Generation of H2O2 in biomembranes

Biochimica et Biophysica Acta, 694 (1982) 69-93

69

Elsevier Biomedical Press

BBA85230

GENERATION OF H zOz IN BIOMEMBRANES T. RAMASARMA

Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 (India) (Received December 12th, 1981)

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Steps in the reduction of oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70 70

II.

Methods of measuring H202 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71

III.

Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Low K m of substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Energy state of mitocbondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Relative rates of H202 formation and oxygen uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Superoxide and ubiquinone as intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Inhibitors of H202 generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Environmental conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Different sources of rnitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Plant mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 71 72 73 73 74 74 76 76 77

IV.

Some intracellular organdies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chloroplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Nuclei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77 77 77 78

V.

Microsomal membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Dependence on flavoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Vanadate stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D, Sulphite and thiol oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

78 78 79 79 79

VI.

Plasma membranes A. Adipocyte plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hepatic plasma membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Erythrocyte membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Bacterial plasma membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Plant cell wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

80 80 81 81 81 82

VII.

Associated cellular phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Parasitism of protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phagocytosis and respiratory burst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Hormonal action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Cyanide-insensitive respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Thermogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

82 82 83 85 87 87

Abbreviations: NBT, neotetrazolium blue; PMN, polymorpho0304-4157/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

nuclear leucocytes; CG'D, chronic granulomatous disease; GRF, granule-rich fraction.

70 Vlll. Concluding remarks Acknowledgements

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Knowledge of the generation of H 2 0 2 in cellular oxidations has existed for many years. It has been assumed that H 2 0 2 is tOxiC tO cells and the presence of catalase is indicative of a detoxication mechanism. Other radicals of oxygen were recently recognized to be more potent destructive agents of biological material than H202. Also catalase and other peroxidases utilize H 2 0 2 in some cellular oxidation processes leading to several important metabolites. Thus, the generation of H202 in cellular processes seems to be purposeful and H 2 0 2 c a n not be dismissed as a mere undesirable byproduct. Biological formation of H202 is not limited to the previously known flavoproteins and some copper enzymes, but other redox systems, particularly heme and non-heme iron proteins, are now found to undergo auto-oxidation yielding H 2 0 2 . The capacity for generation of H202 is now found to be widespread in a variety of organisms and in the organdies of the cells. The reduction of oxygen to H 2 0 by mitochondrial cytochrome oxidase being the predominant oxygen-utilizing reaction had over-shadowed the importance of the quantitatively minor pathways. Under aerobic conditions generation of H202 by a Variety of biomembranes has now been found to be a physiological event interlinked with phenomena such as phagocytosis, transport processes and thermogenesis in some as yet unidentified way. The underlying mechanisms of these processes seem to involve generation and utilization of H 2 0 2 in mitochondria, microsomes, peroxisomes or plasma membranes. This review gives an account of the potential of biomembranes to generate H 2 0 2 and its implication in the cellular processes. IA. Steps in the reduction of oxygen Molecular oxygen has two unpaired electrons each of which goes into separate antibonding ~r-

88 90 90

orbitals with parallel spins giving the molecule the stability and paramagnetic property in the ground state. The reductions of 02 by addition of one, two and four electrons lead to formation of superoxide anion ( O [ ) , H 2 0 : and H20, respectively. e

e

2-

02 --' 0 2 --' O: e

e

0 2 + HO 2 --, H 2 0 2 H+

H+

(a) e ~+

e H20

+ HO --, 2 H 2 0 H+

(b)

Only two electrons can be accommodated by each oxygen atom. The antibonding orbital of molecular oxygen receives the added electrons and each addition weakens and increases the leng.th of O - - O bond, from 1.274A in 02 to 1.480A in H202, leading to rupture [1]. The two-electron reduction of oxygen directly to H202 is restricted by symmetry considerations [2] that can be overcome by binding of 02 to the electron donor and consequent perturbation of the molecular orbitals. 02 + 2 H + + 2 e + H 2 0 2

(c)

0 2 + e --, O [

(d)

0 2 + 0 2 + 2 H + -+ H 2 0 2 + 0 2

(e)

The flavoprotein oxidases appear to follow this type of direct two-electron reduction process (reaction c) with no intermediate step [3]. Other H202-generating systems seem to use one-electron reductions forming superoxide anions (02-) (reaction d) [4] two of which then dismutate yielding a molecule each of H 2 0 2 and 02 either spontaneously or catalyzed by the enzyme, superoxide dismutase (reaction e) [5]. The flavoprotein dehydrogenases and possibly the iron-proteins generating H202 seem to adopt this mechanism and are mostly membrane-localized. It is now found that superoxide formation is a property shared by large number of redox components. In view of the

71 ubiquitous nature of superoxide dismutase and easy non-enzymic dismutation of superoxide, generation of H202 accompanying oxidation of these redox components with molecular oxygen becomes equally widespread. II. Methods of measuring H 202 The rate of H202 generation in biomembranes is small, in the order of n m o l / m i n per mg protein. Therefore only highly sensitive methods could pick up such rates. Direct measurement of H202 by the absorbance change at 240 nm is not sensitive enough as the molar extinction coefficient of H202 at 240 nm (~ = 36 M -1 • cm -1) is low [6]. Release of half the amount of absorbed oxygen back into medium on addition of catalase was used as an indication of the presence of H202 in the reaction mixtures, but this method is indirect and can not be used for studying the rates. The most commonly used method is that of Loschen et al. [7] based on the loss of fluorescence of scopoletin in presence of horseradish peroxidase and H202, originally described by Andrea [8]. Use of dianisidine in place of scopoletin permits spectrophotometric measurement of the oxidized orange product at 436 nm. Owing to broad specificity of horseradish peroxidase interference by a number of substances which can compete with scopoletin for peroxidation was noted. While the relationship of scopoletin/H202 is normally 1.0 [9] this ratio was reduced to 0.25 in presence of N A D H (0.1-0.4 mM) [10]. Studying the recovery of added H202 and standardizing the method under conditions of assay was normally followed. It should also be pointed out that compounds such as azide and trichloroacetic acid [9] and quinacrine and decavanadate i [10] produced nonspecific loss of fluorescence of scopoletin, and the fluorescence intensity of scopoletin depends on pH, being lower in acid pH. In order to avoid these likely interferences, Boveris et al. [11] introduced the highly specific yeast cytochrome c peroxidase and measured the increase in absorbance of this enzyme-H202 complex at 419 nm relative to reference at 407 nm. Another method of determination of H202 is based on oxidation of Fe 2+ of ferrous ammonium

sulfate in presence of potassium thiocyanate. The intense red color of Fe(SCN) 3 formed is read at 480 nm [12]. Oxidation of methanol to formaldehyde by H202 in presence of excess of catalase was employed to measure n202 [13]. This was later modified by using [14C]methanol and estimating the formation of [14C]formaldehyde to eliminate any risk of interference by endogenous formaldehyde. It should be noted that the enzymes like catalase and several peroxidases are closely associated with H20/-generating systems making it difficult to measure the true rates of H202 generation.

III. Mitochondrla Generation of H202 was first shown in pigeon heart mitochondria by Loschen et al. [7] in 1971 who used the method of scopoletin-horseradish peroxidase to detect H202 formed. Since then a variety of mitochondria were found to have this activity. The studies on the properties of the H2OE-generator system in rat liver mitochondria revealed the following salient features: substrates entering the respiratory chain before antimycin A-sensitive site possibly at ubiquinone donate electrons to reduce 02 via superoxide to H202, the relative rate of generation of H202 compared to oxygen uptake is small and normally 1% or less; saturation is obtained at low concentrations of the substrates; maximal rates are obtained only in the presence of antimycin A and an uncoupler; in coupled mitochondria highest rates are obtained in state 4; H202 generation is inhibited by radical quenchers and phenolic compounds and is regulated under conditions of thermal stress on the animals and of varying availability of oxygen. The details of these findings are described below.

IliA. Substrate specificity Succinate was considered the most effective substrate for H202 generation by mitochondria Ill]. It was found that many substrates oxidized by mitochondria are equally effective. In the mannitol/sucrose/phosphate/Tris/morpholinopropanesulphonic acid (225 m M / 7 5 m M / 4 m M / 2 5 mM, pH 7.4) buffer, succinate, malate + glutamate, palnfitoylcarnitine and octonoate were found to

72 TABLE I RATES OF H202 G E N E R A T I O N IN M I T O C H O N D R I A smp: submitochondrial particles; mitochondria from rat heart and kidney, yeast protozoa and some higher plants have also been reported to produce H202 Source of mitochondria

Substrate

Pigeon heart

Succinate Succinate + glutamate Malate + glutamate

0.11 3.90 4.20

9 15 15

Rat liver

Succinate Palmitoyl carnitine Octonoate Malate + glutamate Succinate Choline Glycerol l-phosphate Proline NADH Malate

0.40 0.23 0.22 0.19 0.46 0.43 0.45 0.42 0.04 0.05

11 11 11 11 14 14 14 14 14 14

Ox heart (smp) Ox heart (Complex I)

Succinate NADH NADH

0.83 2.03 9.60

16 16 17

M u n g bean M u n g bean (smp)

