Evidence for the reactivity of fatty aldehydes released from oxidized plasmalogens with phosphatidylethanolamine to form Schiff base adducts in rat brain homogenates

The vinyl ether bond of plasmalogens could be among the first target of free radicals attack. Consequently, because of their location in the membranes of cells, plasmalogens represent a first shield against oxidative damages by protecting other macromolecules and are often considered as antioxidant molecules. However, under oxidative conditions their disruption leads to the release of fatty aldehydes. In this paper, we showed using gas chromatography-mass spectrometry (GC-MS) analyses that fatty aldehydes released from plasmalogens after oxidation (UV irradiation and Fe2+/ascorbate) of cerebral cortex homogenates can generate covalent modifications of endogenous macromolecules such as phosphatidylethanolamine (PE), like the very reactive and toxic malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). These newly formed Schiff base adducts could be responsible for deleterious effects on cells thus making the protective role of plasmalogens potentially questionable.     

Plasmalogen degradation by oxidative stress: production and disappearance of specific fatty aldehydes and fatty alpha-hydroxyaldehydes.

Plasmalogens are often considered as antioxidant molecules that protect cells from oxidative stress. Their vinyl ether bond could indeed be among the first targets for newly formed radicals. However, the long chain aldehydes released from plasmalogens were seldom studied and possible injurious or harmless effects were poorly examined. Thus, the sensitivity of the vinyl ether bond of plasmalogens was investigated in a cerebral cortex homogenate under UV irradiation- or Fe2+/ascorbate-induced peroxidation. Kinetics of aldehyde production was followed by gas chromatography/mass spectrometry. This confirmed that plasmalogens were highly sensitive to oxidative stress (70% cleavage after 90 min UV irradiation and 30% after 30 min of Fe2+/ascorbate). The aldehydes corresponding to sn-1 position 16:0, 18:0, or 18:1 were poorly detected. Conversely, oxidation of plasmalogens yielded preferentially 15:0, 17:0, and 17:1 aldehydes under UV and the alpha-hydroxyaldehydes 16:0-OH and 18:0-OH following a Fe2+/ascorbate oxidation. Kinetics showed that free aldehydes and above all free alpha-hydroxyaldehydes disappeared from the medium as soon as produced. Consequently, the behavior of these released aldehydes in the tissues has to be investigated in order to ascertain the protective effect of plasmalogens against oxidation.     

The protective role of plasmalogens in iron-induced lipid peroxidation

The role of plasmalogens in iron-induced lipid peroxidation was investigated in two liposomal systems. The first consisted of total brain phospholipids with and without plasmalogens, and the second of phosphatidylethanolamine/phosphatidylcholine liposomes with either diacyl- or alkenylacyl-phosphatidylethanolamine. By measuring thiobarbituric acid reactive substances, oxygen consumption, fatty acids and aldehydes, we show that plasmalogens effectively protect polyunsaturated fatty acids from oxidative damage, and that the vinyl ether function of plasmalogens is consumed simultaneously. Furthermore, the lack of lag phase, the increased antioxidant efficiency with time, and the experiments with lipid- and water-soluble azo compounds, indicate that plasmalogens probably interfere with the propagation rather than the initiation of lipid peroxidation, and that the antioxidative effect cannot be related to iron chelation.     

Effects of iron limitation on the degradation of toluene by Pseudomonas strains carrying the tol (pWWO) plasmid

Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P. putida WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.     

The metabolism of halothane by hepatocytes: a comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.     

Gamma-hydroxybutyric acid induces oxidative stress in cerebral cortex of young rats

