Hydroxyl radical formation in chondrocytes and cartilage as detected by electron paramagnetic resonance spectroscopy using spin trapping reagents

Chondrocytes have been shown to produce superoxide and hydrogen peroxide, suggesting possible formation of hydroxyl radical in these cells. In this study, we used electron spin resonance/spin trapping technique to detect hydroxyl radicals in chondrocytes. We found that hydroxyl radicals could be detected as α-hydroxyethyl spin trapped adduct of 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN) in chondrocytes stimulated with phorbol 12-myristate 13-acetate in the presence of ferrous ion. The formation of hydroxyl radical appears to be mediated by the transition metal-catalyzed Haber-Weiss reaction since no hydroxyl radical was detected in the absence of exogenous iron. The hydroxyl radical formation was inhibited by catalase but not by superoxide dismutase, suggesting that the hydrogen peroxide is the precursor. Cytokines, IL-1 and TNF enhanced the hydroxyl radical formation in phorbol 12-myristate 13-acetate treated chondrocytes. Interestingly, hydroxyl radical could be detected in unstimulated fresh human and rabbit cartilage tissue pieces in the presence of iron. These results suggest that the formation of hydroxyl radical in cartilage could play a role in cartilage matrix degradation.     

Kinetic and magnetic properties of cobalt(III) ion in the active site of carbonic anhydrase.

Cobalt(III)bovine carbonic anhydrase B was prepared by the oxidation of the cobalt(II) enzyme with hydrogen peroxide and was purified by affinity chromatography. The oxidation reaction is inhibited by specific inhibitors of carbonic anhydrase. The inhibition is explained by the fact that the Co(II)-enzyme . inhibitor complex cannot be directly oxidized by hydrogen peroxide, but has to dissociate to give free Co(II) enzyme which is then oxidized. The Co(III) ion in Co(III) carbonic anhydrase cannot be directly substituted by zinc ions. It can be reduced by either dithionite or BH-4 ions to give, first, their complexes with the Co(II) enzyme, and upon their removal, a fully active Co(II) enzyme. Cyanide and azide bind to cobalt(III) carbonic anhydrase with similar rate constants of 0.060 +/- 0.005 and 0.070 +/- 0.007 M-1 S-1 respectively. These rates are faster than those found for Co(III) inorganic complexes. The Co(III) ion in both Co(III) carbonic anhydrase and Co(III) carboxypeptidase A was found to be diamagnetic, indicating a near octahedral symmetry.     

Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C(i) Cells of Chlamydomonas: Studies Using O Exchange with C/O Labeled Bicarbonate

By measuring ¹⁸O exchange from doubly labeled CO₂ (¹³C¹⁸O¹⁸O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [C_i]) or air enriched with 5% CO₂ (high C_i). Intact low C_i protoplasts had a 10-fold higher carbonic anhydrase activity than did high C_i protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of ¹⁸O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low C_i cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low C_i than in high C_i preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment.     

The case for chloroplast thylakoid carbonic anhydrase

Washed thylakoid membranes and photosystem II-enriched membrane fragments from cyanobacteria, green algae, and chloroplasts from both C₃ and C₄ plants possess the ability to reversibly hydrate CO₂. That is, the membranes have an intrinsic carbonic anhydrase activity. The present review outlines the discovery of thylakoid carbonic anhydrase and presents the evidence that it is a unique isozyme, distinct from other cellular carbonic anhydrases. It appears that at least some thylakoid carbonic anhydrase is closely associated with photosystem II and may be required for electron transport. This would explain why all inhibitors of carbonic anhydrase also inhibit photosystem II. Several speculative functions of thylakoid carbonic anhydrase are discussed. These include a possible role in carbon metabolism, in the protonation of plastoquinone, and/or in oxygen evolution.     

Accumulation of bicarbonate in intact chloroplasts following a pH gradient

With silicone layer filtering centrifugation the uptake of radioactively labelled bicarbonate into isolated spinach chloroplasts was followed. This uptake was shown to have the following properties:

1. (a) It is so rapid that the kinetics of uptake usually cannot be resolved.

2. (b) Bicarbonate is accumulated in the stroma. The factor between the internal and external concentrations increases greatly when the pH of the medium is lowered from pH 8.5 to pH 7.0.

3. (c) The accumulation factor is independent of the concentration in the medium for a long concentration range.

4. (d) The accumulation of bicarbonate is increased when the chloroplasts are illuminated. This increase is abolished by the addition of uncoupler.

5. (e) Diamox, an inhibitor of carbonic anhydrase, inhibits the rate of bicarbonate uptake.

The activity of carbonic anhydrase was assayed in isolated chloroplasts and in leaf homogenates. In agreement with earlier reports the main activity was found to be located in the chloroplasts. This activity is latent; it can be only assayed if the chloroplasts are osmotically shocked.