Succinate + ATP Succinate + ATP

0.68 17.50

50 50

support H202 generation (Table I). In high-concentration phosphate buffer (0.45 M) which gave maximal rates [14] rat liver mitochondria produced H202 almost equally effectively with succinate, choline, glycerol l-phosphate and proline, but poorly with malate and N A D H and negligibly with ascorbate or xanthine (Table I). The low rates with NAD-linked substrates appears to be due to delinking of these particular dehydrogenases from the H202-generating system. In the initial experiments with pigeon heart mitochondria, similar ineffectiveness of NAD-linked substrates was reported [7]. But in later experiments pigeon heart mitochondria were found to use malate + glutamate as well as succinate+ glutamate for H 202 generation, (Table I) but palmitoyl carnitine, stearoyl-CoA and octonate were less efficient as substrates [15]. In submitochondrial particles having no permeability barrier for NADH, H202 generation with N A D H was even higher than with succinate [16]. The rates were not additive when two substrates, choline and succinate [14] or palmitoyl carnitine and succinate + glutamate [15]

n m o l / m i n per mg protein

Ref.

were added together. At saturating concentration of each substrate nearly the same maximal rate of about 0.5 n m o l / m i n per mg of protein was obtained with intact mitochondria from rat liver [14]. IIIB. Low K m o f substrates

One interesting feature of H202 generation was the low K m values for the substrates. It can be seen from the data in Table II that the K m values for each substrate was higher in experiments on measurement of dye reduction than of H202 generation: succinate, 30-fold; choline, 200-fold and glycerol 1-phosphate, 5-fold [14] and for NADH, 230-fold [17]. There is a possibility that two forms of each dehydrogenase with significantly different K m values are present with the high Km-enzyme being used for main electron transport chain and the low Km-enzyme for H202 generation. Alternatively, two subunits of the dehydrogenase may have differing affinities for the same substrate. Succinate dehydrogenase of rat liver mitochondria seems to have different affinities for succinate; the

73 TABLE II K m VALUES FOR SU'BSTRATES For experiments with succinate, choline, glycerol 1-phosphate rat liver mitochondria were used. H202 was measured by scopoletin-horseradish peroxidase method and phenazine methosulphate was used for the dye reduction [14]. For experiments with NADH. Ox heart mitochondria (complex I) was used. H202 was measured by cytochrome peroxidase method and ferricyanide was used for dye reduction [17]. Substrate

Succinate Choline Glycerol 1-phosphate NADH

K m (mM)

Dye/H 202 ratio

H 202 generation

Dye reduction

0.01 0.03

0.30 6.00

30 200

2.20 0.0006

9.50 0.14

5 233

low K m for activation site and the high K m for active site [18]. In mouse liver plasma membranes a low K m value for N A D H was obtained for H202 generation compared to the dehydrogenase activity [10,19].

IIIC. Energy state of mitochondria In mitochondrial samples, H202 generation could be demonstrated in the absence of antimycin A but these rates increased several fold in its presence [15]. For some unexplained reason the highest rates were obtained in the presence of an uncoupler such as carbonylcyanide-p-trifluoromethoxyphenylhydrazine [11 ] or S 13 (salicylanilide derivative) [20], in addition to antimycin A. Titration of mitochondria with antimycin A under these conditions showed maximum effect at 0.26 n m o l / m g of protein, a concentration similar to that needed for titration of cytochrome bT, or b K [21]. Also, the antimycin A effect points to a redox component on the substrate side of this site - cytochrome b, ubiquinone, non-heine iron proteins or flavoproteins - - acting as the electron donor for H 2 0 2 generator. All these components have the ability to generate superoxide [5,22,23] and H202.

The sensitivity to uncouplers [15,20] had been taken to indicate that the energy coupling mecha-

nism is directly involved in mitochondrial H 2 0 2 generation. Also, addition of ADP decreased markedly the rates of H 2 0 2 generation by pigeon heart mitochondria [ 11] and rat heart mitochondria [20] showing that H2OE-generator works more efficiently in state 4 than state 3. Similar responses were obtained for both H 2 0 2 generation and reduction of cytochrome b-556 on changing the phosphate potential and this was taken to indicate involvement of phosphorylation site II [20]. In antimycin A-supplemented rat heart mitochondria, H202 generation was found to be enhanced in presence of C a 2+ and an ionophore (valinomycin or gramicidin) [24]. Instead of interpreting these data as involvement of the energy coupling mechanism, reduction of a specific electron transport component that donates electrons to the H 2 0 Egenerator in the presence of these uncoupling compounds or ionophores must be considered. When the normal respiration increases on addition of an uncoupler or ADP (state 4--, state 3 transition) H202 generation decreases. Addition of antimycin A under these conditions blocks electron flow to cytochrome chain, and diverts it towards reduction of cytochrome b-556, ubiquinone and H 2 0 2 generation. This diversion of electrons may indeed be the reason for obtaining high rates in buffers containing high concentration of oxyanions [14] in the absence of an uncoupler.

IIID. Relative rates of 11202 formation and oxygen uptake With intact mitochondria from rat liver even

TABLE III COMPARATIVE RATES OF THE DEHYDROGENASES AND H:O 2 GENERATION Rat liver mitochondria were used [14]. Substrate

Succinate Choline Glycerol 1-phosphate

Vnaax (nmol/min per mg protein) H202 generation

Dye reduction

0.47 0.53 0.54

250 75 7.5

74 the maximal rate of about 0.5 n m o l / m i n per mg protein for H202 generation is less than 1% of the oxygen uptake of the state 4 respiration [11]. But in the presence of antimycin A H202 accounts for most of the 02 uptake in pigeon heart mitochondria and 35% of it in submitochondrial particles [16]. The rates of the dehydrogenase activities (Vm,x values in Table III) are 15-500 times higher than the relatively constant Vma~ for H202 generation with succinate, choline or glycerol 1-phosphate as the substrate. Thus O 2 consumption to form H202 is quantitatively a minor pathway in mitochondria.

HIE. Superoxide and ubiquinone as intermediates An active superoxide dismutase is present in mitochondrial matrix [25] which ensures conversion of any 0 2 formed during electron transfer into H202 [26]. Enough superoxide dismutase must be present in intact mitochondria as addition of exogenous enzyme showed only a small increase in the rates of H202 generation [14,27]. Addition of known scavengers of 0 2 such as ascorbic acid, ferricytochrome c and nitroblue tetrazolium [28,29] as well as Mn 2+ inhibited mitochondrial H202 generation [14]. Direct proof that 0 2 was produced by mitochondrial membranes could be obtained in submitochondrial preparations depleted of superoxide dismutase, by measuring the dismutase-sensitive activity of adrenochrome formation from adrenaline [16,27,30], and of reduction of cytochrome c [16,30,31]. All these studies were carried out with mitochondria from ox or rat heart and with succinate as the substrate. Generation of 02 was also supported by N A D H in complex I [17] and by d i h y d r o - o r o t a t e in rat liver mitochondria [32]. In all these experiments presence of antimycin A was essential. The production of 0 2 and H202 showed parallel increases when p H was increased from 7.0 to 8.2 [31] and the ratio of O2-/H202 was close to 2 as expected for the dismutation of superoxide [31]. It is obvious that the donor redox component in mitochondria must be able to donate one electron to oxygen. The known sources of 02 are flavoproteins, iron-sulphur proteins and quinols [5,22,23]. Oxidation of ubiquinol proceeds via a semiquinone intermediate and can produce univalent reduction of 02 to 0 2 . Ubiquinone, therefore,

can fill this role as it is known to be present in mitochondrial and other membranes [33] and its semiquinone was detected in mitochondrial membranes albeit to a small extent [34]. Direct proof of the dependence of H202 generation on ubiquinone was obtained with acetone-extracted mitochondrial preparations [ 15,16]. These preparations, depleted of ubiquinone, showed little activity but when reconstituted with ubiquinone (Ql0) generated 02 , and when further supplemented with superoxide dismutase, H202 [16]. In such reconstituted mitochondrial preparations, ubiquinone at low concentration (1 n m o l / m g protein) restored maximal succinate-cytochrome c reductase activity. But the activity of succinate-dependent H202 generation increased 'monotonously' with increasing ubiquinone (even up to 25 n m o l / m g protein). This ubiquinone stimulation was not due to increasing the activity of succinate dehydrogenase, known to be regulated by the reduced form but not the oxidized quinone [35,36]. Adding increasing concentrations of ubiquinone to intact rat liver mitochondria also increased monotonously the activity of succinate-neotetrazolium reductase [37,38] an activity considered to represent a shunt pathway [38]. Reduced quinones are known to be autooxidized and yield O 2 and H202 [17,39]. Menadiol (nucleus of vitamin K) is very active in generation of both 0 2 and H202 [18] and a system capable of reducing menadione will act as a H2Oz-generator. Similarly the nucleus of ubiquinone (Qo) is highly auto-oxidizable but when associated with the isoprene side-chain the reduced quinol becomes stable. In the hydrophobic membrane environment in mitochondria ubisemiquinone seems to be formed by one-electron transfer whose auto-oxidation generates 0 2 , the precursor of H202 [16].

1HF. Inhibitors of 1120e generation Generation of H202 in mitochondria was dependent on oxygen radicals. Removal of 0 2 by ferricytochrome c, nitroblue tetrazolium and ascorbate, known scavengers of superoxide [28,29], inhibited generation of H202 [14]. Addition of mannitol, benzoate and Tris gave poor rates of H202 generation, suggesting the possible involvement of O H radicals [14].

