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.     

Subcellular distribution and activities of superoxide dismutase,catalase, glutathione peroxidase,and glutathione reductase in the southern armyworm,Spodoptera eridania

In mid-fifth-instar larvae of the southern armyworm, Spodoptera eridania, the subcellular distribution of four antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR)—were examined. Two-thirds (4.26 units ·mg protein^?1) of the SOD activity was found in the cytosol, and one-thirds (2.13 units ·mg protein^?1) in the mitochondria. CAT activity was unusually high and not restricted to the microsomal fraction where peroxisomes are usually isolated. The activity was distributed as follows: cytosol (163 units) mitochondria (125 units) and microsomes (119 units). Similar to CAT, the subcellular compartmentalization of both GPOX and GR was unusual. No activity was detected in the cytosol, but in mitochondria and microsomes, GR levels were 5.49 and 3.09 units. Although GPOX activity exhibited 14–16-fold enrichment in mitochondria and microsomes, respectively, over the 850g crude homogenate, the level was negligible (mitochondria = 1.4 × 10^?3 units; microsomes = 1.6 × 10^?3 units), indicating that this enzyme is absent. The unusual distribution of CAT has apparently evolved as an evolutionary answer to the absence of GR from the cytosol, and the lack of GPOX activity.     

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

Activities of enzymes that detoxify superoxide anion and related toxic oxyradicals in Trichoplusia ni

In third-, fourth-, and fifth-instar larvae of the cabbage looper moth, Trichoplusia ni, the activities of the antioxidant enzymes, superoxide dismutase (SOD*), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR) were examined using 850 g supernatants of whole-body homogenates. The enzyme activities, expressed as units mg⁻¹ protein min⁻¹ at 25°C ranged as follows: SOD, 0.67-2.13 units; CAT, 180.5-307.5 units; GPOX, none detectable; and GR, 0.40-1.19 units. There was a similar pattern of changes for SOD and CAT activities with larval ontogeny, but not for GR. The cabbage looper apparently uses SOD and CAT to form a “defensive team” effective against endogenously produced superoxide anion (O₂⪸). Glutathione may serve as an antioxidant for the destruction of any organic/lipid peroxides formed, and GSH oxidized to glutathione disulfide would be recycled by GR. Bioassays against pro-oxidant compounds exogenous sources of (O₂⪸) show high sensitivity of mid-fifth instars to the linear furanocoumarin, 8-methoxypsoralen (xanthotoxin) primarily from photoactivation (320-380 nm), and auto-oxidation of the flavonoid, quercetin. The LC₅₀s are 0.0004 and 0.0045% (w/w) concentration of xanthotoxin and quercetin, respectively. Both pro-oxidants have multiple target sites for lethal action and, in this context, the role of antioxidant enzymes is discussed.     

Pro-oxidant and antioxidant processes in gas gland and other tissues of cod (Gadus morhua)

Partial reduction of molecular oxygen produces reactive oxyradicals, including the superoxide anion radical (O _- ² ) and hydroxyl radical (·OH). The gas gland functions under hyperoxic and acidic conditions and therefore is likely to be subjected to enhanced oxidative stress. Aspects of pro- and antioxidant processes in gas gland were compared with other tissues likely to be subject to differing degrees of oxyradical production, viz. liver (site of chemically-mediated oxyradical production), gills and skeletal muscle. Antioxidant enzyme activities (superoxide dismutase, catalase, selenium-dependent and total glutathione peroxidase) per g wet weight were highest in liver and lowest in muscle. Catalase and glutathione peroxidase activies per g wet weight were higher in gills than in gas gland, whereas the reverse was seen for superoxide dismutase. Cytosolic superoxide dismutase activities per mg protein were two- and nine-fold higher in gas gland than in liver and gills. The pH characteristics of the antioxidant enzymes were generally similar in all the tissues. Glutathione, vitamin E and unsaturated (peroxidizable) lipid levels were generally highest in liver followed by gas gland. Lipid peroxidation (malonaldehyde equivalents) was evident in all tissues except gas gland. Hydrogen peroxide and O _- ² were involved in the NAD(P)H-dependent ferric/EDTA-mediated formation of ·OH (as measured by 2-keto-4-methiolbutyrate oxidation) by mitochondrial and postmitochondrial fractions of gas gland. Tissue maximal potentials for ·OH production paralled superoxide dismutase but not catalase or glutathione peroxidase activities. Overall, the results confirm the presence of effective antioxidant defences in gas gland and support previous workers' contentions of a central role for superoxide dismutase in this process.Abbreviations EDTA di-sodium ethylenediaminetetra-acetic acid - G-6-P glucose-6-phosphate - GPX total glutathione peroxidase - GSH reduced glutathione - GSSG oxidised glutathione - GST glutathion-S-transferase - HPLC high performance liquid chromatography - KMBA 2-keto-4-methiolbutyric acid - MOPS 3-[N-morpholino] propane-sulphonic acid - PMS postmitochondrial supernatant - Se-GPX selenium-dependent glutathion peroxidase - SOD superoxide dismutase - TCA trichloroacetic acid     

Oxygen metabolism in rabbit bone marrow and liver.