From these results the following conclusions have been drawn:

1. (a) The inner membrane is impermeable to protons. Light-driven proton transport into the thylakoid space causes an alkalisation of the stroma.

2. (b) The uptake of bicarbonate proceeds via diffusion of CO₂ across the inner membrane. There are no indications for a specific transport of bicarbonate.

3. (c) The CO₂ concentration in the chloroplasts may be equal to the CO₂ concentration in the external space. The distribution of bicarbonate between the two compartments is inversely proportional to the distribution of protons.

A possible involvement of carbonic anhydrase and the bicarbonate pool in the stroma in increasing the CO₂ affinity of CO₂ fixation is discussed.     

Uptake of bicarbonate ion in darkness by isolated chloroplast envelope membranes and intact chloroplasts of spinach

Bicarbonate uptake by isolated chloroplast envelope membranes and intact chloroplasts of spinach (Spinacia oleracea L. var. Viroflay) in darkness exhibited a similar dependency upon temperature, pH, time, and concentrations of isolated or attached envelope membranes. This similarity in uptake properties demonstrates the usefulness of the envelope membranes for the study of chloroplast permeability. Maximal rates for dark HCO₃^- uptake by isolated envelope membranes and intact chloroplasts were more than sufficient to account for the maximal rates of photosynthetic CO₂ fixation observed with intact chloroplasts. The active species involved in the uptake process was found to be HCO₃^- and not CO₂. The significance of HCO₃^- uptake and its relationship to carbonic anhydrase and ribulose diphosphate carboxylase is discussed. Conditions for maximal HCO₃^- uptake in darkness by intact chloroplasts were found to be similar to those required for maximal photosynthetic CO₂ fixation, suggesting that HCO₃^- uptake by the envelope membrane may regulate photosynthetic CO₂ fixation.     

Oxygen reduction in chloroplasts and the ascorbate cycle

Current views concerning the generation of superoxide radicals and hydrogen peroxide in chloroplasts as well as their toxic influences on photosynthesis are presented. Systems of H2O2 detoxification including the ascorbate peroxidase reactions and the ascorbate regenerating reactions are described. Data concerning mechanisms of monodehydroascorbate reduction by the photosynthetic electron transport chain are reviewed. The participation of the Mehler-peroxidase reaction in building of a proton gradient across the thylakoid membrane and its possible input in ATP synthesis and in protection from photoinhibition are analyzed. Ascorbate functions in chloroplasts and the need to consider the high concentration of ascorbate in chloroplasts when photosynthetic reactions in vivo are discussed are briefly reviewed.     

The Low CO2-Inducible 36-Kilodalton Protein Is Localized to the Chloroplast Envelope of Chlamydomonas reinhardtii

The localization of the 36-kD polypeptide of Chlamydomonas reinhardtii induced by photoautotrophic growth on low CO2 concentrations (0.03% in air [v/v], low CO2-grown cells) has been investigated. This polypeptide was specifically localized to the chloroplast envelope membranes isolated from low CO2-grown cells and was not present in the chloroplast envelopes isolated from high (5% CO2 in air [v/v]) CO2-grown cells. The 36-kD protein does not show carbonic anhydrase activity and was not present on the plasma membranes isolated from low CO2-grown cells. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO2-grown cells of C. reinhardtii.     

Melanin photoreactions in aerated media: electron spin resonance evidence for production of superoxide and hydrogen peroxide.

Electron spin resonance measurements on aerated melanin suspensions during photoirradiation show changes in the microwave saturation of melanin free radicals and formation of adducts in the presence of spin traps. These observations indicate that oxygen is reduced to superoxide and hydrogen peroxide.     

The nucleotide sequence of a complementary DNA encoding Flaveria bidentis carbonic anhydrase

We have isolated and characterised a cDNA clone encoding the cytosolic form of carbonic anhydrase in the leaves of Flaveria bidentis, a C₄ dicotyledonous plant. The deduced amino acid sequence is similar to the carbonic anhydrase found in the chloroplasts of C₃ dicotyledonous plants. Western blot analysis of crude leaf extracts of F. bidentis indicates that the leader sequence (equivalent to the transit peptide of the chloroplastic form of CA found in C₃ plants) is not removed following translation of mRNA.     

