Heinz Schleyer

Ecdysone 20-monooxygenase: Characterization of an insect cytochrome P-450 dependent steroid hydroxylase ☆

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworm, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the product of the enzyme system by high-pressure liquid chromatography, gas-liquid chromatography and mass spectrometry. Differential centrifugation, sucrose-gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with the mitochondria. The enzymatic properties of ecdysone 20-monooxygenase are that it is most active in a 0.05 M phosphate buffer, is inhibited by Mg2+ and exhibits pH and temperature optima at 7.5 and 30 degrees C, respectively. The enzyme complex has an apparent Km for ecdysone of 1.60 x 10(-7) M and is competitively inhibited by its product, ecdysterone, with an apparent Ki of 2.72 x 10(-5) M. The cytochrome P-450 nature of this insect steroid hydroxylase was initially suggested by its obligate requirement for NADPH and its inhibition by carbon monoxide, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Difference spectroscopy revealed the presence of cytochrome P-450 in the fat-body mitochondrial fraction. A photochemical action spectrum of ecdysone 20-monooxygenase activity confirmed the involvement of cytochrome P-450 in this monooxygenase system.

SOME CHEMICAL PROPERTIES OF CYTOCHROME P‐450 AND ITS CARBON MONOXIDE COMPOUND (P‐450.CO) *

It is particularly fitting to include a discussion of cytochrome P-450 in this conference on the biological effects of carbon monoxide, since it was the ability of this heme protein to combine with CO that led to its discovery in liver microsomes by Klingenbergl and Garfinkel2 and to our demonstration of its function as the “terminal oxidase” of many mixed function oxidases involved in hydroxylation and modification of many lipid-soluble substrates.3~~ Most previous studies on cytochrome P-450 have employed microsomal preparations from liver tissue and endocrine glands or mitochondria1 preparations from endocrine tissue-all rather complex and somewhat ill-defined structure-bound enzyme systems, often with broad chemical specificities. The original observations relied heavily on the spectroscopic properties of the CO compound of P-450 (i.e., P-450.CO). Difference spectroscopy of the CO compound has been used to estimate steady state concentrations of P-450 as well as rates of the overall reactions and was, up to now, the only experimental approach to the study of P-450 in these multienzyme systems. Perhaps the most important application of carbon monoxide to the study of P-450 and related molecules is found in the demonstration of the functional role of the heme protein as terminal oxidase. This demonstration is based on the occurrence of CO inhibition, its light-dependent reversal, and the spectroscopic studies which lead to the action spectrum of the light reversal of inhibition5 in a manner analogous to that which led to the discovery of cytochrome oxidase, as discussed earlier in this monograph. By using these techniques, P-450 has been proven to be the terminal oxidase of at least 14 hydroxylases,6 including the important adrenocortical steroid-1 lp-hydroxylase.

Studies on non-heme iron proteins and the piericidin A binding site of submitochondrial particles from Candida utilis cells grown in media of varying iron concentrations.

EPR spectra of iron sulfur proteins associated with succinate dehydrogenase, glycerol-1-phosphate dehydrogenase, NADH dehydrogenase, and, perhaps, the cytochromes b and c1 in submitochondrial particles from Candidautilis are presented. The piericidin A sensitive site is assigned to the oxygen side of the iron sulfur protein associated with NADH dehydrogenase. Virtually no correlation of piericidin A sensitivity with the EPR signal of the NADH dehydrogenase-linked iron sulfur protein is indicated from comparison of these parameters as a function of iron concentration in the growth medium.

Presence of a low molecular weight inhibitor of succinate-supported cholesterol side chain cleavage in rat ovaries.

1. Low molecular weight fractions (mol. wt. 3500-10 000) prepared from cytosols of luteinized rat ovaries inhibited succinate-supported cholesterol side chain cleavage by intact ovarian mitochondria utilizing endogenous or exogenous sterol as substrate. 2. The low molecular weight fractions inhibited steroid secretion by collagenase-dispersed ovarian cells stimulated with lutropin or dibutyryl cyclic AMP. 3. Steroidogenesis by intact mitochondria incubated with NADPH was enhanced by the low molecular weight ovarian fraction, but cholesterol side chain cleavage carried out by sonicated mitochondria incubated with NADPH was unaffected. 4. Succinate-supported mitochondrial respiration was stimulated by the low molecular weight factor, apparently by uncoupling of oxidative phosphorylation. The uncoupling seems to be the mechanism by which steroid synthesis is inhibited. 5. The low molecular weight factor was heat-labile and not extracted by activated charcoal. Similar heat-labile material capable of inhibiting succinate-supported mitochondrial steroid synthesis was not found in low molecular weight fractions prepared from rat kidney, liver, spleen, brain, plasma and bovine corpus luteum. 6. Treatment of rats with cycloheximide 1 h before killing resulted in a reduction of inhibitory activity in ovarian low molecular weight cytosolic fractions. 7. We conclude that ovarian cytosols contain a low molecular weight factor, presumably a protein, which inhibits mitochondrial cholesterol side chain cleavage by uncoupling oxidative phosphorylation. The physiological function of this factor remains to be determined.