Oxygen metabolism has been quantified in rabbit bone marrow and liver. NADPH-Cytochrome $\text{c}$ reductase activity in bone marrow microsomal and cytosol fractions was about 40% of that found in liver. Superoxide anion and peroxide generation were found to be present in both liver and bone marrow. Catalase and superoxide dismutase activity were measured in liver and in marrow preparations free of erythrocytes; while liver catalase activity was approximately twice that of bone marrow, very low superoxide dismutase activity was observed in erythrocyte free bone marrow homogenates.     

Replacement of media in cell culture alters oxygen toxicity: Possible role of lipid aldehydes and glutathione transferases in oxygen toxicity

Replacement of media in cell cultures during exposure to hyperoxia was found to alter oxygen toxicity. Following 100 hr of exposure to 95% or 80% O₂, the surviving fraction (SF) of Chinese hamster fibroblasts, as assayed by clonogenicity, was less than 1 × 10^?3 when the culture media was replaced only at the onset of the O₂ exposure. Media replacement every 24 hr throughout the hyperoxic exposure resulted in SFs of 1.7 × 10^?1 (95% O₂) and 1.9 × 10^?1 (80% O₂) at 95 hr. Cellular resistance to and metabolism of 4-hydroxy-2-nonenal (4HNE), a cytotoxic byproduct of lipid peroxidation, was examined in cells 24 hr following exposure to 80% O₂ for 144 hr with media replacement. These O₂-exposed cells were resistant to 4HNE, requiring 2.6 times as long in 80 μM 4HNE to reach 30% survival as compared to density-matched normoxia control. Furthermore, during 40 and 60 min of exposure to 4HNE, the O₂-preexposed cells metabolized greater quantities of 4HNE (fmole/cell) relative to control. The activity of glutathione S-transferase (GST), an enzyme believed to be involved with the detoxification of 4HNE, was significantly increased in the O₂-preexposed cells compared with controls. Catalase activity was significantly increased, but no change was found in total glutathione content, glutathione peroxidase, manganese superoxide dismutase, and copper-zinc superoxide dismutase activities at the time of 4HNE treatment in the O₂-preexposed cells relative to density-matched control. The results demonstrate that in vitro tolerance to the cytotoxic effects of hyperoxia can be achieved through media replacement during O₂ exposure. Tolerance to oxygen toxicity conferred resistance to the cytotoxic effects of 4HNE, possibly through GST-catalyzed detoxification. These results provide further support for the hypothesis that toxic aldehydic byproducts of lipid peroxidation contribute to hyperoxic injury.     

The intracellular localization and properties of carnitine acetyltransferase from ram spermatozoa

Although spermatozoa possess a very active carnitine acetyltransferase, there is no satisfactory explanation for such a high activity. In order to help elucidate possible roles for carnitine acetyltransferase in spermatozoa, we examined the intracellular location and properties of carnitine acetyltransferase from ejaculated ram spermatozoa. The spermatozoa were disrupted by hypotonic treatment with 10 mm phosphate buffer (pH 7.4), followed by mild sonication. The resulting homogenate was separated by sucrose step-gradient centrifugation into soluble, plasma membrane, acrosomal membrane, and mitochondrial fractions. These fractions were characterized by electron microscopy and marker enzyme assays. The particulate fractions were made soluble by treatment with 0.1% deoxycholate and then were assayed for carnitine acetyltransferase activity. Carnitine acetyltransferase activity was found exclusively in the mitochondrial fraction with a specific activity of 0.151 μmol CoASH · min^?1 · mg^?1. The apparent K_m values for acetyl-CoA and l-carnitine were 1.1 × 10^?5 and 1.3 × 10^?4m respectively.     

