures a variation of pa,, ionic strength or camphor concentration modifies the spin equilibrium from nearly pure high-spin form to nearly pure low-spin form. Since the apparent pK, of transition is a linear function of log I, the spin state seems to be controlled by the electrostatic potential in the heme proximity. K' is found to have a specific effect on the spin state.The change of enthalpy, AH, of the spin transition depends on the same parameters as the equilibrium constant, in the organic cosolvent as well as in aqueous buffer. As the cosolvent effect is reflected by higher AH values, and KCl and pH tend to lower AH, the cosolvent effect can easily be compensated. Therefore kinetic studies of the spin conversion might well be undertaken at sub-zero temperature in this solvent.Recently [l], we have shown the advantages of studies of the first step of the camphor hydroxylation cycle : the binding of camphor to cytochrome P-450 at sub-zero temperatures in a mixed organic solvent. Simultaneously with results of Sligar [2] above 0 "C in aqueous phosphate buffer, we found that camphorbound cytochrome P-450 (Fe3+.RH) is in thermal spin equilibrium in fluid hydro-organic medium at temperatures well below 0 "C. Some interesting results have been gathered by such a technique, for example the mechanism of camphor binding and the low-spin+high-spin transition of Fe3+ . RH were found to depend on pa, in the physiological pa, range, and the ionization state of a basic residue (possibly histidyl) was shown to be affected by the absence or presence of camphor.However, these results were overshadowed by the fact that the cosolvent, although not denaturing, shifted the [high-spin]/[low-spin] equilibrium constant K and AH to higher values [l].Here we show that K and AH of the low-spin to high-spin equilibrium of Fe3 +. RH are in fact relative values which depend on (a) physiological parameters such as ionic strength, concentration of K + , which is found to be a specific modulator of the high-spin/ low-spin equilibrium, concentration of camphor, pa, and (b) non-physiological parameters such as polyelectrolytes, organic cosolvent.The possibility of modulating K (= [high-spin]/ [low-spin]) at sub-zero temperatures by variation in these factors, namely pa, and ionic strength, enables us to study the transition of nearly pure low-spin to nearly pure high-spin Fe3+. RH and sheds some light on the structural basis of the mechanism of the spin conversion.Furthermore, we have examined the cosolvent effect on AH, a very important term of the mechanism of a reaction. In this paper we show how K may be adjusted to any desired value by simple modification of the parameters (a) or (b) and how the cosolvent effect on AH can be compensated.These various studies are the basis of a kinetic analysis of the Fe3+. RH spin state transition, which will be developed in a future pape...

No abstract

No abstract

We have investigated the CO binding to various reduced hemoproteins by stopped-flow rapid mixing as a function of pressure (from 0.1 to 200 MPa) and temperature (from 4 to 35 degrees C). In particular, we studied several varieties of cytochrome P-450: CYP11A1 (scc), CYP2B4 (LM2), CYP3A6 (LM3c), and Cyp2a (7 alpha), as well as chloroperoxidase and lactoperoxidase, and compared the results to data reported for other hemoproteins. Whereas the CO binding activation enthalpy delta H++ and entropy delta S++ (correlated through a compensation effect) varied greatly between the hemoproteins, with no apparent relation to structural features, the pressure effect depended on the nature of the proximal axial heme ligand: the activation volume was very small for cysteine (S-) ligand hemoproteins (delta V++ = +1 to +6 ml mol-1), and markedly negative for histidine (N) ligand hemoproteins (delta V++ = -3 to -36 ml mol-1). Furthermore, the transition state volume of the histidine ligand class enzymes, but not that of the cysteine ligand enzymes, depended on the solvent composition. These results suggest that the CO-binding transition state of the S-ligand class has a molecular conformation similar to the ground state. In the histidine class, however, the transition state appears to involve protein conformational changes and/or solvation processes.