Electron paramagnetic resonance studies on photosynthetic bacteria. I. Properties of photo-induced EPR-signals of Chromatium D.

Abstract (1) Photo-induced EPR-signals of the free radical type were investigated in a variety of preparations derived from the photosynthetic bacterium Chromatium D. Materials ranged from intact, actively photosynthesizing organisms to cell-free “simplified systems” such as chromatophore-type preparations to synthetic photochemical systems containing bacteriochlorophyll in solution. Closely similar signals were observed in terms of the general properties such as g -value, linewidth and line shape, microwave saturation behaviour, and lack of detectable hyperfine structure. (2) Improvements of the time response capabilities of the instrumentation made fast measurements possible — present time limit a few msec — and allowed detailed kinetic studies on all these materials. Characteristic time courses of both formation and decay of photo-induced EPR-signals were observed. They are seen to fall into two classes of patterns which are described in detail for intact photosynthesizing bacteria and for the classical chromatophore-type preparation. These patterns are termed the “cell pattern” and the “chromatophore-type pattern”, respectively. (3) The “cell pattern” is characterized by a relatively slow and complex time course of formation with a fast initial rise at the onset of irradiation and a much faster, comparatively simple time course of decay. It is only observed when intact bacteria are studied under physiological conditions and is strongly dependent on such metabolic parameters as age of the cultures, substrate levels, and preillumination history. Changes introduced by altering the metabolic situation are reversible. No evidence for the existence of lag periods in either formation or decay has been obtained. (4) The “chromatophore-type pattern” shows a fast monotonic rise with no complexities and a much slower and typically biphasic decay. It is a quite stable phenomenon, strictly repetitive, and relatively independent of the quality and intactness of the preparation used. This pattern is observed with the classical chromatophore-type preparations and also with a large number of further simplified systems. (5) The transition from the cell pattern to the chromatophore-type pattern is readily achieved by unspecific treatments and is found to be irreversible. It occurs at a level of organization very close to that of the intact organism and is accompanied by a gradual loss of the overall photosynthetic capacities of the materials. (6) A comparison of these results with other measurable parameters of bacterial photosynthesis shows that existence and demonstratability of these photo-induced EPR-signals parallel strictly the photochemical activities of the bacteriochlorophyll molecule, including its participation in bacterial photosynthesis. On the other hand, the existence of the two characteristic time course patterns parallels the overall photosynthetic capacities of the materials, but does not correlate with the known capacities of the materials to form ATP in the process of photophosphorylation. The results demonstrate clearly that the paramagnetic species detected by EPR-spectroscopy are closely linked to the overall processes of photosynthesis and are participating in the electron transfer system of these bacteria.

Studies on the electron-transfer systems in photosynthetic bacteria IV. Kinetics of light-induced cytochrome reactions and analysis of electron-transfer paths

Abstract The light-induced cytochrome reactions and dark steady state changes were studied in photosynthetic bacteria, Chromatium and Rhodospirillum rubrum . The effects of external substrates, reducing agents, inhibitors, O 2 tension etc. on the kinetics and the steady state changes of the cytochrome reactions are presented. The pattern of electron transfer in the bacterial systems changed markedly under different conditions. O 3 was one of the key factors to control the patterns. In general, apparently “non-cyclic” cytochrome response was observed under the growing conditions; on the other hand, ‘cyclic” or “closed-chain” type characteristics were observed under non-growing aerobic conditions. Kinetic analysis indicated that heptylhydroxyquinoline- N -oxide had an interaction with the reaction of cytochrome c and cytochrome b as had been postulated from difference spectra. Cross-over of the photochemical oxidation and photochemical reduction was observed in the presence of the inhibitor. It was indicated that ascorbate and trichlorophenolidophenol (added singly or combined) did not compete effectively with the internal photochemical reducing system and did not change the steady state of cytochromes. Under certain conditions, however, ascorbate plus trichlorophenolindophenol shifted the steady state of cytochrome b . The formation of an electron-transfer by-pass by N -methylphenazonium methosulfate was confirmed. Direct interaction of bacteriochlorophyll and cytochrome molecules was postulated in the heptylhydroxyquinoline- N -oxide-insensitive rapid “light-off” reaction or aerobic cells, as well as in the rapid phase of the light-induced oxidation of cytochrome. The possibility of the control of electron transfer by concentration of adenosine phosphates was also discussed.

