Antioxidant Enzymes of Larvae of the Cabbage Looper Moth,Trzchoplusza Ni: Subcellular Distribution and Activities of Superoxide Dismutase,Catalase and Glutathione Reductase

In the mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, the subcellular distribution of total superoxide dismutase was as follows: 3.05 units (70.0%), 0.97 units (22.3%), and 0.33 units (7.6%) mg^?1 protein in the mitochondrial, cytosolic and nuclear fractions, respectively. No superoxide dismutase activity was detected in the microsomal fraction. Catalase activity was unusually high and as follows: 283.4 units (47.3%), 150.1 units (25.1%). 142.3 units (23.8%), and 22.9 units (3.8%) mg^?1 protein in the mitochondrial, cytosolic, microsomal (containing peroxisomes), and nuclear fractions. No glutathione peroxidase activity was found, but appreciable glutathione reductase activity was detected with broad subcellular distribution as follows: 3.86 units (36.1%), 3.68 units (34.0%). 2.46 units (23.0%). and 0.70 units (6.5%) mg^?1 protein in the nuclear, mitochondrial, and cytosolic fractions, respectively. The unusually wide intracellular distribution of catalase in this phytophagous insect is apparently an evolutionary adaptation to the absence of glutathione peroxidase; hence, lack of a glutathione peroxidase-glutathione reductase role in alleviating stress from lipid peroxidation. Catalase working sequentially to superoxide dismutase, may nearly completely prevent the formation of the lipid peroxidizing OH radical from all intracellular compartments by the destruction of H₂O₂ which together with O^?₂ is a precursor of OH.     

Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

Phosphatidylinositol kinase,phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ-³²P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP₂ in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.     

Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects

Iron and copper ions, in their unbound form, may lead to the generation of reactive oxygen species via Haber–Weiss and/or Fenton reactions. In addition, it has been shown that copper ions can irreversibly and non-specifically bind to thiol groups in proteins. This non-specific binding property has not been fully addressed for iron ions. Thus, the present study compares both the pro-oxidant and the non-specific binding properties of Fe³⁺ and Cu²⁺, using rat liver cytosol and microsomes as biological systems. Our data show that, in the absence of proteins, Cu²⁺/ascorbate elicited more oxygen consumption than Fe³⁺/ascorbate under identical conditions. Presence of cytosolic and microsomal protein, however, differentially altered oxygen consumption patterns. In addition, Cu²⁺/ascorbate increased microsomal lipid peroxidation and decreased cytosolic and microsomal content of thiol groups more efficiently than Fe³⁺/ascorbate. Finally, Fe³⁺/ascorbate and Cu²⁺/ascorbate inhibited in different ways cytosolic and microsomal glutathione S-transferase (GST) activities, which are differentially sensitive to oxidants. Moreover, in the absence of ascorbate, only Cu²⁺ decreased the content of cytosolic and microsomal thiol groups and inhibited cytosolic and microsomal GST activities. Catechin partially prevented the damage to thiol groups elicited by Fe³⁺/ascorbate and Cu²⁺/ascorbate but not by Cu²⁺ alone. N-Acetylcysteine completely prevented the damage elicited by Cu²⁺/ascorbate, Fe³⁺/ascorbate and Cu²⁺ alone. N-Acetylcysteine also completely reversed the damage to thiol groups elicited by Fe³⁺/ascorbate, partially reversed that of Cu²⁺/ascorbate but failed to reverse the damage promoted by Cu²⁺ alone. Our data are discussed in terms to the potential damage that the accumulation of iron and copper ions can promote in biological systems.     

Calcium-activated proteolytic activity in rat liver mitochondria

Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca²⁺-activated proteolytic activity using ¹⁴C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca²⁺-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca²⁺, and the other by 750 μM Ca²⁺. Rat liver cytosol contained only a high Ca²⁺-requiring protease peak. This is the first demonstration of Ca²⁺-activated proteases in mitochondria.     

UPD-glucuronosyltransferase,epoxide hydrolase and gultathione S-transferase

The activities of UDPglucuronosyltransferase, microsomal epoxide hydrolas and cytosolic glutathione S-transferase were measured in the liver of spontaneously (db/db and ob/ob) or streptozotocin-induced diabetic mice. An important (2–3-fold) increase of most phase II activities was observed instreptozotocin-treated animals, whereas sligher changes were detected in spontaneously diabetic animals. The latter exhibit physico-chemical modifications of liver microsomal membranes, as shown by the temperature-induced variations of epoxide hydrolase activity.     