75

Copper ions were found to be powerful inhibitors of H 2 0 2 generation at/~M concentrations and both F e E+ and Mn 2+ were effective inhibitors at 0.1 mM concentration [14]. It is difficult to distinguish whether they affect the process of generation of H 2 0 2 o r its degradation as these metal ions are capable of carrying out the dismutation of O~t o H202 as well as the conversion of H 2 0 2 into O H radicals (see Ref. 40 for a discussion). With pigeon heart mitochondria, addition of A D P (0.7 mM) completely suppressed the rate of H 2 0 2 generation for a transient period until state 3 - - , 4 transition occurred [7]. With rat liver mitochondria addition of ADP (0.12 mM) showed 85% inhibition initially, but after a short period 50% of the original rate was reestablished [11]. The earlier interpretation of these effects as reflecting dependence on energy state of mitochondria [7,11] must now be reconsidered because inhibition by ADP, as well as by ATP was observed even in uncoupled state of mitochondria [14]. At 0.5 mM and higher concentration only 20% residual rate was found with both ADP and ATP whereas AMP, cyclic AMP, UTP and adenosine even at 1 mM had little effect [14]. Since even the crystalline preparations of ATP and ADP are known to have contamination of Fe E+ , as judged by the promo-

tion of lipid peroxidation [41], the effects of these nucleotides per se on H202 generation remain to be clarified. A set of phenolic acids were found to be specific inhibitors of H202 generation in rat liver mitochondria [14] (Table IV). The presence of a hydroxyl group in the phenyl ring is necessary for inhibition. The vinyl group in the side chain of phenolic acids (e.g. p-coumaric and ferulic acids) enhanced the potency of inhibition. Also, p-hydroxycinnamic acid is more effective than the misomer. An additional methoxy group (ferulic and isoferulic acids) or hydroxyl group (noradrenaline or catechol) in the phenolic ring conferred increased inhibitory capacity. Thyronine derivatives (T2, T3 and T4), all having phenolic rings, also inhibited a t / t M concentrations. They had no affect on the dehydrogenase activity tested by the phenazine methosulphate-oxygen method [42]. They also had no effect on superoxide dismutase activity of mitochondria or on the generation of Oy by xanthine-xanthine oxidase system. Thus their effect must be on the step of univalent electron transfer from the dehydrogenase to the possible common H202 generator. The purported ironsulphur protein centre or the ubiquinol/cytochrome b system may then be the target of this

TABLE IV INHIBITION OF H202 GENERATION Source of mitochondria

Substrate

Inhibitor

Concentration (#M)

% Inhibition

Ref.

Rat liver

Succinate Malate + glutamate

ADP ADP Rotenone Tyrosine p-OH phenylacetate p-OH phenylpyruvate p-Coumarate Ferulic acid Isoferulic acid Noradrenaline Di(OH)phenylalanine ATP ADP ADP Pentachlorophenol

500 500 0.6 500 200 50 50 2 1 0.67 330 330 330 700 50

85 60 50 55 50 72 76 62 59 45 91 85 30 100 100

11 11 11 14 14 14 14 14 14 14 14 14 14 7 7

Choline

Pigeon heart

Succinate

76 /,02

FLAVOPROTEIN~ DEHYDROGENASES ~ ~'~'[C YTOCHRONES] H20 Q / ~ O - ~ NDISMUTASE ~o~o~,~ 02

H202

Fig. 1. H202 generation in mitochondria. The flavoprotems that converge at ubiquinone (Q) can donate electrons both for cytochrome system and the shunt pathway that branches at Q. In the main respiratory chain, O 2 is reduced to H20 by the cytochrome oxidase involving a 4-electron transfer to a molecule of oxygen. In the shunt pathway (additional Q is shown at this system) responds to added Q which apparently draws out electrons. The formation of H202 involves an initial l-electron reduction of molecular oxygen to superoxide anions ( 0 2 ), two of which dismutate to form H202. It appears possible that the HEO2-generator (dehydrogenases-Q-QH 2 oxidase-superoxide dismutase) may occur as a distinct entity independent of the cytochrome system.

inhibition by phenolic compounds. Rotenone at 0.33 ffM inhibited N A D H ubiquinone reductase nearly completely, but NADH-dependent H 2 0 2 generation decreased only by 50% [17]. The concentration of rotenone required for maximum inhibition of H 2 0 2 generation was about 10 n m o l / m g protein which was much larger than the concentration needed to inhibit the dehydrogenase [43]. Succinate-dependent H 2 0 2 generation was competitively inhibited by malonate. The concentration of malonate relative to succinate required for inhibition of H 2 0 z generation was at least two orders of magnitude higher than that for succinate dehydrogenase (Swaroop, A. and Ramasarma, T., unpublished data). Thus, larger concentrations of the dehydrogenase-specific inhibitors are required for inhibition of H202 generation and this property does not fit with the assumption current in this field that the same dehydrogenase flavoproteins serve as the electron donors to ubiquinone for H202 generation. The reactions involved in the electron transfer leading to H202 generation are shown schematically in Fig. 1.

HIG. Environmental conditions

It is well-known that the rate of cytochrome oxidase will not decrease even at a partial pressure of oxygen as low as 1/10 of the atmospheric pressure. In contrast, saturation of oxygen for H 2 0 2 generation was not obtained even at nearly 20 atmospheres pressure [15]. With both mitochondria from pigeon heart or rat liver subjected to hyperbaric conditions in vitro the rate of H 2 0 2 generation increased several fold. A value of 20 n m o l / m i n per mg protein was obtained for Vma~ and about 0.8 atmosphere as the K m for p O 2 [15]. Conversely at low p O 2, the rates are likely to decrease. In rats exposed to hypoxic conditions simulating 700 m altitude 2 h daily, for 2 weeks, superoxide dismutase was found to decrease [44] and the effect o n H 2 0 2 generation is not yet known. Treatment of rats with thyroxine (1 m g / d a y per kg body wt., two doses, killed at 48 h), or noradrenaline (2mg per kg body wt., one dose, killed at 4 h) increased the rates of H 2 0 2 generation in isolated liver mitochondria by 57 and 84%, respectively [45]. Treatment of rats with propylthiouracil (100 m g / d a y per kg body wt., 7 days, killed at 8th day) or thyroidectomizing rats (killed 7th day), on the other hand, decreased the activity to 59% of the control animals. In the same mitochondrial preparations the dehydrogenase activities measured by dye reduction remained unchanged. An increase in activity was observed in mitochondria of livers from rats exposed to cold stress (9°C, 24h) and a decrease in heat stress (38-39°C, 2 days) [45]. Thus, increases and decreases of H 2 0 2 generation are correlated with corresponding changes in thermogenic conditions of the animals [45]. These studies provide the first examples of physiological alterations of the HzOz-generator system independent of dehydrogenases and the respiratory oxidations. IIIH. Different sources of mitochondria

Most of the work on mitochondrial H 2 0 2 generation was confined to rat liver and pigeon heart tissues [7,11,14,15]. Other mitochondria are known to possess this activity. Among these are: ox heart, rat kidney and heart, yeast, protozoa and some

77 higher plants. The rates found in these preparations were all in the range of a few n m o l / m i n per mg protein" (Table I). Mitochondria obtained from Ehrlich ascites tumor or Morris hepatoma 3924A showed little H202 generation and this was traced to the lack of superoxide dismutase in these proliferating cells and therefore only superoxide was generated [30]. Other types of Morris hepatoma also were found to be low in mitochondrial specific Mn-superoxide dismutase [46]. It must be mentioned here that the presence of superoxide dismutase by itself does not ensure H202 generation as mitochondria from brain tissue despite the presence of the dismutase were found to be ineffective as they were incapable of generating oxygen radicals [47]. IIII. Plant mitochondria

The cyanide and antimycin A-insensitive respiration [48] was considered to be due to tapping electrons at the site of ubiquinone [49]. But in view of large concentrations of catalase and peroxidase no more than 2% of oxygen uptake in presence of antimycin A was recovered as H202 [50]. In submitochondrial particles the antimycin-insensitive oxygen uptake accounted for H202 generation that was sensitive to hydroxamic acids [50,51]. Only N A D H was used as the substrate and succinate could not be tested as its dehydrogenase was found to be low in these particles. It is, however, interesting superoxide generation concomitant with oxygen uptake by whole mitochondria or submitochondrial particles was not unambiguous even in mung bean and arum species known to exhibit high activities of alternate oxidase activity [51]. Ubisemiquinone formed on oxidation of ubiquinol was considered to reduce oxygen to 0 2 which then dismutates to H202. Another possible source of O ; in plant mitochondria had been identified as tyrosinase, found as a contaminant. Rich [52] had solubilized the alternate oxidase system of Arum maculatum and identified quinol oxidase activity using menadiol and ubiquinol (with short side-chain) as substrates. Auto-oxidation of quinols is known to generate H202 directly without an intermediate [17]. However, with the long isoprenoid side-chain the natural ubiquinol has a low auto-oxidation rate and also it interacts with the

respiratory chain in mammalian mitochondria differently from menadiol [53]. It is possible that a ubiquinol oxidase may indeed turn out to be the common H202 generator in all mitochondria. Wheat seedling mitochondria showed a large stimulation of oxygen uptake on oxidation of N A D H in presence of vanadate [54] possibly generating H202. One interesting new possibility arises out of studies on the distribution of cyanide-sensitive and cyanide-insensitive respiration in different populations of mitochondria. It was found that the mitochondrial fraction of wheat seedlings separated into two discrete bands on percoll density gradients but both of these showed oxygen uptake that was sensitive to cyanide and antimycin. Interestingly a third gradient fraction, which contained no mitochondria, possessed cyanide-insensitive respiration inhibited by propylgallate or salicylhydroxamate [55]. These results indicated that this activity was dissociated from mitochondria in the gradient separation or existed in a separate particle. Evidence was obtained that this oxidase is a lipoxygenase [55]. Activities of lipoxygenase and tyrosinase, possibly as contaminants of plant mitochondrial preparations, also contribute to H202 generation.

