Denaturation of cytochrome P-450 by cyclophosphamide metabolites

Cyclophosphamide (CP) metabolites, acrolein and 4-hydroxy-CP, were found to denature rat liver microsomal cytochrome P-450, whereas another metabolite, phosphoramide mustard, CP $\text{per}\text{se}$ or its analog Ifosfamide had no effect. The denaturation produced by CP metabolites could be blocked by cysteine, suggesting an interaction between CP metabolite(s) and sulfhydryl groups in cysteine and probably in cytochrome P-450. These studies might explain the biochemical basis of the specific depression of various microsomal mixed function oxygenase activities produced by high doses of CP.     

Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450

Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450.     

Purification and properties of cytochrome P-450(11beta) from adrenocortical mitochondria.

A cytochrome P-450, which is functional in the steroid methylene 11β-hydroxylation (P-450_11β), has been purified to a protein weight of 85 kg per heme from bovine adrenocortical mitochondria. The purification is accomplished in the presence of deoxycorticosterone as a substrate stabilizer. The procedure involved solubilization of sonicated mitochondrial pellets, ammonium sulfate fractionation, alumina Cγ gel treatment and aniline-substituted Sepharose 4B chromatography.The purified preparation when freed from deoxycorticosterone, has a low spin type absorption spectrum which can rapidly be converted into a typical high spin substrate-bound form by the addition of an 11β-hydroxylatable steroid, either deoxycorticosterone or testosterone. The preparation exhibits high 11β-hydroxylase activity and is free from the cholesterol side-chain cleavage cytochrome P-450 (P-450_scc).The purified P-450_11β, when submitted to SDS-polyacrylamide gel electrophoresis, exhibits a single protein band (molecular weight of 46 kilodaltons) which is clearly distinguished from P-450_scc. As determined by the sedimentation equilibrium method, the molecular weight of the guanidine-treated P-450_11β is estimated to be 43 kilodaltons.     

Ornithine decarboxylase activity in regenerating rat adrenal cortex

The activity of ornithine decarboxylase during the regenerating process of adrenal cortex was evaluated in Sprague-Dawley male rats weighing 140–180 g. In this study, 4 groups of rats were enucleated and another 4 groups were sham operated. Animals were maintained at a temperature of 23 ± 1°C and 12:12 h light:dark cycle. At 7 and 11 days post surgery, animals were sacrificed at 0600 h and 1800 h, respectively. Adrenal glands were immediately removed and assayed for ornithine decarboxylase (ODC). The enzyme activity was found to be significantly elevated in enucleated groups as compared to the sham control groups at the 7th and the 11th day of surgery. ODC activity was found to be about 2 times higher at 1800 h by the 7th day and 5 times higher at 1800 h by the 11th day of adrenal surgery when compared to the activity at 0600 h. From these results, it appears that ODC activity not only increases as the regeneration process of adrenal cortex approaches completion, but also becomes more pronounced towards the end of the light period.     

NMR studies of P-450 model systems: new structural probes for sulfur-containing hemoproteins.

A simple model for ferrous cytochrome P-450 has been investigated by proton and carbon-13 Fourier transform NMR. In the proton spectrum of the β-phenethyl mercaptan-protoheme-CO complex, the protons α and β to mercaptide sulfur are observed 2.62 and 0.62 ppm upfield of tetramethylsilane. The ¹³CO spectra show characteristic shifts at 204.7 and 197.0 δ for neutral and deprotonated mercaptan complexes, respectively.     

Synthesis of liver cytochrome P-450b in a cell-free protein synthesizing system

Live ppolysomes isolated from rats that had been treated with phenobarbital (PB) are able to incorporate [³H]leucine into total protein $\text{in}\text{vitro}$ at a rate almost five times that of polysomes prepared from control animals. Specific immunoprecipitation of translational products has shown that polysomes from induced animals synthesize cytochrome P-450b at a rate almost seven times greater than polysomes from control animals. The increased protein and cytochrome P-450b synthesis can be detected as early as 6 h following phenobarbital administration and reaches a maximum at 12–18 h. The results suggest that PB administration effects an increase in mRNA for cytochrome P-450b.     

Differential control of adrenal drug and steroid metabolism in the guinea pig.