GHB is a naturally occurring compound in the central nervous system (CNS) whose tissue concentration are highly increased during drug abuse and in the inherited deficiency of succinic semialdehyde dehydrogenase (SSADH) activity. SSADH deficiency is a neurometabolic-inherited disorder of the degradation pathway of gamma-aminobutyric acid (GABA). It is biochemically characterized by increased concentrations of gamma-hydroxybutyric acid (GHB) in tissues, cerebrospinal fluid (CSF), blood and urine of affected patients. Clinical manifestations are variable, ranging from mild retardation of mental, motor, and language development to more severe neurological symptoms, such as hypotonia, ataxia and seizures, whose underlying mechanisms are practically unknown. In the present study, the in vitro and in vivo effects of GHB was investigated on some parameters of oxidative stress, such as chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), as well as the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in homogenates from cerebral cortex of 15-day-old Wistar rats. In vitro, GHB significantly increased chemiluminescence and TBA-RS levels, while TRAP and TAR measurements were markedly diminished. In contrast, the activities of the antioxidant enzymes SOD, CAT and GPX were not altered by GHB in vitro. Acute administration of GHB provoked a significant enhance of TBA-RS levels and a decrease of TRAP and TAR measurements. These results indicate that GHB induces oxidative stress by stimulating lipid peroxidation and decreasing the non-enzymatic antioxidant defenses in cerebral cortex of young rats. If these effects also occur in humans, it is possible that they might contribute to the brain damage found in SSADH-deficient patients and possibly in individuals who consume GHB or its prodrug gamma-butyrolactone.     

F2-isoprostanes as biomarkers of lipid peroxidation in patients with chronic renal failure

Chronic renal failure patients on long-term hemolysis are found to be under increased oxidative stress, caused by antioxidant deficiency, neutrophil activation during hemodialysis (HD), platelet activation and/or chronic inflammation. Increased levels of oxidants (e.g. malondialdehyde, 4-hydroxynonenal, hydrocarbons, lipohydroperoxides, oxycholesterols, carbonyls) in HD patients are thought to play an important role in the development of endothelial dysfunction, atherogenesis and cardiovascular disease, which is a frequent condition in end-stage renal disease. F2-isoprostanes have been established as chemically stable, highly specific and reliable biomarkers of in vivo oxidative stress which can very sensitively measured by gas chromatography-mass spectrometry (Morrow et al. [17]). An up to 6-fold increase of plasma F2-isoprostanes in HD patients is accompanied by an enhanced formation of indicators of inflammation (e.g. C-reactive protein) and decreases of endogenous antioxidants (e.g. ascorbate, alpha-tocopherol). In their esterified form F2-isoprostanes may be a useful criteria to evaluate the effectiveness of clinical interventions to diminish oxidant stress and associated inflammation. Furthermore, F2-isoprostanes possess potent biological activities (e.g. 8-iso-PGF2alpha is known as a renal vasoconstrictor) suggesting that they may also act as mediators of the cellular effects of oxidative stress and inflammation.     

The Effect In vitro of High-Density Lipoprotein from Healthy and Infected Humans on the Oxidative Metabolism of Polymorphonuclear Leukocytes

We studied the effects in vitro of high-density lipoprotein from healthy (N-HDL) and from infected humans (AP-HDL) on the oxidative metabolism of human polymorphonuclear leukocytes (PMN). Products of the H₂O₂–MPO–halide system were monitored by luminol-enhanced chemiluminescence and superoxide anion formation was monitored by lucigenin-enhanced chemiluminescence during stimulation of human PMN with phorbol myristate acetate (PMA) or an opsonized stimulus (OS). The results showed that N-HDL and AP-HDL affect the oxidative metabolism of PMN in different ways. The posible role of this effect is discussed. © 1997 John Wiley & Sons, Ltd.     

Determination of choline and ethanolamine plasmalogens in human plasma by HPLC using radioactive triiodide (1-) ion (125I3-)