IV. Some intracellular organeiles IVA. Chloroplasts

Illumination-dependent burst of oxygen uptake and H202 generation was found in intact cells of Anaeystis nidulans [56]. Broken chloroplasts preparations were able to generate H/O 2 through a light-dependent process and the energy source seemed to be glycollate. The oxygen inhibition of photosynthetic CO 2 assimilation, known as 'Warburg Effect', was found to be mimicked by H202 suggesting that its generation in chloroplasts may regulate photosynthesis [57]. Further, H202 was also found to inhibit ribulose diphosphate carboxylase of spinach [58]. If this inhibition proves to be reversible, through dithiol-disulphide formation, a general pattern of H202 effect may emerge. IVB. Nuclei

The nuclear membrane isolated from ascites tumor cells produced superoxide and H202 [59]. A

78 FAD-containing flavoprotein mono-oxygenase in the nuclear preparation from hamster liver produced H202 in the presence of superoxide dismutase [60]. Oxidation of NAD(P)H by nuclear membrane from ascites cells or hepatoma 22a produced 0 2 . Being sensitive to cyanide and azide this activity was conjectured to involve an autooxidizable cytochrome [61 ].

oxygen species that form H202. However, hydroxylation and H202 generation seems to be mutually exclusive processes. Microsomes have a dithioloxidizing system which also can generate oxygen radicals. Further, microsomes have high activities of peroxidation of membrane lipids triggered by iron salts and of N A D H oxidation stimulated by vanadate, and these are related to the H202-generating system.

IVC. Peroxisomes VA. Dependence on flavoproteins

Peroxisomes, found in some animal and plant tissues, possess flavin oxidases which directly reduce oxygen to H202 and use it for peroxidative activity [62]. Peroxisomes also have high proportion of cellular catalase (nearly 60% in the liver tissue). Most of the generated H202 would therefore be degraded in situ. D-Alanine and uric acid, the substrates for peroxisomal oxidases were found to be oxidized and the consequent H202 generation in peroxisomes could be measured with cytochrome c peroxidase method [11]. Addition of azide to this system inhibited catalase and increased the observed rates of H202 generation. From the relative rates of uric acid oxidation, of oxygen uptake and of formation H202 it was calculated that about 40% of H202 generated diffused out of peroxisomes and was available for detection by cytochrome c peroxidase. Sonication or deoxycholate treatment disrupted the peroxisomal structure and yielded peroxisomal membranes which oxidized uric acid, with 90% of oxygen uptake being accounted as H202. Rates of the order of 20 n m o l / m i n per mg protein were obtained which are the highest among all the cellular organelles in the same tissue [11]. The oxidases in plant microbodies (see Ref. 63 for a review) are known to yield HzO 2 but more work is needed on direct measurements of H202 generation with these plant organelles. V. Microsomal membranes

Microsomal membranes have oxidation reactions specific to NADPH, primarily utilized for drug hydroxylations, and H202 is excluded as an intermediate in the mixed function oxidase reaction. But the redox component of the system, cytochrome P-450, can also produce reduced

Generation of H202 consequent to oxidation of N A D P H by microsomes was first reported by Gillette et al. [64] with the detection system of methanol-catalase. This was confirmed by other laboratories subsequently [65,66] using rat liver microsomes and a variety of methods for measuring H202 [9]. It was found that carbon monoxide inhibited H202 generation and the involvement of cytochrome P-450 was thus indicated. Treatment of animals with phenobarbital increased P-450 and also stimulated generation of H202 by 2.5-fold [65]. Auto-oxidation of cytochrome P-450 was found to generate superoxide [67,68]. Liver microsomal NADPH-cytochrome P-450 system was found to reduce neotetrazolium blue (NBT) and this reduction being sensitive to superoxide dismutase was interpreted to represent generation of superoxide [69]. The use of superoxide dismutaseinhibitable reduction of NBT for the measurement of superoxide was questioned by Auclair et al. [70] because the primary reaction in the microsomal system was reduction of these electron acceptors, NBT or cytochrome c, rather than oxygen. Definitive evidence for the formation of H202 by the reconstituted mono-oxygenase system was provided by Kuthan et al. [71] who showed that the partially purified NADPH-cytochrome P-450 reductase-flavoprotein by itself generated H202 on oxidation of NADPH. The rate was enhanced by addition of purified cytochrome P-450 and further increased under conditions of dealkylation process. Corresponding changes were found in the formation of 0 2 measured by detection systems using succinylated cytochrome c or lactoperoxidase. That the flavoproteins themselves are responsible for a considerable proportion of H202 gener-

79 ated in microsomes was indicated by the studies on developmental changes [72]. Increase in the generation of H202 and of 0 2 followed the developmental pattern of the NADPH-cytochrome c reductase, and not of the microsomal cytochromes (P-450 or bs). It was calculated that of the total H202 generated by microsomes about 50% was accounted by the flavoproteins and the remaining by cytochrome P-450.

VB. Lipid peroxidation Coupled to oxidation of N A D P H by the flavoprotein of NADPH-cytochrome c reductase [73] originally described by Horecker [74], microsomes generate active oxygen species that can be used for lipid peroxidation, H202 generation and possibly hydroxylations [75]. NADPH-dependent lipid peroxidation in microsomes increased in the presence of iron salts [41,76], and this effect was further enhanced by ATP and ADP but not by AMP [76]. Thus, caution should be exercised in interpreting any effect obtained both by ATP and ADP in terms of protein phosphorylation until the possible effect of Fe, found as a contaminant even in the crystalline A T P / A D P samples [41,76], is dissociated. In these experiments, it was found that the increased lipid peroxidation on addition of Fe was also accompanied by a large increase in oxygen uptake. This excessive use of oxygen which was 15-fold higher than the rate of formation of malonaldehyde [76] indicates its conversion to H202. Lipid peroxidation seems to depend on an auto-oxidation chain, possibly involving O f and H202, as it is sensitive to added cytochrome c or superoxide dismutase and catalase or glutathione, respectively [77,78].

VC. Vanadate stimulation It must however be pointed out that H202 does not participate independently in either lipid peroxidation or hydroxylation occurring in microsomes. In fact stimulation of H202 generation occurs when these systems become partially uncoupled. This is exemplified by the effect of vanadate on microsomal oxidation of NAD(P)H. There is a very active N A D H dehydrogenase in microsomes (measured by the reduction of ferri-

cyanide) which seems to be of common occurrence in the endomembranes [79]. The oxygen uptake accompanying oxidation of N A D H by microsomes was very low and this was stimulated over 20-fold on addition of decavanadate [80]. Both microsomes and decavanadate were required for this increased oxygen uptake and the products of the reaction were identified as NAD and H202. The rates of N A D H oxidation (and H202 generation) increased another 10-fold when the p H was decreased from 7.0 to 5.0 [81]. This activity was inhibited by noradrenaline, adriamycin, ascorbate and dihydroxyphenylalanine as well as by superoxide dismutase, MnC12 and cytochrome c indicating that oxygen radicals, particularly O [ , were involved in a chain reaction. In the native state N A D H dehydrogenase in microsomes was poorly accessible to molecular oxygen and its maximum activity was measurable only with the artificial electron acceptor, K3Fe(CN)6. Vanadate links the electron flow to oxygen. But it remains unclear whether this is achieved through its participation as an intermediate or by supporting the formation of a radical in the chain reaction. Vanadium occurs in animal tissues but in low quantities and orthovanadate was inactive in the above reaction. The physiological relevance of this capability of decavanadate for massive H 2 0 z generation and whether this stimulation can be obtained by other agents in the cell remain uncertain [82].

