Conformational control of ovoperoxidase catalysis in the sea urchin fertilization membrane

The ovoperoxidase-catalyzed oxidation of iodide has been investigated as a function of pH for the homogeneous enzyme and for ovoperoxidase incorporated into several forms of the egg fertilization membrane. The pH dependent hysteresis previously observed in purified ovoperoxidase (Deits, T. L., Shapiro, B. M. (1985) J. Biol. Chem. 260, 7882-7888) is entirely absent in ovoperoxidase incorporated into the mature fertilization membrane, where the enzyme is bound noncovalently in vivo. The pH activity profile of ovoperoxidase incorporated into the mature fertilization membrane closely resembles the profile observed only transiently in purified ovoperoxidase subjected to a rapid downward pH shift. These observations can be accounted for by our previously presented mechanism for ovoperoxidase hysteresis (ibid.). We hypothesize that ovoperoxidase, upon incorporation into the fertilization membrane, is restricted to a limited subset of the conformational states available to the purified enzyme. This matrix-dependent conformational restriction is a novel control mechanism that serves to enhance the catalytic activity of ovoperoxidase upon its assembly into the fertilization membrane and thereby modulates ovoperoxidase catalysis in the vicinity of the developing egg.     

Assembly of the sea urchin fertilization membrane: isolation of proteoliaisin, a calcium-dependent ovoperoxidase binding protein

Fertilization of the sea urchin egg is accompanied by the assembly of an extracellular glycoprotein coat, the fertilization membrane. Assembly of the fertilization membrane involves exocytosis of egg cortical granules, divalent cation-mediated association of exudate proteins with the egg glycocalyx (the vitelline layer), and cross- linking of the assembled structure by ovoperoxidase, a fertilization membrane component derived from the cortical granules. We have identified and isolated a new protein, which we call proteoliaisin, that appears to be responsible for inserting ovoperoxidase into the fertilization membrane. Proteoliaisin is a 250,000-Mr protein that binds ovoperoxidase in a Ca2+-dependent manner, with half-maximal binding at 50 microM Ca2+. Other divalent cations are less effective (Ba2+, Mn2+, and Sr2+) or ineffective (Mg2+ and Cd2+) in mediating the binding interaction. Binding is optimal over the physiological pH range of fertilization membrane assembly (pH 5.5-7.5). Both proteoliaisin and ovoperoxidase are found in isolated, uncross-linked fertilization membranes. We have identified several macromolecular aggregates that are released from uncross-linked fertilization membranes after dilution into divalent cation-free buffer. One of these is an ovoperoxidase- proteoliaisin complex that is further disrupted only upon the addition of EGTA. These results suggest that a Ca2+-stabilized complex of ovoperoxidase and proteoliaisin forms one structural subunit of the fertilization membrane.     

Superoxide dismutase, peroxidase, and germin-like protein activity in plasma membranes and apoplast of maize roots

Summary. The analysis of plasma membranes from maize roots by native gel electrophoresis revealed the existence of Mn-containing 120 kDa and CuZn-containing 70, 40, and 15 kDa superoxide dismutase (SOD) isoform activities. Isoelectric focusing of the plasma membranes differentiated anionic SOD isoforms with a pI of about 5 and cationic SOD isoforms at pI 8.6. Solubilization of the plasma membrane proteins further separated the cationic SOD into pI 8.6, 8.2, 8.4, and 7.2 isoforms. Double staining for both SOD and peroxidase activities showed an overlap of these activities only in the case of the high-molecular-mass (ca. 120 kDa) isoforms. High-temperature treatments demonstrated that the 120 kDa isoform was active even at 100 °C, indicating that it was a germin-like protein with superoxide-dismutating activity, different from the peroxidase with a similar molecular mass and the lower-molecular-mass CuZn-containing superoxide dismutases. These results are compared to those obtained from whole-tissue extract and apoplastic fluid. Correspondence and reprints: Maize Research Institute, POB 89-Zemun, 11081 Belgrade, Serbia and Montenegro.     