Studies were carried out to compare the effects of several physiological variables on adrenal microsomal drug (ethylmorphine demethylation) and steroid (21-hydroxylation) metabolism in guinea pigs. The rate of adrenal ethylmorphine (EM) metabolism increased with maturation in males but not females, resulting in a sex difference (M > F) in adrenal enzyme activity in adult guinea pigs. Twenty-one hydroxylase activity, in contrast, was similar in adrenals from males and females. The concentration of adrenal microsomal cytochrome P-450 was unaffected by age or sex. ACTH administration decreased adrenal EM demethylase activity but did not affect 21-hydroxylation. Testosterone, when given to female guinea pigs, increased the rate of EM metabolism and decreased 21-hydroxylase activity. Various compounds known to interact with adrenal microsomal cytochrome P-450 had divergent effects on EM metabolism and 21-hydroxylation $\text{in}$$\text{vitro}$. Prostaglandins E₁ and F_2α, spironolactone, and canrenone inhibited EM demethylation but not 21-hydroxylation. Simple aromatic hydrocarbons (benzene, toluene), in contrast, inhibited 21-hydroxylation but did not affect EM metabolism. The results indicate that adrenal drug and steroid metabolism are independently regulated and that different terminal oxidases (cytochrome P-450) are probably involved in adrenal 21-hydroxylation and EM demethylation.     

Cumene hydroperoxide and yeast cytochrome P-450: spectral interactions and effect on the genetic activity of promutagens.

Cells of $\text{Saccharomyces}\text{cerevisiae}$, harvested from log phase cultures, contain cytochrome P-450 and are capable of activating promutagens to products that are genetically active in the same cell. The effect of cumene hydroperoxide, a compound known to support cytochrome P-450-mediated reactions, on the activation of a variety of the promutagens was investigated. In all cases the genetic activity of the promutagens was increased. With dimethyl-nitrosamine as the promutagen, the increased rate of gene conversion was linear for at least 1 hr. Yeast cytochrome P-450 was stable in intact cells in the presence of cumene hydroperoxide. However, in microsomal preparations the cytochrome was rapidly destroyed. When cumene hydroperoxide was added to a suspension of intact yeast cells, a spectrum with a Soret maximum at 455 nm — indicative of an interaction with cytochrome P-450 — was observed.     

The rabbit pulmonary monooxygenase system: characteristics and activities of two forms of pulmonary cytochrome P-450.

Two forms of rabbit pulmonary cytochrome P-450 have been characterized spectrally and their activities in reconstituted monooxygenase systems investigated. The presence of both microsomal phospholipids and sodium cholate was required to obtain optimum activity. Only one of the cytochromes (I) was active in the N-demethylation of benzphetamine and the O-deethylation of 7-ethoxycoumarin. However, cytochrome II was 20% more active than cytochrome I in the metabolism of benzo[a]pyrene. The profile of the metabolites formed from benzo[a]pyrene indicated that metabolism at the 9 and 10 positions was insignificant in the case of cytochrome I but represented about 40% of the metabolites produced by cytochrome II. The two forms of the cytochrome are present in pulmonary microsomes in approximately equal amounts.     

Metabolism of cyclophosphamide by purified cytochrome P-450 from microsomes of phenobarbital-treated rats

Incubation of [³H]-sidechain-labeled and [¹⁴C]-C(4)-ring-labeled cyclophosphamide (CPA) with purified cytochrome P-450 from liver microsomes of rats treated with phenobarbital resulted in the production of a major metabolite that contained both labels, was unaffected by diazomethane, possessed high polarity, was identical in TLC and HPLC behavior to a synthetic standard, didechlorodihydroxy-CPA, and was converted to CPA and bis(2-chloroethyl)amine by thionyl choloride. These results indicate that phenobarbital-inducible cytochrome P-450 is able to dechlorinate CPA and may account, in part, for the inability of phenobarbital to enhance the therapeutic activity and toxicity of this important anticancer and immunosuppressive agent.     

Studies on cytochrome P-450-dependent lipid hydroperoxide reduction

A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction.     