Detection and identification of sulfhydryl conjugates of p-benzoquinone in microsomal incubations of benzene and phenol

The glutathione and cysteine conjugates of p-benzoquinone are detected and conclusively identified in microsomal incubations of benzene and phenol using liquid chromatography/electrochemistry (LCEC). Identification of the compounds is based on retention time, electrochemical behavior and acid hydrolysis. The fact that both of these compounds can be detected easily in a benzene incubation provides further evidence that p-benzoquinone or the corresponding semiquinone is a product of benzene metabolism in vivo. The conjugation of p-benzoquinone with glutathione is predominantly a nonenzymatic process. This is illustrated by the fact that the addition of cytosolic glutathione-S-transferases do not significantly increase the amount of glutathione conjugate produced in a phenol incubation containing glutathione.The kinetic constants for phenol metabolism to hydroquinone by microsomal protein are calculated. As suspected, the rate of metabolism of phenol is significantly higher than the rate of benzene metabolism. The V_max for phenol metabolism was calculated to be 7.1 nmol/min/mg protein and the K_M was found to be 0.38 mM.The further oxidation of hydroquinone to p-benzoquinone appears to be primarily an enzymatic process. Incubations of just hydroquinone with glutathione at 37°C produced only a small amount of the glutathione conjugate. The addition of cytosolic protein increases the amount of p-benzoquinone produced about 10-fold. This could be due to the peroxidases found in that medium. The addition of microsomal protein and NADPH increases the amount of glutathione conjugate produced to over 100-fold of that produced nonenzymatically. This indicates that a microsomal enzyme is responsible for the oxidation of hydroquinone to p-benzoquinone in vitro and the subsequent covalent binding to macromolecules.     

Cytosolic and microsomal glutathione-S-transferases from bovine filarial worms Setaria cervi

The work presented here deals with the status of glutathione-S-transferase (GST; E.C. 2.5.1.18), the major enzyme of the phase II detoxification pathway, in bovine filarial worms Setaria cervi. GST activity was determined in various subcellular fractions of bovine filarial worms S. cervi (Bubalus bubalis Linn.) and was found to be mainly associated with cytosolic and microsomal fractions. The respective specific activities of the enzyme from cytosolic and microsomal fractions of S. cervi females were determined to be 0.122 +/- 0.024 and 0.010 +/- 0.0052 micromol/min/mg protein, respectively. Cytosolic enzyme was found to possess optimal activity between pH 6.5 and 7.5, whereas the microsomal enzyme showed a broad pH optima, centered at pH 6.0. Kinetic studies on the cytosolic and microsomal forms of the enzyme revealed significant differences between them, thereby indicating that microsomal GST from S. cervi is quite distinct to the cytosolic protein catalyzing the same reaction.     

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems

A pathway for the synthesis of dimethyl seledine from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30 000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO₂ (93% inhibition by 10^?6 M arsenite in the presence of a 20 000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.     

Properties of cytosolic components activating rat hepatic 5'' [corrected]-deiodination in the presence of NADPH.

The effects of cytosol, NADPH and reduced glutathione (GSH) on the activity of 5'-deiodinase were studied by using washed hepatic microsomes from normal fed rats. Cytosol alone had little stimulatory effect on the activation of microsomal 5'-deiodinase. NADPH had no stimulatory effect on the microsomal 5'-deiodinase unless cytosol was added. 5'-deiodinase activity was greatly enhanced by the simultaneous addition of NADPH and cytosol (P less than 0.001); this was significantly higher than that with either NADPH or cytosol alone (P less than 0.001). GSH was active in stimulating the enzyme activity in the absence of cytosol, but the activity of 5'-deiodinase with 62 microM-NADPH in the presence of cytosol was significantly higher than that with 250 microM-GSH in the presence of the same concentration of cytosol (P less than 0.001). The properties of the cytosolic components essential for the NADPH-dependent activation of microsomal 5'-deiodinase independent of a glutathione/glutathione reductase system were further assessed using Sephadex G-50 column chromatography to yield three cytosolic fractions (A, B and C), wherein A represents pooled fractions near the void volume, B pooled fractions of intermediate Mr (approx. 13 000), and C of low Mr (approx. 300) containing glutathione. In the presence of NADPH (1 mM), the 5'-deiodination rate by hepatic washed microsomes is greatly increased if both A and B are added and is a function of the concentrations of A, B, washed microsomes and NADPH. A is heat-labile, whereas B is heat-stable and non-dialysable. These observations provide the first evidence of an NADPH-dependent cytosolic reductase system not involving glutathione which stimulates microsomal 5'-deiodinase of normal rat liver. The present data are consistent with a deiodination mechanism involving mediation by a reductase (other than glutathione reductase) in fraction A of an NADPH-dependent reduction of a hydrogen acceptor in fraction B, followed by reduction of oxidized microsomal deiodinase by the reduced acceptor (component in fraction B).     