For the purpose of developing highly sensitive and convenient determination of plasmalogens, the high-performance liquid chromatography (HPLC) method using radioactive iodine ((125)I) was investigated. Radioactive triiodide (1-) ion ((125)I(3)(-)), which is an actual iodine form capable of reacting with vinyl ether bond ([bond]CH(2)[bond]O[bond]CH[double bond]CH[bond]) of plasmalogens, could be safely and efficiently produced by oxidizing a commercial radioactive sodium iodine (Na(125)I) with hydrogen peroxide (H(2)O(2)) under acid condition (pH 5.5-6.0), which is called iodine-125 reagent. I(3)(-) specifically reacted with plasmalogens at the molar ratio of 1:1 in methanol, and 1 or 2 mol of plasmalogens was involved in the binding with iodine per iodine atom, resulting in the formation of stable iodine-binding phospholipids. The HPLC system with Diol column and acetonitrile/water as a mobile phase was available for separating iodine-binding phospholipids from nonbinding free iodine and for separately eluting iodine-binding phospholipids derived from choline and ethanolamine plasmalogens. Using iodine-125 reagent (1.85 MBq/ml), plasmalogens were detectable at high sensitivity of 10,000-15,000 cpm/nmol, which is more than 1000-fold higher sensitivity than the classical determination with nonradioactive iodine. Plasmalogen concentrations in human plasma were measured with the HPLC system and determined as, on average, 129.1+/-31.3 microM (n=8) in a 1.2 content ratio of choline to ethanolamine plasmalogens, a concentration that nearly agrees with the value reported previously.     

Identification of plasmalogens in the cytoplasmic membrane of Bifidobacterium animalis subsp. lactis

Plasmalogens are ether-linked lipids that may influence oxidative stress resistance of eukaryotic cell membranes. Since bacterial membrane composition can influence environmental stress resistance, we explored the prevalence of plasmalogens in the cytoplasmic membrane of Bifidobacterium animalis subsp. lactis. Results showed plasmalogens are a major component of the B. animalis subsp. lactis membrane.     

Reactive chlorinating species produced during neutrophil activation target tissue plasmalogens: production of the chemoattractant, 2-chlorohexadecanal.

Recently alpha-chloro fatty aldehydes have been shown to be products of reactive chlorinating species targeting the vinyl ether bond of plasmalogens utilizing a cell-free system. Accordingly, the present experiments were designed to show that alpha-chloro fatty aldehydes are produced by activated neutrophils and to determine their physiologic effects. A sensitive gas chromatography-mass spectrometry technique was developed to detect pentafluorobenzyl oximes of alpha-chloro fatty aldehydes utilizing negative ion chemical ionization. Phorbol 12-myristate 13-acetate activation of neutrophils resulted in the production of both 2-chlorohexadecanal and 2- chlorooctadecanal through a myeloperoxidase-dependent mechanism that likely involved the targeting of both 16 and 18 carbon vinyl ether-linked aliphatic groups present in the sn-1 position of neutrophil plasmalogens. 2-Chlorohexadecanal was also produced by fMLP-treated neutrophils. Additionally, reactive chlorinating species released from activated neutrophils targeted endothelial cell plasmalogens resulting in 2-chlorohexadecanal production. Physiologically relevant concentrations of 2-chlorohexadecanal induced neutrophil chemotaxis in vitro suggesting that alpha-chloro fatty aldehydes may have a role in neutrophil recruitment. Taken together, these studies demonstrate for the first time a novel biochemical mechanism that targets the vinyl ether bond of plasmalogens during neutrophil activation resulting in the production of alpha-chloro fatty aldehydes that may enhance the recruitment of neutrophils to areas of active inflammation.     

Kinetic properties of plasmalogenase from bovine brain.

Abstract— Plasmalogenase was assayed by measuring the disappearance of the plasmalogen by two-dimensional thin-layer chromatography. The enzyme was present in a glycerol-bicarbonate extract of an acetone-dried powder from bovine brain. With ethanolamine plasmalogens as the substrate, the K_m was 180 μM. Diacyl glycerophosphorylcholines, diacyl glycerophosphorylethanolamines and choline plasmalogens were competitive inhibitors. With choline plasmalogens as the substrate, the K_m was 208 μM and competitive inhibition was observed with diacyl glycerophosphorylcholines and ethanolamine plasmalogens. The same enzyme may be responsible for the hydrolysis of the alk-1-enyl moiety from both plasmalogens. Plasmalogenase activity was 5.1 μmol/h/g of dog brain, 3.9 μmol/h/g of rat brain and 3.4 μmol/h/g of gerbil brain. A lysophospholipase was also found in the glycerol-bicarbonate extract from the acetone-dried powder. The lysophospholipase was more active in hydrolysing acyl groups from 2-acyl-sn-glycero-3-phosphorylethanolamines than the plasmalogenase was active in hydrolyzing alk-1-enyl groups from 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines.     