Catalase modifies yeast Saccharomyces cerevisiae response towards S-nitrosoglutathione-induced stress

Abstract

Nitric oxide is known to be a messenger in animals and plants. Catalase may regulate the concentration of intracellular ^?NO. In this study, yeast Saccharomyces cerevisiae cells were treated with 1–20 mM S-nitrosoglutathione (GSNO), a nitric oxide donor, which decreased yeast survival in a concentration-dependent manner. In the wild-type strain (YPH250), 20 mM GSNO reduced survival by 32%. The strain defective in peroxisomal catalase behaved like the wild-type strain, while a mutant defective in cytosolic catalase showed 10% lower survival. Surprisingly, survival of the double catalase mutant was significantly higher than that of the other strains used. Incubation of yeast with GSNO increased the activities of both superoxide dismutase (SOD) and catalase. Pre-incubation with cycloheximide prevented the activation of catalase, but not SOD. The concentrations of oxidized glutathione increased in the wild-type strain, as well as in the mutants defective in peroxisomal catalase and an acatalasaemic strain; it failed to do this in the mutant defective in cytosolic catalase. The activity of aconitase was reduced after GSNO treatment in all strains studied, except for the mutant defective in peroxisomal catalase. The content of protein carbonyls and activities of glutathione reductase and S-nitrosoglutathione reductase were unchanged following GSNO treatment. The increase in catalase activity due to incubation with GSNO was not found in a strain defective in Yap1p, a master regulator of yeast adaptive response to oxidative stress. The obtained data demonstrate that exposure of yeast cells to the ^?NO-donor S-nitrosoglutathione induced mild oxidative/nitrosative stress and Yap1p may co-ordinate the up-regulation of antioxidant enzymes under these conditions.     

Some kinetic properties of Mucor rouxii phosphofructokinase. Effect of cyclic adenosine 3', 5'-monophosphate.

Intact and pure parenchymal and nonparenchymal cells were isolated from rat liver. The activities of Superoxide dismutase in these cell types were determined by two different methods. With both methods the specific activity of this enzyme is 1.5 times higher in parenchymal than in nonparenchymal liver cells. It can be calculated that about 7% of the total rat liver Superoxide dismutase activity is localized in the nonparenchymal liver cells. Electrophoresis on polyacrylamide gels indicates that the isolated parenchymal cells contain both cytosolic and mitochondrial isoenzymes, whereas with nonparenchymal cells only the cytosolic enzyme could be detected. The mitochondrial band observed in isolated parenchymal cells is absent in the original total liver homogenate. This isoenzyme seems to be activated during the parenchymal cell isolation procedure. Isoelectrofocusing indicates that the cytosolic Superoxide dismutase consists in four different isoelectric forms in both parenchymal and nonparenchymal cells. With the mitochondrial isoenzyme two bands are obtained. The possibility that O₂^? is an important intermediate in H₂O₂ formation in nonparenchymal liver cells is discussed. In this respect, Superoxide dismutase might not only protect the cell against a toxic reagent as O₂^t-, but might also help to regulate the level of the important antimicrobial agent, H₂O₂.     

Action of antioxidant enzymes and cytochrome P-450 monooxygenases in the cabbage looper in response to plant phototoxins

Effects of two biosynthetically distinct plant phototoxins—xanthototoxin, a furanocoumarin, and harmine, a β-carboline alkaloid, which are known to produce toxic oxygen species—on the food utilization efficiencies and enzymatic detoxification systems of the polyphagous cabbage looper. Trichoplusia ni (Lepidoptera: Noctuidae), were studied. Newly molted fifth-instar larvae were allowed 36 h to ingest diets containing these two phototoxins at 0.15% wet weight in the presence of near ultraviolet (UVA). The growth and development of the larvae, as well as the corresponding activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR) and the detoxification enzyme cytochrome P-450, were measured. Xanthotoxin reduced rates of relative growth and consumption and efficiencies of conversion of ingested and digested food to biomass. Harmine reduced rates of growth and consumption without affecting efficiencies of conversion. Specific activities of SOD, CAT, GPOX, and GR of whole-body homogenates in the absence of compounds were 0.88 units, 153μmol H₂O₂ decomposed·mg protein^?1·min—¹, 38.3 nmol NADPH oxidized·mg protein^?1·min^?1, and 0.56 nmol NADPH oxidized·mg protein^?1·min^?1, respectively. SOD activity was induced 2.9-fold and 3.8-fold by dietary xanthotoxin and harmine, respectively. CAT and GPOX activities were induced 1.2-fold by harmine only, and GR activity was not changed by either chemical. The P-450 activity toward xanthotoxin in the microsomal fraction of midguts was low (0.15 nmol xanthotoxin metabolized·mg protein^?1·min^?1) and was not induced by xanthotoxin ingestion. These studies indicate that P-450 and antioxidant enzyme systems may be independent but consequential, the induction of antioxidant enzymes by phototoxins occurring when low P-450 activity toward the phototoxin permits the accumulation of oxidative stress from unmetabolized phototoxin, which in turn induces antioxidant enzymes.     