25-Hydroxylation of 1-α-hydroxyvitamin D-3 in rat and human liver

1α-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1α-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent K_m value of 2·10^?5 M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100°C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1α-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1α-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.     

Interaction of 1,4-benzoquinone and 2,4-dichlorophenoxyacetic acid with microsomal glutathione transferase from rat liver

Glutathione transferase (GST) was purified from the microsomes of rat liver by glutathione affinity chromatography. The interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1,4-benzoquinone with microsomal GST was investigated and compared with cytosolic GST. The kinetic inhibition pattern of 1,4-benzoquinone towards microsomal GST was found to be different from that towards cytosolic GST. Microsomal GST purified by affinity chromatography was inhibited by 2,4-D in a non dose-dependent manner, while the crude microsomal GST was inhibited in a dose-dependent manner. This difference was shown to be induced by a reaction on the affinity column, and not by Triton X-100 (also shown to be a GST inhibitor), glutathione, or the elution buffer 0.2% Triton X-100 and 5 mM glutathione in 50 mM Tris-HCl, pH 9.6. The binding of microsomal GST to the affinity matrix caused a partial inactivation of the active site for 2,4-D interaction. The results show that the properties of soluble GST enzymes may not be extrapolated to the microsomal ones.     

Antioxidant Enzymes of Larvae of the Cabbage Looper Moth, Trzchoplusza Ni: Subcellular Distribution and Activities of Superoxide Dismutase, Catalase and Glutathione Reductase

In the mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, the subcellular distribution of total superoxide dismutase was as follows: 3.05 units (70.0%), 0.97 units (22.3%), and 0.33 units (7.6%) mg^-1 protein in the mitochondrial, cytosolic and nuclear fractions, respectively. No superoxide dismutase activity was detected in the microsomal fraction. Catalase activity was unusually high and as follows: 283.4 units (47.3%), 150.1 units (25.1%). 142.3 units (23.8%), and 22.9 units (3.8%) mg^-1 protein in the mitochondrial, cytosolic, microsomal (containing peroxisomes), and nuclear fractions. No glutathione peroxidase activity was found, but appreciable glutathione reductase activity was detected with broad subcellular distribution as follows: 3.86 units (36.1%), 3.68 units (34.0%). 2.46 units (23.0%). and 0.70 units (6.5%) mg^-1 protein in the nuclear, mitochondrial, and cytosolic fractions, respectively. The unusually wide intracellular distribution of catalase in this phytophagous insect is apparently an evolutionary adaptation to the absence of glutathione peroxidase; hence, lack of a glutathione peroxidase-glutathione reductase role in alleviating stress from lipid peroxidation. Catalase working sequentially to superoxide dismutase, may nearly completely prevent the formation of the lipid peroxidizing OH radical from all intracellular compartments by the destruction of H₂O₂ which together with O^-₂ is a precursor of OH.     

Detection of NAD: arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

¹²⁵I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. And Romig, W.R. (1979) J. Biol. Chem. 254, 5894-5854) was utilized to identify NAD: arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and ¹²⁵I-labelled guanyltyramine as ADPribose acceptor, formation of ADPribosyl ¹²⁵-I-guanyltyramine was linear with time and enzyme concentration. The product was distinguishable on both thin-layer and high-performance liquid chromatography from that formed by cholerangen. Using ¹²⁵I-guanyltyramine, ADPribosyltransferase acitivity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractioned, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free ¹²⁵I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl ¹²⁵I-guanyltyramine and other metabolites of ¹²⁵I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl ¹²⁵I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, ¹²⁵I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Detection of NAD: arginine ADPribosyltransferases in animal tissues using I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