Detoxification of SO2 derivatives in chloroplasts ofHardwickia binata

Intact chloroplasts isolated from sulphur dioxide fumigatedHardwickia binata leaves showed inhibition of PS II electron transport activity without any significant effect on photosystem I. Sulphur dioxide exposed leaves accumulated more hydrogen peroxide than those from non-fumigated plants and this was caused by increase in superoxide radical production. Hydrogen peroxide formation was inhibited by addition of cytochrome C and superoxide disrnutase. In sulphur dioxide fumigated leaves, increase in superoxide dismutase activity showed resistance to sulphite toxicity. The localization of ascorbate peroxidase, glutathione reductase and dehydroascorbate reductase activities in chloroplasts provide evidence for the photogeneration of ascorbate. The scavenging of hydrogen peroxide in chloroplast due to ascorbate regenerated from DHA by the system: PS I → Fd → NADP → glutathione. The system can be considered as a means for preliminary detoxification of sulphur dioxide by chloroplasts     

The metal-mediated formation of hydroxyl radical by aqueous extracts of cigarette tar

Aqueous extracts of cigarette tar produce hydroxyl radicals that are spin trapped by 5,5-dimethyl-1-pyrroline-N-oxide. The addition of catalase almost completely inhibits and superoxide dismutase partially inhibits spin adduct formation. The addition of ethylenediamine tetraacetic acid greatly increases the amount of hydroxyl radical adduct observed; in contrast, diethylenetriamine pentaacetic acid causes complete inhibition of spin adduct formation. We suggest that the hydroxyl radical arises from the metal-mediated decomposition of hydrogen peroxide, and that hydrogen peroxide is formed from the reduction of dioxygen by the semiquinones present in the cigarette tar.     

Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II

Under strong illumination of a photosystem II (PSII) membrane, endogenous superoxide anion, hydrogen peroxide, and hydroxyl radical were successively produced. These compounds then cooperatively resulted in a release of manganese from the oxygen-evolving complex (OEC) and an inhibition of oxygen evolution activity. The OEC inactivation was initiated by an acceptor-side generated superoxide anion, and hydrogen peroxide was most probably responsible for the transportation of reactive oxygen species (ROS) across the PSII membrane from the acceptor-side to the donor-side. Besides ROS being generated in the acceptor-side induced manganese loss; there may also be a ROS-independent manganese loss in the OEC of PSII. Both superoxide anion and hydroxyl radical located inside the PSII membrane were directly identified by a spin trapping-electron spin resonance (ESR) method in combination with a lipophilic spin trap, 5-(diethoxyphosphoryl)-5-phenethyl-1-pyrroline N-oxide (DEPPEPO). The endogenous hydrogen peroxide production was examined by oxidation of thiobenzamide.     

Prokaryotic carbonic anhydrases

Carbonic anhydrases catalyze the reversible hydration of CO(2) [CO(2)+H(2)Oright harpoon over left harpoon HCO(3)(-)+H(+)]. Since the discovery of this zinc (Zn) metalloenzyme in erythrocytes over 65 years ago, carbonic anhydrase has not only been found in virtually all mammalian tissues but is also abundant in plants and green unicellular algae. The enzyme is important to many eukaryotic physiological processes such as respiration, CO(2) transport and photosynthesis. Although ubiquitous in highly evolved organisms from the Eukarya domain, the enzyme has received scant attention in prokaryotes from the Bacteria and Archaea domains and has been purified from only five species since it was first identified in Neisseria sicca in 1963. Recent work has shown that carbonic anhydrase is widespread in metabolically diverse species from both the Archaea and Bacteria domains indicating that the enzyme has a more extensive and fundamental role in prokaryotic biology than previously recognized. A remarkable feature of carbonic anhydrase is the existence of three distinct classes (designated alpha, beta and gamma) that have no significant sequence identity and were invented independently. Thus, the carbonic anhydrase classes are excellent examples of convergent evolution of catalytic function. Genes encoding enzymes from all three classes have been identified in the prokaryotes with the beta and gamma classes predominating. All of the mammalian isozymes (including the 10 human isozymes) belong to the alpha class; however, only nine alpha class carbonic anhydrase genes have thus far been found in the Bacteria domain and none in the Archaea domain. The beta class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants as well as enzymes from phylogenetically diverse species from the Archaea and Bacteria domains. The only gamma class carbonic anhydrase that has thus far been isolated and characterized is from the methanoarchaeon Methanosarcina thermophila. Interestingly, many prokaryotes contain carbonic anhydrase genes from more than one class; some even contain genes from all three known classes. In addition, some prokaryotes contain multiple genes encoding carbonic anhydrases from the same class. The presence of multiple carbonic anhydrase genes within a species underscores the importance of this enzyme in prokaryotic physiology; however, the role(s) of this enzyme is still largely unknown. Even though most of the information known about the function(s) of carbonic anhydrase primarily relates to its role in cyanobacterial CO(2) fixation, the prokaryotic enzyme has also been shown to function in cyanate degradation and the survival of intracellular pathogens within their host. Investigations into prokaryotic carbonic anhydrase have already led to the identification of a new class (gamma) and future research will undoubtedly reveal novel functions for carbonic anhydrase in prokaryotes.     

Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes.     