Acute Hexachlorocyclohexane-Induced Oxidative Stress in Rat Cerebral Hemisphere

Hexachlorocyclohexane (HCH) is reported to induce oxidative stress in liver and testis of rat. With an objective to examine its effect on brain tissue acute toxicity of HCH (10 and 20 mg/kg body wt, i.p.) on the antioxidant defense system of cerebral hemisphere of rat was evaluated. Lipid peroxidation (LPX) was elevated after 24 h in the crude homogenate and sub-cellular fractions (nuclear and mitochondrial) except the microsomal fraction in which LPX was induced after 6 h and remained elevated till 24 h. The pesticide elicited decrease in the activities of cytosolic total, CN^–-sensitive (not at 24 h) and CN-resistant superoxide dismutases; total, Se-dependent and Se-independent glutathione peroxidases; and catalase throughout the measurement period. In contrast, glutathione reductase activity was elevated till 24 h after a fall at 6 h of pesticide exposure. Cerebral contents of glutathione and ascorbic acid were decreased in response to HCH. The results suggest the possible involvement of reactive oxygen species in the mechanism of HCH-induced neurotoxicity in rat.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves

We investigated the relationship between H₂O₂ metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H₂O₂ content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O₂^·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.     

The enzymatic reaction of chlorotrifluoroethylene with glutathione

The nephrotoxic gas chlorotrifluoroethylene is a substrate for glutathione S-transferase activity in rat hepatic cytosolic and microsomal fractions. The rates of reaction, determined by measuring glutathione disappearance, were 5–15 or 35–70 nmol/min/mg of cytosolic or microsomal protein, respectively. Glutathione disappearance was completely abolished by heat-denaturing the subcellular fractions. A product of the cytosolcatalyzed reaction between chlorotrifluoroethylene and glutathione was isolated and shown by amino acid analysis and ¹H- and ¹⁹F-NMR to be S-(2-chloro-1,1,2-trifluoroethyl)glutathione. This appears to be the first demonstration of a glutathione S-transferase-catalyzed addition reaction with a halogenated olefin, and this reaction may be of toxicological significance.     

Taurine Stimulation of Isolated Hamster Brain Na+, K+-ATPase: Activation Kinetics and Chemical Specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na⁺, K⁺-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na⁺, K⁺-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na⁺, K⁺-ATPase by taurine. Normal whole brain homogenate Na⁺, K⁺-ATPase activity is 5.1 ± 0.4 (4) μmol P_i± h^?1± mg^?1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na⁺, K⁺-ATPase activity of 204.6 ± 5.8 (4) mol P_i± h^?1± mg^?1 Lowry protein. Taurine activates the Na⁺, K⁺-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K_1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na⁺, K⁺-ATPase. Its action is mediated by a membrane-bound protein.     

Increase in glutathione peroxidase activity in erythrocytes from trisomy 21 subjects.

Glutathione peroxidase activity has been measured in erythrocytes from normal subjects and from trisomy 21 patients. The latter cases show about 50 % increase of this enzyme similar to the increase observed for superoxide dismutase (erythrocuprein) suggesting either localisation of the gene for glutathione peroxidase on chromosome 21 (as is the case for erythrocuprein) or regulation of this enzyme by intracellular levels of O₂^$\text{?}$, H₂O₂ or superoxide dismutase.     

Occurrence and some properties of carbonic anhydrases from legume root nodules

Carbonic anhydrase activity (hydration of CO₂ was found in homogenates of leaves (116–500 units.mg^?1 protein) and root nodules (27–255 units.mg^?1 protein) from 8 legume genera inoculated in each case with a host specific Rhizobium. No enzyme, or only trace amounts (2–7 units.mg^?1 protein), were detected in root extracts, The enzymatic activity was inhibited in all cases by azide and acetazolamide. The sizes of nodule and leaf carbonic anhydrases, estimated by gel filtration of partially purified preparations from Phaseolus vulgaris, were around 45 000 and 205 000 respectively. These enzymes also differed in sensitivity to inhibitors. More than 99% of the activity present in Vicia faba nodules was recovered as a soluble enzyme and only a trace was located in the isolated bacteroids.     

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.