STUDIES ON THE PARTIALLY PURIFIED HEME PROTEIN P‐450 FROM THE ADRENAL CORTEX*

The reconstituted steroid 1 lp-hydroxylase is a particularly suitable system for studying the individual reactions involved in hydroxylations catalyzed by mixedfunction oxidase. This system is that described elsewhere by our laboratory and is composed of a specific flavoprotein (Fp), an iron-sulfur protein (ISP), and a partially purified heme protein P-450 preparation.’ The electron transport scheme and the oxidation-reduction properties found in our previous work are outlined in FIGURE 1. These studies furthermore have demonstrated that TPNH and the flavoprotein are dispensable and that the electrons for 11/3-hydroxylation can be supplied by reduced ISP.’*8 In this paper we wish to describe in some detail the results of recent studies on the properties of the individual components of this system.

The metabolism and excretion of enzyme-inducing doses of phenobarbital by rats with bile fistulas

The metabolism of enzyme-inducing doses of 14C-phenobarbital injected i. p. into bile duct-cannulated rats has been studied using improved chromatographic separation and quantification techniques. In animals with bile fistulas most of the 14C-phenobarbital was excreted in bile as p-hydroxyphenobarbital conjugated with glucuronic acid. In urine the main substance found was phenobarbital, with significant amounts of p-hydroxyphenobarbital and varying amounts of its glucuronide conjugate. Animals without bile fistulas excreted 80% dose of phenobarbital in the urine; metabolites were free phenobarbital, p-hydroxyphenobarbital and conjugated material. Approx 90% of the conjugated material was the glucuronide. Only free phenobarbital and p-hydroxyphenobarbital were found in the faeces. Animals drinking plain water excreted 50-65% dose of phenobarbital (80 mg/kg) in bile and the remainder mainly in the urine, whereas superhydrated animals (drinking 5% glucose and 0.9% NaCl) excreted 90% of the dose as free phenobarbital in the urine. Phenobarbital is the only labelled material detectable in hepatic tissue and portal, vena caval or aortic blood, which indicates that phenobarbital is the enzyme-inducing substance and that liver and kidney rapidly eliminate all metabolites. Metabolism of phenobarbital in vivo is a complex process involving interaction of hepatic and intestinal metabolism, partial readsorption from the intestinal tract and renal elimination.

Comparison of the Induction Course, Biophysical Chemical Interactions and Photochemical Action Spectra of Phenobarbital and 3-Methylcholanthrene Induced Hepatic Microsomal P-450

A comparison has been made of the physical and chemical properties of hepatic microsomal P-450 and associated enzyme systems from rats treated with phenobarbital or with 3-methylcholanthrene and other polycyclic aryl hydrocarbons. The results of these studies, though preliminary in nature, indicate clearly that the aryl-induced mixed-function oxidase systems differ significantly from the PB-induced ones in time course of induction, spectral properties, hyroxylase and demethylase activities, CO-inhibition of these reactions and light-reversal of the inhibition. The results support and extend the findings of other investigators regarding the differential biophysical and biochemical properties of aryl-induced systems and provide an experimental design for studying these properties in greater depth at the maximum of aryl induction.

Comparative kinetic studies of the dealkylation reaction and steroid hydroxylations in various oxygen and carbon monoxide:oxygen atmospheres.

The time-course kinetics of the cytochrome P-450-catalyzed dealkylations of the exogenous compounds benzphetamine, ethylmorphine, codeine, and 7-ethoxycoumarin were compared to the hydroxylation of the endogenous compound testosterone. Using liver microsomes from phenobarbital-induced rats, the time course of the demethylations of ethylmorphine, codeine, and especially benzphetamine was characterized by a fast initial phase of enzymatic activity and then a steady decline in the rate throughout the remainder of the reaction. In contrast, under the same experimental conditions, both the dealkylation of 7-ethoxycoumarin and the hydroxylation of testosterone showed no initial fast phase of activity and a constant rate of product formation for most of the remainder of the time course. The difference also held for the carbon monoxide inhibition studies in which the degree inhibition of the demethylation reactions by a variety of CO:O2 mixtures was time dependent, in contrast to the constant, time-independent degree of CO inhibition of the other two reactions. The kinetics of the demethylation reactions could not be explained by enzyme destruction, back reaction, or product adduct formation and were further confirmed by measurements of the rate of O2 utilization and NADPH oxidation. The complexity of the demethylation reaction should be taken into consideration in any detailed studies of the monooxygenation reaction system.