Ethanolamine plasmalogens protect cholesterol-rich liposomal membranes from oxidation caused by free radicals

The aim of the present study is to investigate the effect of ethanolamine plasmalogens on the oxidative stability of cholesterol-rich membranes by comparing it with that of diacyl glycerophosphoethanolamine, using bovine brain ethanolamine plasmalogen (BBEP) or egg yolk phosphatidylethanolamine (EYPE)-containing large unilamellar vesicles (LUVs) and the water-soluble radical initiator AAPH. Electron microscopic observation and particle size measurement visually demonstrated that ethanolamine plasmalogens protect cholesterol-rich phospholipid bilayers from oxidative collapse. Lipid analyses suggested that the effect of ethanolamine plasmalogens in stabilizing membranes against oxidation is partly due to the antioxidative action of plasmalogens involved in scavenging radicals at vinyl ether linkage.     

Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes

ABSTRACT: BACKGROUND: Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. RESULTS: The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation. CONCLUSIONS: In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.     

Plasmalogen synthesis is regulated via alkyl-dihydroxyacetonephosphate-synthase by amyloid precursor protein processing and is affected in Alzheimer's disease

Lipids play an important role as risk or protective factors in Alzheimer's disease, which is characterized by amyloid plaques composed of aggregated amyloid-beta. Plasmalogens are major brain lipids and controversially discussed to be altered in Alzheimer's disease (AD) and whether changes in plasmalogens are cause or consequence of AD pathology. Here, we reveal a new physiological function of the amyloid precursor protein (APP) in plasmalogen metabolism. The APP intracellular domain was found in vivo and in vitro to increase the expression of the alkyl-dihydroxyacetonephosphate-synthase (AGPS), a rate limiting enzyme in plasmalogen synthesis. Alterations in APP dependent changes of AGPS expression result in reduced protein and plasmalogen levels. Under the pathological situation of AD, increased amyloid-beta level lead to increased reactive oxidative species production, reduced AGPS protein and plasmalogen level. Accordingly, phosphatidylethanol plasmalogen was decreased in the frontal cortex of AD compared to age matched controls. Our findings elucidate that plasmalogens are decreased as a consequence of AD and regulated by APP processing under physiological conditions.     

Neuroprotective role of lipoic acid against acute toxicity of N-acetylaspartic acid

N-Acetylaspartic acid (NAA) accumulates in Canavan disease, a severe inherited neurometabolic disorder clinically characterized by mental retardation, hypotonia, macrocephaly, and seizures. The mechanisms of brain damage in this disease remain poorly understood. Recent studies developed by our research group showed that NAA induces oxidative stress in vitro and in vivo in cerebral cortex of rats. Lipoic acid is considered as an efficient antioxidant which can easily cross the blood–brain barrier. Considering the absence of specific treatment to Canavan disease, this study evaluates the possible prevention of the oxidative stress promoted by NAA in vivo by the antioxidant lipoic acid to preliminarily evaluate lipoic acid efficacy against pro-oxidative effects of NAA. Fourteen-day-old Wistar rats received an acute administration of 0.6 mmol NAA/g body weight with or without lipoic acid (40 mg/kg body weight). Catalase (CAT), glutathione peroxidase (GPx), and glucose 6-phosphate dehydrogenase activities, hydrogen peroxide content, thiobarbituric acid-reactive substances (TBA-RS), spontaneous chemiluminescence, protein carbonyl content, total antioxidant potential, and DNA–protein cross-links were assayed in the cerebral cortex of rats. CAT, GPx activities, and total antioxidant potential were significantly reduced, while hydrogen peroxide content, TBA-RS, spontaneous chemiluminescence, and protein carbonyl content were significantly enhanced by acute administration of NAA. Those effects were all prevented by lipoic acid pretreatment. Our results clearly show that lipoic acid may protect against the oxidative stress promoted by NAA. This could represent a new therapeutic approach to the patients affected by Canavan disease.     

