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J B Schenkman

Publications and source records attributed to J B Schenkman.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of cytochrome-P450 reductase by polyols has an electrostatic nature.

The present study was undertaken to examine the nature of the inhibitory action of glycerol on the liver microsomal monooxygenase system. In agreement with earlier observations, glycerol inhibited benzphetamine N-demethylation by liver microsomes of the phenobarbital-treated rabbit. The presence of glycerol in the medium did not affect binding of the substrate to cytochrome P450. Another polyol, ethylene glycol, was equally efficient in inhibiting benzphetamine N-demethylation. Both also inhibited reduction of rabbit cytochrome P450 LM2, cytochrome c and potassium ferricyanide by NADPH-cytochrome-P450 reductase in microsomes. Recently, we showed that the stimulation of electron transfer by increased ionic strength is due to neutralization of electrostatic interaction between NADPH-cytochrome-P450 reductase and its charged redox partners [Voznesensky, A. I. & Schenkman, J. B. (1992) J. Biol. Chem. 267, 14669-14676]. Polyols have an opposite effect to that of salt on ionic properties of a solution. They decrease the dielectric constant, thereby promoting electrostatic interactions between proteins. Addition of polyols decreased the conductivity of the medium. When rates of electron transfer to charged acceptors, cytochrome P450, cytochrome c and potassium ferricyanide, at various salt and polyol concentrations, relative to activities in 200 mM sodium phosphate, were plotted as a function of the conductivity the data for each acceptor fit on the same line. In contrast, neither alteration of ionic strength nor polyol addition affected the rate of electron transfer from NADPH-cytochrome-P450 reductase to an uncharged acceptor 1,4-benzoquinone. The data obtained is consistent with our earlier suggestion that charge repulsion limits redox interactions between rabbit cytochrome P450 LM2 and its reductase at low ionic strength, and suggest that the observed action of polyols is the result of enhancement of electrostatic interactions that inhibits electron transfer between NADPH-cytochrome-P450 reductase and its charged redox partners. In congruence with the hypothesis, the Km of rabbit cytochrome P450 LM2 for NADPH-cytochrome-P450 reductase was increased almost one order of magnitude by elevating the glycerol content from 5% to 25% (by vol.) without a change in Vmax.

Animals↗
PubMed1992

The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing.

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.

Animals↗
PubMed1992

Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation.

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.

Animals↗
PubMed1992

Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene.

1. A polyclonal, monospecific antibody to a constitutive, diabetes-inducible and insulin-reversible cytochrome P-450 isozyme (RLM6) was used to screen a male rat liver cDNA library in lambda gt 11. Six clones harbouring the RLM6 cDNA insert were isolated initially from the expression library and three of these were further plaque-purified and sub-cloned. A 1.1 Kb cDNA insert, representing approximately 65% of the expected full length cDNA was characterized by restriction endonuclease mapping and sequenced by the dideoxy chain-termination method. Comparison of the nucleotide sequence of RLM6 cDNA to that of ethanol-inducible P4502E1 rat cDNA showed the two cDNAs to be identical, the RLM6 cDNA corresponding to nucleotides 310-1402 of the P4502E1 sequence. 2. RLM6 cDNA probe was used in Northern blot and RNA dot blot hybridization analysis to demonstrate that both streptozotocin-induced diabetes and fasting significantly elevated the steady-state level of RLM6 mRNA in male rat liver. Increased RLM6 mRNA level in the diabetic rat resulted in increased RLM6 apoprotein synthesis when polysomal RNA was used in a cell-free, protein-synthesizing system, indicating that the elevated RLM6 level observed in diabetic rats was correlated directly with the increased RLM6 mRNA concentration. 3. Daily insulin treatment of diabetic rats reversed the diabetes-dependent increase in RLM6 mRNA in a time-dependent manner, returning to control values after approximately 2 weeks of continuous insulin treatment. This insulin-dependent decrease of the RLM6 mRNA level was paralleled by a similar time-dependent decrease in serum acetone concentration. 4. Treatment of the male diabetic rat with testosterone also resulted in a decrease in both RLM6 mRNA and in vitro translated apoprotein. 5. Modulation of RLM6 mRNA level in the diabetic rat by insulin and testosterone, and the nucleotide sequence similarity with that of P4502E1 confirms that diabetes-inducible P450RLM6 and ethanol-inducible P4502E1 are coded for by the same gene.

Amino Acid Sequence↗
PubMed1991

Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes.

In intact rats, ethanol treatment has been associated with increases in hepatic levels of both P450IIB1/2 and P450IIE. When rat hepatocytes were cultured on an extracellular tumor matrix (Matrigel), exposure to ethanol from 48 to 96 h in culture resulted in increases in cytochromes P450IIE, IIB1/2, and IIIA. Cytochrome P450IIE was detected immunologically and enzymatically, using two activities associated with cytochrome P450IIE, p-nitrophenol hydroxylation, and acetaminophen activation to a metabolite that binds to glutathione. The content of cytochrome P450IIE in freshly isolated cells decreased when the cells were placed in culture. Exposure of the cultured hepatocytes to ethanol from 48 to 96 h after inoculation resulted in an increase in cytochrome P450IIE compared to untreated cultured cells. In addition, in culture, the amount of enzymatically active protein after ethanol treatment was equal to that in hepatocytes freshly isolated from intact animals. Ethanol treatment resulted in increases in cytochrome P450IIB1/2 compared to untreated cells, as shown immunologically and by increased benzyloxyresorufin dealkylase activity. However, phenobarbital induced cytochrome P450IIB1/2 to higher levels, compared to ethanol. Ethanol and phenobarbital treatments both increased P450IIIA, as determined immunologically and by the amount of propoxycoumarin depropylase activity that is inhibited by triacetyloleandomycin. However, the amount of P450IIIA increased after ethanol treatment was less than that increased after treatment with dexamethasone in these cells. The ethanol-mediated increases in all four forms of cytochrome P450 in culture suggest that these increases in the intact animal result from direct effects of ethanol on the liver.