VD. Sulphite and thiol oxidation Microsomes have two other oxidative enzyme systems that are potential H202 generators. A portion of cellular oxidation of sulphite occurs in microsomes which has the distinction of being partly cryptic, released on treatment with a detergent or removal of lipids by acetone [83]. Sulphite oxidase was found to use oxygen, cytochrome c and other dyes as electron acceptors [84]. Microsomal oxidation of sulphite also supported lipid peroxidation in presence of vanadate [85] and is intrinsically a H202 generator, like N A D H oxidase. Also, microsomes have the capability of refolding to the native, active form of pancreatic ribonuclease from the random coil form unfolded by reducing the disulphide bridges and this was considered to be a dithiol exchange between proteins [86]. This

80

process of reactivation of reduced ribonuclease was found to be inhibited by adding sulphite, sulphydryl and thiol reagents as well as by cadmium indicating the involvement of a dithiol protein [87]. A dithiol protein of microsomes must then be auto-oxidized to regenerate the disulphide form and such a process would produce 0 2 [88] and H202. A reinterpretation of the stimulatory effect observed with oxidized cytochrome c, but not its reduced form [89], suggest that the generated 0 2 may be deleterious to the protein renaturation process. As catalase or H202 addition produced no significant effect, the processes of refolding and dithiol oxidation were neither dependent on H202 nor affected by it. Microsomes were shown to have such disulphide proteins capable of exchanging disulphide bridges with other proteins and enzymes and thereby exerting a regulatory influence on metabolism [90]. Dithiol-disulphide exchange as a mode of enzyme regulation is gaining support in a number of systems [91,92]. Thus, the process of oxidation of dithiols and H202 generation may be interdependent. Microsomal preparations have ubiquinone as a natural constituent [33]. This ubiquinone was reducible by added N A D H or N A D P H and reoxidizable by either cytochrome c or ferricyanide. None of the known microsomal oxidative reactions seems to depend on ubiquinone in contrast to mitochondria [93]. There is no proof yet that microsomal ubiquinone has any role in the generation of oxygen radicals or H202. The reactions

CYTOCHROME c

NADPH

= FLAVOPROTEIN

02

H202

leading to H202 generation in microsomes are shown in Fig. 2. VI. Plasma membranes

Plasma membranes of a variety of cells have to carry out the basic function of selective, specific and regulated transport of material. It is now recognized that plasma membranes intrinsically posseses dehydrogenase activities, particularly NAD(P)H-acceptor oxidoreductase [79]. Direct measurement of H202 generated accompanying oxidation of N A D ( P ) H by isolated plasma membranes showed small but significant rates (Table V). Plasma membranes in general seem to have the NADH-oxidase in a dormant state. While the reduction of ferricyanide is highly active, activity with oxygen as acceptor leading to H202 formation is low. In a review on the dehydrogenases of plasma membranes Crane et al. [79] identified the sources possessing the activity of N A D H oxidase: rat liver, rat skeletal muscle, rat adipocyte, mouse liver, bovine milk fat globule, bovine adrenal chromaffin granule, squid retinal nerve, pea stem particles, cauliflower bud walls, corn coleoptile and horseradish cell wall. The activities with oxygen as the electron acceptor were low, in the range of 1.5-40 n m o l / m i n per mg protein. With ferricyanide as the artificial electron acceptor, the rates of N A D H oxidation increased up to 2000 n m o l / m i n per mg protein. The dehydrogenase involved was found to be different from the mitochondrial variety and possibly also from the microsomal N A D H - c y t o c h r o m e b 5 reductase in view of several differences of effects with selective inhibitors [94,95] and hormones [96].

VIA. Adipocyte plasma membrane

f

-',,,--<'P'°

02

H202

Fig. 2. H202 generation in microsomes. The electron transport sequence from N A D P H to activated Fe2+-oxygen complex is shown. This can be used either for hydroxylation of substrates (RH), for reduction of molecular oxygen to H202 possibly via superoxide, or for peroxidation of lipids. Also shown are the direct reduction of cytochrome c and H202 generation at the flavoprotein level

N A D P H oxidation by isolated rat adipocyte plasma membranes was found to produce H202 at the rate of 14 n m o l / m i n per mg protein, with a ratio of 0.8 for H 2 0 2 / N A D P H [97]. The enzyme in the plasma membrane, considered to be dormant in the basal state, was found to be exposed or activated about 5-fold on treatment of the cells by insulin (0.24 m U / m l of medium). It showed a p H optimum of 6.0, and had a K m for N A D P H of 58

81 TABLE V H202 GENERATION BY PLASMA MEMBRANES -- COMPARATIVESTUDY OF ACCEPTORS OF NAD(P)H OXIDO-REDUCTASE Source of plasma membranes

Substrate

Rat adipocytes

NADPH

02

NADH

02 02 02 ( + Vanadate)

02 uptake H 202 340 340

02

H202

Cytochrome c Ferricyanide

340 340

Mouse liver

Acceptor

/~M and m a x i m u m rate of 230 n m o l / m i n per mg protein. Such a high rate is unusual and not obtained even with the peroxisomes in the liver [15].

VIB. Hepatic plasma membranes Mouse liver plasma membranes possess a well characterized N A D H oxidoreductase distinctive from the microsomal enzyme [95]. The activity of this enzyme was high with ferricyanide and low with oxygen as electron acceptor. Using the horseradish peroxidase-scopoletin method, the formation of H202 was directly demonstrated [10]. The rate of H202 formation was about 1/30 of oxygen uptake showing only a small fraction was available for reduction to H202. This small rate of about 0.3 n m o l / m i n per mg protein is in normal range of H202 generation by other cellular organelles [15]. Both N A D H and N A D P H were equally active for H 2 0 2 generation whereas N A D P H was a poor electron donor for the reduction of ferricyanide or cytochrome c. The optimum p H of 7.0 and a low g m of 3 ttM for N A D H are similar to those of mitochondria [14]. Vanadate stimulated the enzyme activity by 20-fold measured both by N A D H disappearance and oxygen uptake [81], but H202 generation remained unaffected [ 19].

VIC. Erythrocyte membranes Microsomal contamination poses a problem in evaluating the potential of plasma membranes for

Assay method

K m for

NADH (gM) --

-200 3 40 40

Vm~, (nmol/min per mg protein)

Ref.

14 12 10 455 0.3 35 500

97 97 10 10 10 20 20

N A D H oxidation and H 2 0 2 generation. The preparation of ghosts from erythrocytes, which are free of microsomal membranes, have been helpful in clarifying the situation. Active NADH-ferricyanide reductase was found in human erythrocyte membranes [98,99] pig erythrocytes [100] and also rat erythrocytes [101]. These preparations showed low activity of oxygen uptake and little H 2 0 2 generation. Erythrocyte membranes could generate H 202 corresponding to the rate of increased oxygen uptake obtained on adding vanadate. This nearly 100-fold stimulation was specific for the deca-form of vanadate and for oxygen as electron acceptor, but not for ferricyanide. Implication of superoxide and hydroxyl radicals was also indicated. Therefore it seems that the ubiquitous N A D H dehydrogenase in plasma membranes appears to be adapted to the generation of H 2 0 2 in presence of decavandadate. It must be pointed out that the enzyme system of the plasma membrane was being used and the large activation of oxidation of N A D H was not due to a non-enzymic mechanism or an indirect effect of lipid peroxidation (Vijaya, S. and Ramasarma, T., unpublished data).

VID. Bacterial plasma membranes In bacteria, plasma membranes carry out redox functions similar to those of mitochondria in eukaryotes. A plasma membrane preparation from Paracoccus denitrificans was able to generate superoxide using succinate or N A D H as the substrate and this activity was sensitive to cyanide

82 and externally added superoxide dismutase [102]. The properties of this system resembled those of mitochondria including the involvement of antimycin-sensitive site. The evidence pointed to a low-potential component of the b-type cytochrome as the source of electrons to oxygen in bacteria and is sensitive to carbon monoxide and cyanide. Definitive evidence was obtained that cytochrome O purified from Vitreoscilla cells was able to support formation of H : O 2 in presence of N A D H [102]. In microorganisms little work was done on the capacity to generate H202 although presence of superoxide dismutase was well documented. In this connection it is of interest to note that Mycoplasma pneumoniae, a prokaryote that lacks superoxide dismutase as well as catalase, produces 0 2 , and not H202, equivalent to the consumed oxygen [ 104].

VIE. Plant cell wall The cell wall fractions of horseradish [105] and

Forsythia xylem [106] were able to oxidase NAD(P)H and generate H202 and this activity was attributed to a peroxidase. Horseradish peroxidase was capable of oxidizing N A D H [107] which was sensitive to catalase and superoxide dismutase. Initial triggering of the reaction by HzO 2 generates N A D radical which then propagates a chain-reaction through oxygen radicals, forming large amounts of H 202 stoichiometric with N A D H oxidized [108]. As expected this activity was inhibited by superoxide, but not hydroxyl radical scavengers [108]. Strangely, Mn 2÷ (a purported O 2 scavenger) and p-coumaric acid stimulated N A D H oxidation. These effects may have bearing on the lignin formation which depends on a cell-wall-bound peroxidase for the H202-dependent peroxidation and polymerization of phenolates [109]. In cell walls of tobacco three peroxidases were found each with different Kapp values for H 202 and possibly different catalytic function. Of these two with high Kapp values found only in cell wall, were concerned with H202-dependent process of polymerization of coniferyl alcohol or p-coumaryl alcohol, and a third one for generating H 2 0 z by oxidation of N A D H that was stimulated by Mn 2+ and phenols [110].

VIII. Associated cellular phenomena Cellular H202 generation is a result of combination of a number of membrane and soluble enzyme systems. In the rat liver it had been estimated that 10% of oxygen uptake accounted for H202 generation [11,111]. Such estimates in other cells are not available. Invariably, the capacity for generation of H202 is accompanied by the presence of highly active H202-scavenging systems, catalase and peroxidases. It is increasingly being realized that this complex metabolic capability has functions to perform in the cellular activities. A number of cellular phenomena are now recognized in which generation or utilization of H202 seems to play a central role. Among these are the respiratory burst in phagocytosis and in fertilization, maturation of protozoal parasites, hormonal response of cell membranes and transport processes, cyanide-resistant respiration in plants, initiation of lignification, alternative oxidations and thermogenesis. Studies on stimuli for generation and utilization of H202 in a variety of associated cellular phenomena are revealing a significant physiological role for H202.