Partial characterization of a new isoenzyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii

A new isoenzyme of carbonic anhydrase has been isolated and purified from Chlamydomonas reinhardtii. This carbonic anhydrase is composed of two nonidentical subunits with apparent molecular masses of 39 and 4.5 kDa and is located in the periplasmic space. This is the second periplasmic carbonic anhydrase found in C. reinhardtii. Two genes, CAH1 and CAH2, which code for carbonic anhydrase, have been recently described by Fujiwara et al. (Fujiwara, S., Fukuzawa, H., Tachiki, A., and Miyachi, S. (1990) Proc. Natl. Acad, Sci. U.S.A. 87, 9779-9783). The CAH1 gene codes for a periplasmic carbonic anhydrase which is induced under low CO2 conditions and is well characterized. The carbonic anhydrase characterized in this report was isolated from a mutant that is unable to synthesize the CAH1 gene product. Amino acid sequencing demonstrates that this newly isolated carbonic anhydrase is the CAH2 gene product. This is the first report of another functional carbonic anhydrase in C. reinhardtii.     

Oxygen reduction in chloroplast thylakoids results in production of hydrogen peroxide inside the membrane

Hydrogen peroxide production in isolated pea thylakoids was studied in the presence of cytochrome c to prevent disproportionation of superoxide radicals outside of the thylakoid membranes. The comparison of cytochrome c reduction with accompanying oxygen uptake revealed that hydrogen peroxide was produced within the thylakoid. The proportion of electrons from water oxidation participating in this hydrogen peroxide production increased with increasing light intensity, and at a light intensity of 630 micromol quanta m(-2) s(-1) it reached 60% of all electrons entering the electron transport chain. Neither the presence of a superoxide dismutase inhibitor, potassium cyanide or sodium azide, in the thylakoid suspension, nor unstacking of the thylakoids appreciably affected the partitioning of electrons to hydrogen peroxide production. Also, osmolarity-induced changes in the thylakoid lumen volume, as well as variation of the lumen pH induced by the presence of Gramicidin D, had negligible effects on such partitioning. The flow of electrons participating in lumen hydrogen peroxide production was found to be near 10% of the total electron flow from water. It is concluded that a considerable amount of hydrogen peroxide is generated inside thylakoid membranes, and a possible mechanism, as well as the significance, of this process are discussed.     

Evidence for the presence of carbonic anhydrase in the plasma membrane of rat hepatocytes

The cellular distribution of carbonic anhydrase is a key characteristic for the role of the enzyme in cell function. In several epithelia involved in bicarbonate transport this enzyme is located in the plasma membrane. Because bicarbonate secretion is an important mechanism in bile formation by the liver, we investigated the presence of carbonic anhydrase activity in isolated plasma membranes from rat hepatocytes. Carbonic anhydrase activity was enriched 1.79-fold in plasma membrane preparations. This activity was inhibited by acetazolamide and activated by Triton X-100, but was insensitive to Cl- or CNO-. It is highly unlikely that the low contamination of cytoplasm and intracellular membranes could account for the presence of carbonic anhydrase activity in plasma membrane preparations. Moreover, the results from resuspension/washing of plasma membrane fractions in ionic media suggest an absence of soluble carbonic anhydrase adsorption upon plasma membrane. Accordingly, the present findings provide strong evidence for the presence of carbonic anhydrase in the plasma membrane of rat hepatocytes.     

Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells

The important role of plant peroxisomes in a variety of metabolic reactions such as photorespiration, fatty acid beta-oxidation, the glyoxylate cycle and generation-degradation of hydrogen peroxide is well known. In recent years, the presence of a novel group of enzymes, mainly involved in the metabolism of oxygen free-radicals, has been shown in peroxisomes. In addition to hydrogen peroxide, peroxisomes can generate superoxide-radicals and nitric oxide, which are known cellular messengers with a variety of physiological roles in intra- and inter-cellular communication. Nitric oxide and hydrogen peroxide can permeate the peroxisomal membrane and superoxide radicals can be produced on the cytosolic side of the membrane. The signal molecule-generating capacity of peroxisomes can have important implications for cellular metabolism in plants, particularly under biotic and abiotic stress.     

Detection of active oxygen in rat hepatocyte suspensions with the chemiluminigenic probe lucigenin

The chemiluminigenic probe lucigenin has been employed to detect the production of active oxygen species in suspensions of intact rat hepatocytes. Light emission from lucigenin arises from oxygenation by superoxide anion; hydrogen peroxide or a species derived from it may contribute to the reaction. The inhibitory action of antioxidants on the availability of active oxygen species produced by hepatocytes was tested. Propyl gallate was the most potent inhibitor, butylated hydroxyanisole and butylated hydroxytoluene were less active. The latter compounds cause an alteration of the cell membrane at high concentrations.