Acetaminophen↗
PubMed1991

Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain.

We used immunocytochemical staining to localize the RLM6 form of cytochrome P-450 in rat brain. Immunofluorescence staining in vibratome sections was positive in cells that resembled oligodendrocytes, which are the cells that synthesize and maintain myelin. Double immunofluorescence staining with anti-RLM6, plus mouse monoclonal antibodies (MAb) against 2',3'-cyclic nucleotide-3'-phosphohydrolase or galactocerebrosides, showed localization of each of these oligodendrocyte "markers" in the same cells as RLM6. In vibratome sections from brains of adult rats there was faint RLM6 immunostaining in some of the myelinated fibers as well as in oligodendrocytes. In paraffin sections from adult rat brains, myelinated tracts were RLM6 positive, as were oligodendrocytes and myelinated fibers in the gray matter. Oligodendrocytes were also shown to contain glucose-6-phosphate dehydrogenase. We suggest that RLM6, which is constitutive to liver, is also constitutive to brain and, via the acetone monooxygenase reaction, which also utilizes NADPH, may contribute to the conversion of ketone bodies to substrates that could provide energy for the synthesis and maintenance of myelin.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1990

Relationship between phosphorylation and cytochrome P450 destruction.

In our previous report we showed cytochrome b5 to be a competitive inhibitor of cAMP-dependent protein kinase (PKA) for interaction with cytochrome P450 (P450). While P450 was phosphorylated, cytochrome b5 was not. The phosphorylation of P450 resulted in an inhibition of its catalytic activity. In this report we attempt to determine the relationship between phosphorylation of P450 from phenobarbital-induced rat and its destruction. The results indicate there is a considerable alteration of P450 IIB1 when it is put into the phosphorylation medium. This includes destruction, i.e., loss of the hemoprotein nature (Soret peak), as well as denaturation, conversion of a proportion of the P450 to P420. The extent of phosphorylation correlated best with the amount of destroyed hemoprotein, and not with the formation of P420. There did not appear to be phosphorylation-dependent formation of apo-P450. Further, prior conversion of the P450 to P420 using sodium deoxycholate showed the same extent of phosphorylation as before the conversion. Thus, intact P450 is not required for phosphorylation nor is phosphorylation a prerequisite for hemoprotein destruction. P450 CAM (CIA1), which has the PKA substrate recognition sequence internalized, likewise undergoes conversion to P420 but this denaturation does not result in phosphorylation. Destruction of CIA1 with 6 M urea, however, did permit phosphorylation by PKA. P450 IIB1 destruction was greatly diminished by cytochrome b5. This stabilization resulted in a decreased degree of phosphorylation as well as an increase in negative ellipticity in circular dichroism, indicative of an increase in the proportion of alpha-helical content in the P450. Suggestions are made that this structural modification caused by cytochrome b5 stabilizes the P450 against denaturation as well as against destruction and phosphorylation. Further, when the P450 IIB1 was kept stable as P450 in the absence of cytochrome b5 and without loss of hemoprotein during the incubation period, using phosphate-glycerol buffer containing 0.4% Emulgen 911, the phosphorylation of the P450 was greatly diminished, with only minor effects on the protein kinase reaction itself. These results suggest that the protein kinase reaction itself. These results suggest that the protein kinase substrate recognition sequence is not readily accessible to PKA in mammalian P450 IIB1 but requires a destabilization of the protein for phosphorylation to take place.

Animals↗
PubMed1990

Effects of testosterone and growth hormone treatment on hepatic microsomal P450 expression in the diabetic rat.

The profile of hepatic microsomal cytochrome P450 expressed in the male and female rat was dramatically altered by streptozotocin-induced diabetes. In the diabetic male, P450 forms IIC11, IIC13, IIA2, and IIIA2 were suppressed and forms IIA1 and IIC12 were induced to the levels observed in the immature male rat. A 6- to 8-fold induction of P450 IIE1 was detected in both male and female diabetic rats. A member of the P450 IIIA family was also induced in the diabetic female rat. Accompanying the change in P450 profile in the diabetic male rat was reduction in circulating testosterone and tetraiodothyronine concentrations and a sharp diminution of the normally pulsatile pattern of growth hormone secretion. In contrast to the male rat, the growth hormone secretion pattern in the diabetic female rat was unchanged from control. The hormone and P450 profiles detected in the diabetic male rat suggest a reversion to an immature physiological state. Testosterone replacement treatments carried out for 2 weeks slightly but significantly affected the suppression of P450 IIC11 and reversed the changes in P450 IIA2, IIIA2, and IIC12 in the diabetic male, without altering the suppressed state of growth hormone secretion. However, 1 week of human growth hormone, administered intravenously every 4 hr to diabetic male rats, failed to significantly reverse the diabetes-induced changes in hepatic cytochromes P450, in particular forms IIC11 and IIE1, despite the presence of an episodic plasma hGH profile. An induction of P450 IIE1 in diabetic female rats, without a reduction in growth hormone secretion, suggests that its induction in diabetes in both sexes is not related to changes in growth hormone. In addition, the results of testosterone treatment on the expression of IIC12, IIA2, and IIIA2 in the diabetic male rat suggest a regulatory role for this hormone that does not involve the pituitary secretion of growth hormone. However, the lack of effect of human growth hormone treatment in the diabetic male on levels of individual P450 forms indicates that in diabetes there may be a change in the ability of the male rat hepatocyte to respond to a somatic signal, possibly as a result of the changes in other hormone factors.

Animals↗