VIIA. Parasitism of protozoa The agent of Chagas' disease, Trypanosoma cruzi, provides an interesting case of an organism which can not utilize HzO 2. It lacks both catalase [112] and glutathione peroxidase [113] and thus is defenseless against H202. It showed cyanide-insensitive respiration which could be stimulated by fl-lapachone (an O-naphthoquinone derivative) [114] and by nifurtimox (a nitrofuran compound) [112]. The stimulation of oxygen uptake by these compounds was accompanied by formation of H202, possibly via their radical intermediates. Nifurtimox was able to generate 0 2 with isolated microsomes and mitochondria, using either N A D P H or N A D H [112].-Thus, H202, which is toxic to trypanosomes and normally produced in limited quantities, would have been greatly increased in the presence of these drugs that proved to be trypanocides. While this capacity of different compounds to stimulate endogenous H202 generation gave an excellent experimental tool to develop beneficial drugs, its existence must also be pur-

83

poseful in trypanosomal metabolism or its parasitism. Malarial parasite, Plasmodium falciparum, provides another example where H202 generation found as a normal metabolic trait [115], possibly by the action of N A D P H oxidase of the parasite membrane [116]. In its life cycle the parasite undergoes maturation within the red blood cell. In the normal red cells, survival of the plasmodium parasites was found to be less at higher oxygen tension implying an 'oxidant damage' [117]. This effect was more pronounced in red cells obtained from thalassaemia and glucose-6-phosphate dehydrogenase deficiency cases, suggesting greater oxidant stress in these red cells [117]. Supplements of menadione and riboflavin, which are known to generate oxygen radicals, also decreased multiplication of parasite in the thalassaernic and glucose6-phosphate dehydrogenase-deficient red cells and protection against the damage to parasites could be obtained with vitamin E, and with dithiothreitol only in glucose-6-phosphate dehydrogenase-deficient cells [117]. These results can be interpreted as follows: parasite membrane N A D P H dehydrogenase generates H202 as essential metabolic requirement; H202 a n d / o r oxygen radicals are toxic to plasmodium; as part of its parasitism plasmodium depends on the host red cell to detoxify these by peroxidation of glutathione which can be regenerated through NADPH; absence of this regeneration/protection system in glucose-6phosphate dehydrogenase-deficient and thalassaemic cells results in killing the parasites and thus confers the protection against malaria in such populations [ 118,119]. The parallelism in two protozoal parasites with respect to H202 toxicity is striking and therapeutic methods through metabolic control are likely to be complementary. It is of paramount importance to have a better understanding of the need and the mechanism of this suicidal formation of H202 in plasmodium to help in the discovery of suitable H 202-stimulants that can act as anti-malarial drugs (Fig. 3).

VIIB. Phagocytosis and respiratory burst The first observation on increase in H202 accompanying phagocytosis was by Iyer et al.

peroxidase ~

GSH

,

Glutathi°ne ~ ' ~ reductase~w~ ~

k

NADPH

|

~

GSSG

I

-,,~r~

I _ I G6PD-promote, I

N
o6p 6-Po

................I

/

aac Fig. 3. Generation and removal of H202 during maturation of plasmodium in red blood cells (RBC). Plasmodium membranes generate H202 as a metabohcnecessity.This is removedby the host cell glutathione peroxidase,with glutathione being regenerated by NADPH supplied through the action of glucose-6phosphate (G-6-P)dehydrogenase.If H202 was not removed,it generates radicals which cause oxidant damage to the parasite and destroys it. Tocopheroland dithiols prevent this damage. In 'thalassaemia trait' foetal type hemoglobin (HbF) present promotes formationof radicals and thereby the oxidant damage. In glucose-6-phosphate dehydrogenase deficiency (G6PD-), since H202 can not be removed, the parasite is killed. Resistance to malaria is observed in thalassaemia and glucose-6phosphate dehydrogenase-deficientcases. [ 120,121] who found that conversion of labelled formate to CO 2 increased due to peroxidative action of catalase in presence of H202 generated. In polymorphonuclear leucocytes (PMN) a striking phenomenon of a burst of oxygen uptake was observed on internalizing bacteria or other particles [122]. This was termed 'respiratory burst'. The uptake of oxygen is insensitive to 1 mM cyanide and H202 is a product [123]. This formation of H202 utilized, as its reducing source, N A D P H obtained from hexose monophosphate shunt pathway which also increased as indicated by the conversion of [1-]4C]glucose to CO 2 [123]. The implication of H202 generation in ingesting and killing of bacteria was also studied. Bactericidal potency obtained by H202 was found to be indirect through oxygen radicals derived, in combination of myeloperoxidase and iodide (or a halide) [124]. Desialylation of the PMN did not

84 affect phagocytosis per se but impaired markedly killing of internalized bacteria, and also of H202 generation [125]. This defective H202 generation is more than a coincidence. In chronic granulomatous disease (CGD) of childhood characterized by greater susceptibility to bacterial infections despite the normal compliment of immunoglobulins [126], the granules in PMN were found to be defective in H202 generation [127]. The phagocytic cells, neutrophils, when stimulated by serum-treated (referred to as 'opsonized') bacteria or Zymosan (a yeast-cell wall factor), become triggered for the respiratory burst. The ingested particles are enveloped by plasma membrane, become internalized and are recovered in the 'Granule-Rich Fraction' (GRF) [127]. Also, this G R F showed oxidation of N A D P H and H202 generation but only on prior stimulation by the serum-treated particles [128]. This dormant enzyme in the plasma membrane of the G R F was found [ 129-131 ] to be a flavin-dependent N A D P H oxidase whose activity was stimulated only when the neutrophils were exposed to the stimulus of opsonized zymosan. This effect being absent in the neutrophils obtained from patients with C G D [ 128,130-132], further supported the importance

POLYMORPHON UCLEAR LEUKOCYTE

[

G

®

Fig. 4. H202 generation in phagocytosisand respiratory burst. The sequence of events shown schematically represents the processes of internalization, killing and dissolution of bacterial particles by polymorphonuclearleucocytes. (1) The particle is to be activated by a serum component. This is essential for the respiratory burst occurringlater. (2) The particle is recognized by the PMN plasma membrane. (3) Internalization occurs. (4) The phagosomeis formed which produces H202 on oxidation of NAD(P)H that accounts of the large consumption of oxygen in respiratory burst. Phagocytosiscan occur without this but killing of the bacteria needs H202 generation. (5) Lysosomes fuse with the phagosomes. (6) The destruction of the bacterial components is completed.

of H202 generation in bactericidal action in phagocytosis (Fig. 4). The G R F isolated from PMN was found to possess NAD(P)H oxidase activity and had been considered the site of respiratory burst and H202 generation. It is however debatable whether the purpose of the respiratory burst is to generate H2O2 or the oxygen radicals and which of these account for the toxicity. Several investigations showed that superoxide was formed [133-135] (possibly through mediation of ubiquinone [136] found in neutrophils [137]) and liberated into the medium [138]. Its time course was similar to that of the uptake of oxygen and formation of H202 [139]. The evidence was based on the capability of reduction of ferricytochrome c, inhibited by superoxide dismutase [ 133,140]. Doubts were expressed as to whether this was sufficient evidence to accept superoxide as an intermediate because addition of superoxide dismutase, which was expected to divert the electrons from 02 to H202 and cause little change in oxygen uptake, in fact inhibited the whole process [141]. There is no inconsistency in this as the phenomenon of respiratory burst is dependent on a chain-reaction involving 02 and O H ' . Trapping of any of the components in the chain would inhibit the oxygen uptake [29]. This feature appears to be common with the plasma membrane H202-generating N A D H oxidation systems [ 10,81]. Using a variety of radical-quenchers, Torres et al. [29] had found evidence that the first event in GRF-mediated aerobic oxidation of N A D H was the enzyme-mediated formation of OH" from H202 and suggested the following sequence of reactions: Protein (reduced form) + H2O2 Protein (oxidized form) + OH" + OH OH" + N A D H ~ NAD" + H 2 0 NAD" + 02 ---,O [ + N A D + 0 2 + N A D H + H + --, NAD" + H202 But the identification of OH" was based on the oxidation of ferrocytochrome c. It is significant

85 however to note that the activities of N A D H disappearance, oxygen uptake and H202 generation were absent in 'boiled G R F ' which retained 20% of activity of ferrocytochrome c oxidation, an indication of a component capable of non-enzymic OH" generation in GRF. But the simplistic view of the sequence of reactions will be generation of 0 2 by NAD(P)H oxidation followed by dismutation of O 2 to H202. But this leaves unexplained the inhibitory effect of superoxide dismutase unless another function is invoked for this enzyme in the formation of 0 2 itself [40]. The enzyme catalyzing the reaction N A D P H + 202 ~ N A D P + + 202 had been obtained in soluble form from opsonized-zymosan-activated human neutrophils by treatment of G R F with Triton X100 and filtration through a membrane filter [142]. The activity of O z-formation with N A D P H was 3-fold higher than NADH. It required phosphatidylethanolamine to show full activity whereas phosphatidylocholine and phosphatidylserine were ineffective. The soluble O~--forming enzyme could be obtained only from normal (but not from C G D patients), neutrophils treated with opsonized zymosan and not from resting cells, correlating with the respiratory burst phenomenon [143]. The soluble enzyme was highly specific for oxygen and could not use artificial electron acceptors including ferricyanide. The enzyme requires FAD ( K m, 61 nM) and also a free-SH group for activity. The solubilized enzyme was found to be highly unstable and its inactivation was speeded up by salts, EDTA and ATP [144]. Another enzyme with N A D H as the preferred substrate and producing both 0 2 and H202 [145] was extracted from guinea-pig PMN and partially purified. The apparent molecular weight was 310000. It has a rather large K m of 0.4 mM for NADH. The enzyme was able to release the electrons both in an univalent pathway to form O2", and a divalent pathway to form H202 with an approximate distribution of 15 and 85%, respectively. This activity was also inhibited by ATP as well as by ADP, GDP, GTP, UTP, TTP and pyrophosphate at 0.3 mM. The inhibition by ATP was reversed by Mg 2+, Ca 2+ or Mn 2+. Rate calculations showed that this enzyme was also capable of explaining the oxygen uptake in respiratory burst [145]. This N A D H oxidase seemed