¹²⁵I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. And Romig, W.R. (1979) J. Biol. Chem. 254, 5894-5854) was utilized to identify NAD: arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and ¹²⁵I-labelled guanyltyramine as ADPribose acceptor, formation of ADPribosyl ¹²⁵-I-guanyltyramine was linear with time and enzyme concentration. The product was distinguishable on both thin-layer and high-performance liquid chromatography from that formed by cholerangen. Using ¹²⁵I-guanyltyramine, ADPribosyltransferase acitivity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractioned, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free ¹²⁵I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl ¹²⁵I-guanyltyramine and other metabolites of ¹²⁵I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl ¹²⁵I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, ¹²⁵I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Huanglongbing pathogen Candidatus Liberibacter asiaticus exploits the energy metabolism and host defence responses of its vector Diaphorina citri

Asian citrus psyllid Diaphorina citri is the vector of the citrus Huanglongbing (HLB) associated bacterial agent ‘Candidatus Liberibacter asiaticus’ (CLas). The molecular interactions between CLas and D. citri remain unclear. In the present study, protein profiles of mitochondrial, microsomal and cytosolic fractions from uninfected and CLas‐infected adult D. citri are investigated using two‐dimensional gel electrophoresis. The comparative analysis reveals a total of 18, 24 and 20 protein spots that are unique or differentially expressed in mitochondrial, microsomal and cytosolic proteins fractions respectively. These proteins are successfully identified by mass spectrometry. Among the 62 identified proteins, 30 are up‐regulated, whereas 32 are down‐regulated. These proteins include important components in energy metabolism such as ATP synthase, ATPase, ATP/ADP carrier protein, etc.; host stress responses such as heat shock proteins; host detoxification processes (i.e., cytochrome P450 and glutathione S‐transferase); and the cytoskeleton (such as actin, tubulin, myosin and tropomyosin). These data suggest that, after CLas infection, several proteins of D. citri, especially energy metabolism and protein biosynthesis, are altered, and extensive host defence responses are induced. In conclusion, the present study reports proteomic information that is helpful in understanding the vector–pathogen relationship between CLas and D. citri, and could be used to identify potential targets for limiting the spread of CLas, as well as to provide new insights into HLB management.     

Functional and structural membrane topology of rat liver microsomal glutathione transferase

The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11–35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2′-dithiobis(5-nitrobenzoic acid))) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42–154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.     

Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells

We recently reported that nitrogen dioxide (NO₂), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO₂ resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO₂ exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO₂ for 1–24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p < 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO₂-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar¹ Ile⁸] Ang II. Exposure of 5 ppm NO₂ for 1–24 hr provoked rapid increases in [³H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO₂ increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.     

Induction of cytosolic and microsomal epoxide hydrolases in mouse liver by peroxisome proliferators, with special emphasis on structural analogues of 2-ethylhexanoic acid

Using dietary administration, mice were exposed to eight substances known to cause peroxisome proliferation (i.e. clofibrate clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, ICI-55.897, S-8527 and Wy-14.643) or the related substance p-chlorophenoxyacetic acid (group A). Other animals received di(2-ethylhexyl)phthalate, mono(2-ethylhexyl)phthalate, 2-ethylhexanoic acid, or one of 12 other metabolically and/or structurally related compounds (group B). The effects of these treatments on liver cytosolic and microsomal epoxide hydrolases, microsomal cytochrome P-450, cytosolic glutathione transferase activity, the liver-somatic index and the protein contents of the microsomal and cytosolic fractions prepared from liver were subsequently monitored. In general, peroxisome proliferation was accompanied by increases in cytosolic epoxide hydrolase activity. Many peroxisome proliferators also caused increases in microsomal epoxide hydrolase activity, although the correlation was poorer in this case. Immunochemical quantitation by radial immunodiffusion demonstrated that the increases observed in both of these enzyme activities reflected equivalent increases in enzyme protein, i.e. that induction truly occurred. Induction of total microsomal cytochrome P-450 was obtained after dietary exposure to clofibrate, clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, Wy-14.643, di(2-ethylhexyl)phthalate and di(2-ethylhexyl)phosphate. The most pronounced effects on cytosolic glutathione transferase activity were the decreases obtained after treatment with clofibrate, clofibric acid and Wy-14.643. Our results, together with those reported by others, suggest that the processes of peroxisome proliferation and induction of cytosolic epoxide hydrolase are intimately related. One possible explanation for this is presented.