Localization and developmental fate of ovoperoxidase and proteoliaisin, two proteins involved in fertilization envelope assembly

Fertilization of the sea urchin egg leads to the assembly of an extracellular matrix, the fertilization envelope. Ovoperoxidase, the enzyme implicated in hardening the fertilization envelope, is inserted into the assembling structure via a Ca2+-dependent interaction with the protein proteoliasin (P. Weidman and B. M. Shapiro, 1987, J. Cell Biol. 105, 561-567). In the present report, polyclonal antisera were raised to ovoperoxidase and proteoliasin (purified from eggs of Strongylocentrotus purpuratus) and characterized by Western blot analysis and an enzyme-linked immunoabsorbent assay (ELISA). By indirect immunofluorescence microscopy all cortical granules of unfertilized eggs, as well as the fertilization envelope, contained both proteoliasin and ovoperoxidase. At the ultrastructural level both proteins are localized to the electron-dense spiral lamellae of the cortical granules. Western blot analysis revealed that ovoperoxidase and proteoliasin persist in early embryos until hatching, but are absent from later developmental stages. Homogenates of eggs of several other echinoderm species (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, Pisaster ochraceus, Dendraster excentricus, and Lytechinus pictus) also contain proteins antigenically similar to ovoperoxidase and proteoliaisin, indicating that many echinoderms utilize a similar strategy for assembly of the fertilization envelope. The results underline the need for postsecretory controls in the extracellular matrix modifications that accompany the cortical reaction.     

Serum selenium,glutathione peroxidase,lipids, and human liver microsomal enzyme activity

This study evaluated selenium status in relation to lipid peroxidation, liver microsomal function, and serum lipids in humans. Serum selenium concentration, glutathione peroxidase (GSH-Px) activity, liver microsomal enzyme activity, assessed by plasma antipyrine clearance (AP-CL) rate, and serum lipids were determined in 23 healthy subjects in a double-blind placebo-controlled trial of selenium supplementation. The low selenium concentration (74.0±14.2 μg/L, mean±SD) is attributable to the low selenium content of the diet. Subjects with the lowest selenium levels (n=11) had reduced serum GSH-Px activity, AP-CL rate, high-density lipoprotein cholesterol (HDL-C), and total cholesterol (T-C) as compared with subjects with higher selenium concentrations (n=12). Low AP-CL rates were associated with low HDL-C: T-C ratios. Selenium supplementation, 96 μg/d for 2 wk, increased serum selenium, GSH-Px activity, and the HDL-C: T-C ratio. The results suggest that a low serum selenium level is associated with a decrease in liver microsomal enzyme activity and serum HDL-C and T-C concentrations. Selenium supplementation in subjects with low serum selenium may favorably influence relations between serum lipoproteins connected with the development of atherosclerotic vascular disease.     

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Sea urchin ovoperoxidase: oocyte-specific member of a heme-dependent peroxidase superfamily that functions in the block to polyspermy

Ovoperoxidase is one of several oocyte-specific proteins that are stored within sea urchin cortical granules, released during the cortical reaction, and incorporated into the newly formed fertilization envelope. Ovoperoxidase plays a particularly important role in this process, crosslinking the envelope into a hardened matrix that is insensitive to biochemical and mechanical challenges and thus providing a permanent block to polyspermy. Here we present the primary structures of two ovoperoxidases as predicted from cDNAs cloned from the sea urchins Strongylocentrotus purpuratus (AF035380) and Lytechinus variegatus (AF035381). We also present a proposed scheme for the post-translational processing of ovoperoxidase based upon comparisons between the cDNA and protein structures and taking into account previously published reports. The sea urchin ovoperoxidase sequences conform to a profile shared by members of a heme-dependent animal peroxidase family, including the mammalian myelo-, lacto-, eosinophil, and thyroid peroxidases. Using in situ RNA hybridizations, we showed that the mRNA of S. purpuratus ovoperoxidase (4 kb) is present exclusively in oocytes, and is turned over rapidly following germinal vesicle breakdown. Taking into account our immunoblot and N-terminal sequencing data along with reports from similar peroxidases, we propose that ovoperoxidases are synthesized in a pre-pro form and proteolytically processed to result in the 70 and 50 kDa forms that are found in the fertilization envelope. The sequence and structural data presented here will facilitate our continuing studies of the biogenesis of cortical granules and the fertilization envelope. Additionally, since ovoperoxidase activities have been reported in a wide range of animals, these cDNAs will be useful in uncovering similar peroxidases used in the fertilization reactions of other metazoan eggs.     

Superoxide regulation of endothelin-converting enzyme

Reactive oxygen species (ROS) act as signaling molecules in the cardiovascular system, regulating cellular proliferation and migration. However, an excess of ROS can damage cells and alter endothelial cell function. We hypothesized that endogenous mechanisms protect the vasculature from excess levels of ROS. We now show that superoxide can inhibit endothelin-converting enzyme activity (ECE) and decrease endothelin-1 synthesis. Superoxide inhibits ECE but hydrogen peroxide and nitric oxide do not. Superoxide inhibits ECE by ejecting zinc from the enzyme, and the addition of exogenous zinc restores enzymatic activity. Superoxide may inhibit other zinc metalloproteinases by a similar mechanism and may thus play an important role in regulating the biology of blood vessels.     