to be attached to the cytoplasmic surface of the phagosomal membrane [146] which is the same as the inside of the plasma membrane. Therefore, the H202 generated must diffuse into the phagosome in order to exert its bactericidal action. It was not clear whether in these experiments the zymosandependent stimulation was essential. Opsonization, an essential requirement for zymosan (yeast cell wall) to be an effective stimulant, is a process yet to be defined. Little information is available on what serum treatment does to zymosan and how the combination of the serum and cell-wall preparation stimulates and sustains the high NAD(P)H oxidase :activity even after solubilization. One interesting clue on the serum factor was obtained by its stability in storage. Human AB serum stored frozen at - 2 0 ° C was more effective in O 2 production than the sample stored unfrozen at 0°C, while both had similar effect on oxygen uptake [147]. The O 2 generation factor had been conjectured to be the complement fragment C3b believed to function in particle recognition (see Ref. 148 for a review). This indeed gives credence to the view that it is the serum factor, not the particles or bacteria, that is responsible for the respiratory burst [149]. Since serum alone was ineffective, a contact of the serum factor with phagocyte surface may be made possible through the presentation of the particle. Production of superoxide in human neutrophils was observed on contact of cytochalasin B with the membrane in the absence of phagocytosis [150]. Respiratory burst is also activated by soluble agents. T h e c h e m o t a x i c p e p t i d e , N-formylmethionine-leucyl-phenyl-alanine, produced a brief but rapid burst of oxygen uptake in neutrophils [151] and alveolar macrophages [152]. Other soluble activators that produced sustained stimulation of oxygen uptake are complement fragment C5a and phorbol myristate acetate, a promotor in carcinogenesis. VIIC. Hormonal action

Evidence for the presence of an enzyme, N A D P H oxidase, producing H202 accompanying oxidation of NADPH, in the plasma membranes, and its stimulation on exposure of intact rat

86

adipocytes to insulin and its biologically active derivatives was first shown by Mukherjee and co-workers [97,154] and confirmed by May and de Haen [155]. This activity was insensitive to cyanide and azide. It had a low K m for N A D P H (58 ktM, N A D H was not tested). This demonstration of H202 generation by intact cells and its stimulation by a hormone, gave a clue of possible mechanism of insulin action using H202 as an intermediate [156]. The sugar transport system in the muscle [157], erythrocytes [158] and brown fat cells [159] was found to be active only in the 'oxidized state' of plasma membrane. This led to the proposal that oxidation of sulphydryls to disulphide in the proteins of plasma membranes may form an important step in the mechanism of insulin action (see Ref. 159 for a review). Then the insulinresponsive N A D H dehydrogenase generating H 202 which can be used in this oxidation assumes the role of a transducer of the hormonal signal. Added H 202 was shown to have insulin-mimetic action, fulfilling another necessary criterion for a messenger role. The list of insulin effects that are obtained with H202 is growing and includes enhanced glucose transport [159], preferential glucose-carbon 1 oxidation [ 161] increased glycogen synthesis from glucose [162], selective incorporation of glucose-carbons into lipids [163], activation of pyruvate dehydrogenase [163] and inhibition of glucagon-stimulated lipolysis [164]. It is interesting to note that vanadate also exhibited insulin-like effects on glucose transport [165] and on glucose oxidation [166]. These effects are not related to the powerful, inhibitory action on (Na + + K + ) ATPase of vanadate [165]. This insulin-mimetic action of vanadate may turn out to be due to H202, generated in presence of vanadate and N A D ( P ) H [82]. Hormone-induced generation of H202 may also be applicable to other hormones. Generation of H 2Oz in uterus was shown to depend on oestrogen [167]. H 202 production and the oxygen-radical-dependent chemiluminiscence seem to depend on hormonal changes during the sea-urchin development [168]. In this system a respiratory burst occurs after fertilization which may indeed be a consequence of the general phenomenon of internalization of particulate matter. All these observations strengthen the concept

that H202 may act as another second messenger [ 156]. While the adenyl cyclase-cyclic A M P system gave a powerful impetus to the understanding of the mechanism of hormone action, this is limited to some hormones. The action of steroid hormones is totally different being of chromosomal locale. Generation of H202 through N A D ( P ) H oxidation could form an additional transducer system for hormone action either at the plasma membrane or intracellular level. The parallelism of systems of adenylcyclase-cyclic A M P and of N A D H oxidaseH202 are brought to focus in relation to the stimulation by vanadate [82]. It is instructive to notice the common-features (location in plasma membranes) stimulation of low basal activities by hormones and other agents (e.g. vanadate, fluoride, serum factors), substrates used are 'energy rich' (ATP or NAD(P)H), products are degraded by ubiquitous enzymes (cyclic A M P by phosphodiesterase and H202 by catalase or peroxidase), the products act by modification of enzymes by reversible phosphorylation or oxidation. This places the N A D ( P ) H dehydrogenases of plasma membrane in a role similar to adenyl cyclase and the available information summarized by Crane [79] showed the coincidental nature of the two. A model for plasma membrane based N A D ( P ) H oxidase-H202 in metabolic control in response to hormones is shown in Fig. 5.

.

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~N

"~

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.

.

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.

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Fig. 5. A model for N A D ( P ) H

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in metabolic

control. The model draws the analogy between cyclicAMP and H202 systems. Both are generated by plasma membrane enzyme systems triggered by hormones. Both use 'energy-rich' compounds, ATP and NAD(P)H. The products affect protein modifications -- cyclic AMP, phosphorylations; H202 oxidation of thiols. The modifications are known to alter enzyme activities and provide metabolic control. The products are removed rapidly by enzymes -- cyclic AMP by phosphodiesterase; H202 by catalase or peroxidases.

87

VIID. Cyanide-insensitive respiration The classical potent respiratory inhibitor, cyanide, was known to inhibit respiration in certain higher plants only partially. This has come to be known as 'cyanide-insensitive respiration' implying that oxygen uptake occurs in these cells that does not depend on cytochrome oxidase. The first explanation of this phenomenon was given by Bendall and Bonner [48] who postulated an alternative oxidase which uses the same dehydrogenases of the respiratory chain but branches from it at the cytochrome b region. Variations in the degree of cyanide-sensitivity of respiration of mitochondria were noted-arum species mostly insensitive, mung bean intermediate, and potato tuber mostly sensitive [169]. Much of the electron transport through the alternative oxidase occurs in the resting state 4 [170]. A distinguishing property of this 'alternate oxidase' is its sensitivity to aromatic hydroxamic acids [ 171,172]. Using these inhibitors it was possible to identify the following features: an equilibration exists between the respiratory chain and alternative oxidase [171]; ubiquinone is the site of interaction [170]; intensity of electron flux rather than the energy state of mitochondria is the controlling factor for the flow of electrons into alternative oxidase [173]. These inhibitors act by competition with the reduced substrate for the active site on the oxidase [174]. The alternative oxidase seems to be identical with a p-diquinol oxidase. A partially purified preparation from Arum maculatum mitochondria was capable of oxidizing menadiol and ubiquinol and producing H202 as a product. This activity was insensitive to cyanide but was inhibited by m-chlorobenzhydroxamic acid [50,52]. The diversion of electrons to alternative oxidase and their transfer to oxygen seem to involve ubiquinone present ubiquitously in all the mitochondria. The cyanide-insensitive respiration was found to occur also in animals, algae and bacteria [175] but more studies are needed to generalize on its widespread occurrence. In Escherichia coli cyanide-insensitive respiration increased in presence of paraquat, menadione, phenazine methosulphate and alloxan [176] possibly by diverting electrons towards the alternative pathway. The fungus Sternphylium loti utilizes this pathway to

live as a pathogen on the cyanogenic plant Lotus corniculatus [ 177]. Even animal tissues are known to show residual oxygen uptake in presence of cyanide. This was more evident when homogenates of liver, brain, heart and kidney were tested, which showed 42, 36, 37 and 19%, respectively, of the rates of oxygen uptake with succinate as substrate were insensitive to cyanide (0.1 mM) [178]. Isolated liver mitochondria, however, were more sensitive to cyanide. It was found that the liver cytosol had a factor which will prevent cyanide inhibition in a concentration-dependent manner. At constant cyanide concentration, increase in concentration of either mitochondria or homogenate (with equivalent amount of mitochondria) the resistance to cyanide inhibition increased suggesting sequestering of the inhibitor by a protein or a factor. Addition of redox compounds, ascorbate, menadione, phenazine methosulphate or neotetrazolium chloride, increased the rate of cyanide-resistant oxygen uptake. This potential of increasing electron flow through the bypass may be used under conditions requiring H202 in the cell.