Differences in the mechanism of NADPH- and cumene hydroperoxide-supported reactions of cytochrome P-450

A study has been carried out on the association of aldolase with the human erythrocyte membrane. It has been shown that the conditions employed during hypotonic hemolysis affect the amount of aldolase that remains bound to the cell membrane. Thus, the in vivo nature of this binding cannot be ascertained by this technique. Therefore, a method has been developed in which aldolase is crosslinked with glutaraldehyde to the inner surface of the membrane in intact red blood cells. Under the specified conditions, over 90% of the intracellular aldolase can be crosslinked to the membrane with less than 10% of the hemoglobin becoming bound. These results suggest that the localization of aldolase in situ is on or near the inner surface of the membrane. The amount of aldolase bound to the membrane following crosslinking can be decreased by preincubating the cells with cytoskeletal agents such as cytochalasin B, colchicine, and vinblastine sulfate. The in vitro binding of aldolase to the purified spectrin-actin and F-actin complexes was studied. Aldolase bound both complexes very tightly (K_D ? 10^?9m) and this binding could be inhibited by cytochalasin B, but not by colchicine. A competition binding study was carried out to determine if the binding of aldolase to F-actin involved specific interactions. Neither bovine serum albumin nor cytochrome c significantly inhibited the binding of aldolase to F-actin when each was present at equimolar concentrations with aldolase. However, glyceraldehyde 3-phosphate dehydrogenase inhibited aldolase binding to F-actin and when present at equimolar concentrations with aldolase completely blocked the association. The association of aldolase and other glycolytic enzymes with the erythrocyte membrane is discussed and it is postulated that aldolase could be localized in vivo on the inner surface of the membrane by attachment to actin or a spectrin-actin complex.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

Properties of the low-temperature Photosystem I primary reaction in the P-700-chlorophyll a-protein

The Photosystem I primary reaction, as measured by electron paramagnetic resonance changes of P-700 and a bound iron-sulfur center, has been studied at 15°K in P-700-chlorophyll a-protein complexes isolated from a blue-green alga. One complex, prepared with sodium dodecyl sulfate shows P-700 photooxidation only at 300°K, whereas a second complex, prepared with Triton X-100, is photochemically active at 15°K as well as at 300°K. Analysis of these two preparations shows that the absence of low-temperature photoactivity in the sodium dodecyl sulfate complex reflects a lack of bound iron-sulfur centers in this preparation and supports the assignment of an iron-sulfur center as the primary electron acceptor of Photosystem I.     

Constancy of proline hydroxylation in collagen synthesized at different temperatures by poikilotherms.

Previous experiments have suggested that the body temperature of an organism may in part regulate the hydroxyproline content of its collagen. In order to test this hypothesis, the hydroxyproline content of the collagen synthesized at different temperatures by fish cells in culture and by intact amphibian larvae was measured. High levels of prolyl hydroxylation were found at both low and high temperatures, which eliminates the possibility that temperature can directly modulate prolyl hydroxylation in vivo. Synthesis and hydroxylation of collagen appear to be coordinated so that unstable underhydroxylated molecules are not produced under normal circumstances.     

Aging-related decrease in hepatic cytochrome oxidase of the Fischer 344 rat

The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa₃ content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa₃ content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and $\text{ADP}\text{O}$ ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa₃ content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components.     

Selective inactivation of cytochrome P-450 isozymes by suicide substrates

The autocatalytic destruction of cytochrome P-450 by the following six substrates has been investigated in vivo and in vitro with microsomal and purified, reconstituted rat liver enzymes: 2-isopropyl-4-pentenamide (AIA), 1-ethinylcyclopentanol, 17α-propadienyl-19-nortestosterone, fluroxene, 5,6-dichloro-1,2,3-benzothiadiazole (DCBT), and 1-aminobenzotriazole (ABT). Administration of the first three substrates to rats pretreated with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC), or their incubation with hepatic microsomes from such rats, produced a larger decrease in cytochrome P-450 levels in the membranes from Pb- than 3-MC-treated rats. Comparable losses, however, were observed in microsomes from rats pretreated with both Pb and 3-MC when the last three agents were used. Similar experiments were carried out using the major cytochrome P-450 isozymes purified from liver microsomes of Pb- or 3-MC-treated rats. The Pb isozyme was inactivated during catalytic turnover of all six substrates while only three substrates (DCBT, ABT, and fluroxene) were found to inactivate the 3-MC isozyme. Oxygen consumption studies with purified enzymes have shown that AIA is not a measurable substrate for the 3-MC isozyme, a fact which explains its failure to inactivate this isozyme. Similar studies with the Pb isozyme establish that one enzyme molecule is inactivated for approximately every 230–320 AIA molecules processed by the enzyme.