Superoxide dismutase, glutathione peroxidase, and glutathione reductase in sheep organs

The enzyme activities of the superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase (GR) and thiobarbituric acid reactive substances (TBARS) content were measured in tissue extracts of the liver, kidney and lung of sheep in a nonpolluted control area (C), a polluted area pasture (PP) and those from polluted areas but fed in the laboratory with an experimental emission supplement diet (EEF). Compared with the control SOD, activity was significantly increased (1.75 times) only in the liver of the PP group. In the EEF group there was a tendency toward lower activities in all organs. The Cu,Zn-SOD isoenzymes pattern analyzed by isoelectrofocusing was different in the organs of the animals exposed to pollutants when compared with those of the controls. In the liver, two new isoenzymes with pI 5.30 and 5.70 were found in the PP group and an additional isoenzyme with pI 5.10 in the EEF group. The kidney isoenzymes with pl 5.30 and 5.40 were inhibited in the EEF group. In the lung, two new isoenzymes appeared with pl 5.30 and 5.40 in the PP group and two new isoenzymes with pI 6.10 and 6.50 in the EEF group. GSHPx activity was inhibited in the liver and kidney of the sheep exposed to pollutants. GR activity was significantly changed only in the liver. The activity in the PP group was 2.30 and 2.10 times higher than in the C and EEF groups, respectively. TBARS content was increased in the liver and kidney of the EEF group compared with the control.     

Construction of a fusion enzyme exhibiting superoxide dismutase and peroxidase activity

A chimeric gene construct encoding human peroxiredoxin 6 and Mn-superoxide dismutase from Escherichia coli was developed. Conditions for expression of the fusion protein in E. coli cell were optimized. Fusing of the enzymes into a single polypeptide chain with peroxiredoxin 6 at the N-terminus (PSH) did not affect their activities. On the contrary, the chimeric protein with reverse order of enzymes (SPH) was not obtained in a water-soluble active form. The active chimeric protein (PSH) exhibiting both peroxidase and superoxide dismutase activities was prepared and its physicochemical properties were characterized.     

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.     

Engineered selenium-containing glutaredoxin displays strong glutathione peroxidase activity rivaling natural enzyme

Insertion of selenocysteine (Sec) into protein scaffolds provides an opportunity for designing enzymes with improved and unusual catalytic properties. The use of a common thioredoxin fold with a high affinity for glutathione in glutaredoxin (Grx) and glutathione peroxidase (GPx) suggests a possibility of engineering Grx into GPx and vice versa. Here, we engineered a Grx domain of mouse thioredoxin/glutathione reductase (TGR) into a selenium-containing enzyme by substituting the active site cysteine (Cys) with selenocysteine (Sec) in a Cys auxotrophic system. The resulting selenoenzyme displayed an unusually high GPx catalytic activity rivaling that of several native GPxs. The engineered seleno-Grx was characterized by mass spectrometry and kinetic analyses. It showed a typical ping-pong kinetic mechanism, and its catalytic properties were similar to those of naturally occurring GPxs. For example, its second rate constant (k(cat)/K(mH2O2)) was as high as 1.55x10(7) M(-1) min(-1). It appears that glutathione-dependent Grx, GPx and glutathione transferase (GST) evolved from a common thioredoxin-like ancestor to accommodate related glutathione-dependent functions and can be interconverted by targeted Sec insertion.     

Superoxide dismutase and peroxidase: a positive activity stain applicable to polyacrylamide gel electropherograms.

The oxidation of dianisidine, photosensitized by riboflavin, is accelerated by superoxide dismutase. Polyacrylamide gel electropherograms soaked in riboflavin plus dianisidine and subsequently illuminated develop stable brown bands at positions bearing superoxide dismutase activity. This constitutes a new, convenient, and advantageous activity stain for this class of enzymes. Peroxidases are also stained by this procedure due to the photochemical production of H₂O₂. This does not constitute an interference with the specificity of the stain, since peroxidase bands develop more slowly than superoxide dismutase bands and can be further identified through the use of inhibitors or of independent staining for peroxidase. The new, positive activity stain for superoxide dismutases can be applied to crude extracts of cells.     