VILE. Thermogenesis Generation of H202 and its subsequent destruction by catalase being exergonic reactions yield heat. Thus the cyanide-insensitive respiration by alternative oxidases may be used for thermogenic purposes. In fact such a system in the bombardier beetle is elegantly used for production of vapourized secretion as a defensive mechanism [179]. Their reactor glands possess a mixture of H202 and hydroquinone which are squeezed into an outer compartment containing peroxidase and catalase that results in production and degradation of H202, generating sufficient heat to vapourize water. A controlled operation of the same system has the potential of forming the basis of intracellular thermogenesis. In cold acclimation of homeotherms, increased thermogenesis sans shivering occurs that is characterized by increased food consumption, metabolic rate and oxygen uptake. This process of heat production becomes reversibly operational when the ambient temperature goes below the thermoneutral zone and is known as 'regulatory non-

88 shivering thermogenesis' [180]. A variety of explanations are offered for the mechanism of this heat production: uncoupling of oxidative phosphorylation, activation of pumps, of N a - K or Ca, and futile cycles involving an increased turnover of ATP (see Ref. 181 for review). However, in cold exposed animals, the rates of mitochondrial oxidations in most tissues are increased to a small extent and the phosphorylation system remains unaffected [182]. Presently the concept of uncoupling of proton gradient is being actively pursued and evidence for the occurrence in brown-adipose mitochondria of a GTP-binding protein (32000 daltons) that discharges proton gradient was obtained [183]. Increased thermogenesis is not confined to the brown-adipose or the muscle but is shared by most visceral organs [180]. Extra heat can be generated by augmentation of alternative pathways or 'calorigenic shunts' [184] by tapping electrons at the dehydrogenase level. This provides the basis of linking H202 generation with thermogenesis. A model of chemical thermogenesis in the liver [185] regulated by ubiquinone and implicating H 2 0 z had been developed. The evidence is summarised below: hepatic concentration of ubiquinone increased in cold acclimation and concomitantly the activity of the shunt pathway represented by ubiquinone-dependent reduction of neotetrazolium also increased [38]; this transfer of reducing power to the ubiquinone pool and the shunt pathway increases H202 generation; H202 generation decreased in heat stress and conditions that decrease thermogenesis [45]; these responses of H202 generation were dependent on a-adrenergic receptors which also control non-shivering thermogenesis. The rates of H202 generation in mitochondria are too small to account quantitatively for the heat p r o d u c e d . T h u s the mitochondrial system may respond to the initial signals and H202 generated may then transduce the signal to the more rapid generation of H202 in the microsomal system for amplification [185]. The model is given in Fig. 6. It becomes more relevant in plants since the established cyanide-insensitive respiration primarily yields H202. Photorespiration, the phenomenon of light-dependent reoxidation of part of photosynthetic carbon in some temperate zone plants, also generates H202 (see Ref. 63 for a recent review). The design of these

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i Fig. 6. A model for chemical thermogenesis. This is a generalized scheme depicting the participation of the cell components in generation of H202 which on catalatic action generates heat. At the mitochondria level H202 is generated by a number of substrates whose flavoproteins converge at ubiquinone (Q) that forms the link between respiratory chain and H202-generator. This system responds to treatments of noradrenaline (NA), thyroxine (T4) and cold by increasing and to treatments of thiouracil and heat by decreasing. These changes are controlled by the ct-adrenergicreceptors known to be involvedin the process of non-shivering thermogenesis.The H2O 2 generation being small in mitochondria, it can only act as another signal which needs to be amplified. The component is indicated as X and remains unidentified. Vanadate, if present, can fulfill this function since it has the capacity to stimulate oxidation of NAD(P)H by microsomal membranes and generate large quantities of 11202. The diversion of (NAD(P)H from glycolysis and HMP shunt provides the energy. Microbodies-peroxisomeswill destroy the H202 and generate heat. In some plants H202 generated through cyanide-resistant pathway by mitochondria can produce heat.

systems may ensure local intracellular thermogenesis to keep the temperature high enough for sufficient rates of metabolic activity. Indeed the inflorescence of a common garden philodendron [186] and the spadix of Arum maculatum and Sanromatum qattulum [184] exhibit core temperatures of 10-15°C higher than the ambient. It appears that having a high content of ubiquinone [ 187] and high cyanide-insensitive respiration [ 170], both implicated in H202 generation, in Arum species is more than a coincidence.

VIII. Concluding remarks Generation of H202 appears to be a natural process and is of universal occurrence in a variety of aerobic organisms. Contribution of the intracellular components in the rat liver [ll] was

89 found to be distributed as follows: mitochondria (15%), microSomes (45%), peroxisomes (35%) and cytosol (5%). Of these peroxisomal H202 seemed to be destroyed in situ but other sources provide H202, albeit small amounts, for cellular use. A variety of oxidases and dehydrogenases in the cell are capable of generating H202. Only the endomembranes, particularly microsomal or plasma membranes, have the special property of dormant NAD(P)H oxidation that can lead to very high rates of oxidation of NAD(P)H and generation of H202 on stimulation during phagocytosis or treatment with vanadate. Under normal conditions the rates of H202 generation are small (of the order of n m o l / m i n per mg protein) in most of the membranes. The H202 thus generated represents hardly 1-2% of the total oxygen consumption in animal tissues. The concentrations of catalase and glutathione peroxidase are far too much in excess to permit this small quantity of H202 even a remote chance of exhibiting its purported toxicity. The cell is provided with multiple mechanisms of defence against H202 and these over-protective measures are somewhat surprising (see Ref. 188 for a review). During the seventies it was increasingly realized that H202 is not a mere wasteful byproduct but has a functional, metabolic role. The surprising finding that the mitochondrion is a meaningful H202 generator has led Chance et al [188] to propose that this system "affords the conceptual basis for a revolution in our consideration of the problem of hydrogen-transfer reactions in mitochondria". The sequestering of protons in the form of membrane-permeable H202 may play a significant role in controlling proton gradients. Indeed this forms a new concept and deserves further work. With respect to mitochondria the accumulated data seem to anticipate that the H2OE-generator system will be a distinct entity from the respiratory chain. The parallel utilization of substrates has provided a false facade of sharing the dehy. drogenases of the electron transport systems. The cell may not have evolved totally independent enzymes and components but may have used some units of these in a different milieu of the membranes to develop the capability of H202 generation. The differential responses between the activi-

ties of the dehydrogenases and of the reduction of 02 to H202, especially in thermogenic conditions, also vouch for a physiologically meaningful role for this system. Moreover its response to partial pressure of oxygen would also make this system the focal point of oxygen effects, especially the oxygen-dependent toxicity. The widespread occurrence of H202 generation in cellular membranes points to a more general use of this mode of reduction of oxygen. It is hard to decide whether localized H202 has a metabolic role or is utilized for lipid peroxidation and breakdown of the membranes as part of their turnover. More attention is needed on the study of the interrelationship of hormone --, H202 --, dithiol proteins ~ metabolic control. The theoretical base for this seems well-prepared. The hormonal response of N A D H dehydrogenase of plasma membranes already indicated [79,97] needs further substantiation, especially at the cellular level. Its potential in bacteria and plant cells is hardly explored. A specific need for H202 in killing the phagocytosed bacteria had been established. While lysosomes undertake the task of dissolving out the components of the ingested particles, the killing of pathogenic bacteria seem to require H202-derived radicals. This process utilizes the latent capacity of NAD(P)H oxidation of the plasma membrane (which is unmasked by a serum component) and requires the phagosome structure. The explanation for these peculiar requirements is not available. Intrinsic high rate of H202 generation, an apparent metabolic necessity, seems to be a characteristic of protozoa. Parasitism in the case of trypanosoma and plasmodium may indeed be the removal by the host cells of the large amounts of H202 produced during their metabolism, otherwise self-destructive in view of the absence of enzymes that utilize H202 in these organisms. This is exemplified by the decreased survival of these disease-causing parasites in the host cells with defective H2OE-Scavenging mechanism or on treating with agents that increase H202 generation. The crucial requirement of NAD(P)H oxidation despite the product, H202, being toxic to the parasites requires further analysis. Their very survival seems to depend on the capability of the

90

host cells to detoxify H 2 0 2. The vignette of this characteristic is becoming apparent from the study of enteric parasitic protozoa, Entamoeba histolytica and Giardia larnblia (see Ref. 189 for a recent summary). These protozoa, presumed to be anaerobic, lack mitochondria and are deficient in tricarboxylic acid cycle enzymes, lactate dehydrogenase, catalase and peroxidase. Yet they consume oxygen (sensitive to iron-chelators but not to cyanide, antimycin and azide) and purportedly produce H202. It is obvious that the recycling of NAD(P)H for continued glycolysis in the absence of a suitable substrate-dependent dehydrogenases can only happen by direct oxidation of NAD(P)H. The parasite then depends on the host to scavenge H202. This essential feature of parasitism also provides approaches for control measures; on the one side by interfering with the removal of the toxic H202 by the host cell, and on the other by finding specific inhibitory agents of the activity of the plasma membrane NAD(P)H dehydrogenase responsible for H 2 0 2 generation. Respiration by alternative cyanide-resistant pathway and photorespiration is not insignificant in certain plant tissues. The energy in these cases will not be conserved but will be converted to heat. These H202-generating systems serve as localized heat-generators. But more evidence is becoming available for direct use of H 202 through peroxidases that will make H 2 0 2 a n essential metabolite.

Acknowledgements The work in the author's laboratory is supported by University Grants Commission and Indian National Science Academy, New Delhi, India. Thanks are due to Mr. Anand Swaroop and Miss S. Vijaya for help in the preparation of this article.

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