Destructive effect of anticancer oxali-palladium on heme degradation through the generation of endogenous hydrogen peroxide

The interaction between human hemoglobin (Hb) and oxali-palladium was studied using different spectroscopic methods of UV–vis, fluorescence, circular dichroism (CD), and chemiluminescence at two temperatures of 25 and 37°C. The experimental results showed that both dynamic and static quenching is occurred simultaneously when oxali-palladium quenches the fluorescence of Hb. According to the fluorescence quenching method, the binding site number, apparent binding constant, and corresponding thermodynamic parameters were measured at two temperatures. The values of ΔH°, ΔS°, and ΔG° indicate that process of the formation of oxali-palladium–Hb complex is a spontaneous interaction procedure in which electrostatic interaction plays a major role. In addition, UV–vis and CD results showed that the addition of oxali-palladium changes the conformation of Hb. To evaluate the functional changes of Hb via destruction of the heme structure, fluorescence studies were performed. The results demonstrated that two fluorescent heme degradation products are found during the interaction of oxali-palladium with Hb. Also, the amount of hydrogen peroxide produced in the solution of Hb due to the interaction of oxali-palladium with Hb using chemiluminescence method indicated heme degradation in the protein is occurred. Structural and functional changes induced in Hb via heme degradation are considered as side effects of this synthesized anticancer drug.     

Singlet oxygen is part of a hyperoxidant state generated during spore germination

We show that singlet oxygen is generated in asexual spores (conidia) from Neurospora crassa at the onset of germination. Oxidation of N. crassa catalase-1 (Cat-1) was previously shown to be caused by singlet oxygen (Lledías et al. J. Biol. Chem. 273, 1998). In germinating conidia, increased protein oxidation, decrease of total protein, Cat-1 oxidation and accumulation of cat-1 mRNA was detected. These changes were modulated in vivo by light intensity, an external clean source of singlet oxygen, and by carotene amount and content of coordinated double bonds. Conditions that stimulated singlet oxygen formation increased Cat-1 oxidation and accumulation of cat-1 mRNA. Germinating conidia from mutant strains altered in carotene synthesis showed increased levels of protein degradation, Cat-1 oxidation and accumulation of cat-1 mRNA. During germination Cat-1a was oxidized, oxidized Cat-1c-Cat-1e conformers disappeared and Cat-1a was synthesized de novo. Furthermore, spontaneous oxygen-dependent chemiluminescence increased as soon as conidia absorbed dissolved oxygen. Low-level chemiluminescence is due to photon emission from excited electrons in carbonyls and singlet oxygen as they return to their ground state. H2O2 added to conidia under Ar caused a peak of chemiluminescence and germination of 20% of conidia, suggesting that a hyperoxidant state suffices to start germination under anaerobic conditions. Taken together, these results show that singlet oxygen is part of a hyperoxidant state that develops at the start of germination of conidia, in consonance with our proposal that morphogenetic transitions occur as a response to a hyperoxidant state.     

Inhibition of the degradation of receptor-bound human choriogonadotropin by lysosomotropic agents, protease inhibitors, and metabolic inhibitors.

A previous report from this laboratory showed that binding of iodine-labeled human choriogonadotropin to Leydig tumor cells is not a reversible process (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 4892--4899). Most of the cell-bound hormone was found to be degraded to 3'-monoiodotyrosine before being released from the cells, and the degradation process could be inhibited by the lysosomotropic agents NH4Cl, chloroquine, and Triton WR-1339. It is reported herein that the degradation of receptor-bound human choriogonadotropin is an energy-dependent process, which can be inhibited by compounds that interfere with glycolysis or oxidative phosphorylation (e.g. NaF, NaN3, NaCN, and 2-deoxyglucose). Hormone degradation is also inhibited by some protease inhibitors such as the chloromethyl ketones of lysine and phenylalanine, but not by specific trypsin inhibitors (e.g. p-aminobenzamidine and p-tosyl-L-arginine methyl ester). With the exception of NH4Cl, it was found that the compounds which inhibit hormone degradation also inhibit hormone-stimulated steroidogenesis. However, the present results involving dose dependency, and those given in the following paper (Ascoli, M. (1978) J. Biol. Chem. 253, 7839--7843), indicate that these two phenomena are not related.