Superoxide dismutase, glutathione peroxidase and catalase in developing rat brain.

The specific activities of Cu,Zn- and Mn-superoxide dismutases, of glutathione peroxidase and of catalase, the enzymes considered to be specifically involved in the defence of the cell against the partially reduced forms of oxygen, were determined as the function of postnatal age in the early (up to 60 days) period of rat brain development. The enzymes were assayed in the cytoplasmic fraction, in the crude mitochondrial fraction including peroxisomes, and in the mitochondria. The results show that the temporal changes of these enzymes cannot be correlated with each other, thus indicating that they do not concertedly parallel the increasing activity of aerobic brain metabolism during development. Specifically the cytoplasmic fraction shows a gradual increase of the Cu,Zn-superoxide dismutase activity with age, whereas the glutathione peroxidase activity is constant from birth. Furthermore the increase of the mitochondrial Mn-superoxide dismutase as a function of postnatal age is more remarkable than that of the cytoplasmic Cu,Zn-enzyme. Higher activities of catalase in adult animals are detectable only in the subcellular fraction containing peroxisomes, because of the modest catalase activity of the brain. These results indicate independent regulation of the expression of these enzyme activities in the process of brain differentiation and point to a relative deficiency of enzymic protection of the brain differentiation and point to a relative deficiency of enzymic protection of the brain against potentially toxic oxygen derivatives. This situation is similar to the pattern already described in the rat heart and in rat and mouse ascites-tumour cells, at variance with the much more efficient enzyme pattern present in rat hepatocytes.     

A trifunctional enzyme with glutathione S-transferase,glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

The relationship between a novel NAD(P)H oxidase activity of ovoperoxidase and the CN- -resistant respiratory burst that follows fertilization of sea urchin eggs

The extracellular protein coat of the sea urchin egg is cross-linked after fertilization via dityrosyl linkages made by an exocytosed ovoperoxidase. The source of oxidant for this reaction is unknown, but eggs produce H2O2 in amounts equivalent to the cyanide-insensitive O2 uptake "respiratory burst" that follows fertilization. Several possible H2O2-forming oxidase activities, including glucose, xanthine, fatty acyl, and fatty-acyl CoA oxidases, were absent from the egg cortex. However, an NAD(P)H-O2 oxidoreductase activity was found in the egg cortex and was completely accounted for by ovoperoxidase. Homogeneous ovoperoxidase exhibits two types of NAD(P)H oxidase activity. One of these activities is similar to that of horseradish peroxidase and lactoperoxidase; it is dependent on Mn2+ ions and catalytic amounts of phenols, such as 2,4-dichlorophenol and N-acetyltyrosinamide, and is greater than 95% inhibited by 0.1 mM cyanide. A second, novel oxidase activity utilizes Ca2+ and an unidentified, heat-stable, Mr less than 1000 factor that can be extracted by ethanol from egg homogenates. This NADH oxidase activity is only 40% inhibited by 0.1 mM cyanide and is maximally stimulated by 10 mM Ca2+. It has an apparent Km for NADH of 50 microM. The stoichiometry of NADH:O2 consumption is 1.6:1, but approaches 2:1 in the presence of 20 micrograms/ml superoxide dismutase or 200 micrograms/ml catalase. This indicates that complete reduction of O2 to water occurs and that the reaction does not produce H2O2 stoichiometrically. However, nearly complete inhibition of the reaction by higher catalase concentrations suggests that H2O2 is an intermediate. The properties of this novel oxidase activity suggest that it may play such a role in vivo.     

Superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation in the major organs of the aging rats

Glutathione peroxidase (GSh-Px), superoxide dismutase (SOD), catalase (CAT) activities and malon-dialdehyde (MDA) content were determined in heart, liver, kidney and brain of rats. Two different age groups (4 months; 24 months) were considered. GSH-Px and SOD activities decrease significantly for the aged liver and kidney. During aging, the activity of catalase increase in cardiac muscle and, in contrast, decrease in other organs. Lipids peroxidation, expressed in term of MDA formation, decrease in all the organs of the aged rats. The results indicate that: 1) the liver and kidney antioxidative defense decrease with age; 2) the enzymatic activities evolve in a different manner for different enzymes and organs. Furthermore, the results suggest that there is not any correlation between the SOD, CAT, and GSH-Px activities and the peroxidative status of the organs; thus, the age-related increase in the MDA content proposed as a criterion of aging process should be considered with caution.