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PubMed1999

Acetylcholine-induced arteriolar dilation is reduced in streptozotocin-induced diabetic rats with motor nerve dysfunction.

1. Diabetes mellitus produces marked abnormalities in motor nerve conduction, but the mechanism is not clear. In the present study we hypothesized that in the streptozotocin (STZ)-induced diabetic rat impaired vasodilator function is associated with reduced endoneural blood flow (EBF) which may contribute to nerve dysfunction. 2. We examined whether diabetes-induced reductions in sciatic nerve conduction velocity and EBF were associated with impaired endothelium-dependent dilation in adjacent arterioles. We measured motor nerve conduction velocity (MNCV) in the sciatic nerve using a non-invasive procedure, and sciatic nerve nutritive blood flow using microelectrode polarography and hydrogen clearance. In vitro videomicroscopy was used to quantify arteriolar diameter responses to dilator agonists in arterioles overlying the sciatic nerve. 3. MNCV and EBF in 4-week-STZ-induced diabetic rats were decreased by 22% and 49% respectively. Arterioles were constricted with U46619 and dilation to acetylcholine (ACh), aprikalim, or sodium nitroprusside (SNP) examined. All agonists elicited dose-dependent dilation in control and diabetic rats, although ACh-induced dilation was significantly reduced in diabetic rats. Treating vessels from normal or diabetic rats with indomethacin (INDO) alone did not significantly affect ACh-induced relaxation. However, ACh-induced vasodilation was significantly reduced by treatment with KCl or Nomega-nitro-L-arginine (LNNA) alone. Combining LNNA and KCl further reduced ACh-induced dilation in these vessels. 4. Diabetes causes vasodilator dysfunction in a microvascular bed that provides circulation to the sciatic nerve. These studies imply that ACh-induced dilation in these vessels is mediated by multiple mechanisms that may include the endothelial-dependent production of nitric oxide and endothelial-derived hyperpolarizing factor. This impaired vascular response is associated with neural dysfunction.

Acetylcholine↗

PubMed2002

Photochemistry of 2-chloropyrimidine.

The photochemistry of 2-chloropyrimidine (ClPy) was investigated by means of nanosecond laser flash photolysis, HPLC, mass spectrometry, polarography and absorption spectroscopy. Two major products were formed on low-intensity UV irradiation (lambda = 254 nm) of ClPy in anaerobic aqueous solution: 2-hydroxypyrimidine (quantum yield approximately 0.01) and a compound identified as 2-chloro-4,2'-bipyrimidine (quantum yield approximately 0.005). Only the former of these products was obtained under aerobic conditions. Investigation by nanosecond flash photolysis revealed the occurrence of efficient intersystem crossing to the ClPy triplet state; the deactivation processes from this state were determined. Photosensitised generation of the ClPy triplet state showed that the triplet is involved in the formation of the bipyrimidine. A reaction scheme is proposed comprising two reaction channels: heterolytic rupture of the C-Cl bond in the excited singlet state of ClPy leading to formation of 2-hydroxypyrimidine, and homolytic C-Cl rupture in the triplet state with creation of pyrimidinyl radicals, which react with excess ClPy to give 2-chloro-4,2'-bipyrimidine.

Journal Article↗

PubMed2003

Hydrogen-bond forming ionophore for highly efficient transport of phosphate anions across the nitrobenzene-water interface.

Thiourea-based hydrogen-bond forming ionophore 2, alpha,alpha'-bis(N'-p-nitrophenylthioureylene)-m-xylene, is synthesized and investigated by using ion transfer polarography for the facilitated transfers of H2PO4-, HPO42- and Cl- across the nitrobenzene-water interface. Bis-thiourea 2 has a significant ability to assist H2PO4- transfer across the interface whereas its counterpart, N-(p-nitrophenyl)-N'-propylthiourea (ionophore 3), cannot facilitate the transfer of this hydrophilic anion. The H2PO4- transfer assisted by 2 is based on the formation of a 2:1 complex between H2PO4- and ionophore, and the transfer reaction is more stable by over -12 kJ mol(-1) than the case of 3. The stabilization of the H2PO4- transfer for 2 is even stronger by -11 kJ mol(-1) than that for bis-thiourea 1, 2,7-di-t-butyl-4,5-bis(N'-butylthioureylene)-9,9-dimethylxanthene, which forms a 1:1 complex through the formation of four hydrogen bonds. Bis-thiourea 2 is also able to facilitate transfers of HPO42- and Cl- by the formation of 1:1 complex. As compared to bis-thiourea 1, HPO42- transfer by 2 is significantly stabilized by -27 to -31 kJ mol(-1) while the stabilization of the Cl- transfer is relatively moderate (-6.1 kJ mol(-1)). These binding properties of bis-thiourea 2 are discussed for the design of phosphate-selective ionophores for use in two-phase distribution systems such as ion-selective electrodes.

Journal Article↗

PubMed1989

A role of metallothionein in zinc regulation after oestradiol induction of vitellogenin synthesis in rainbow trout, Salmo gairdneri.

The regulation of metallothionein (MT) biosynthesis in rainbow-trout liver was studied after a single intraperitoneal injection of oestradiol-17 beta. Sampling was performed after 2, 7, 14, 21, 28 and 35 days. Following induction of vitellogenin synthesis in the liver, liver somatic index (LSI) rose from 1.25 to 2.00 in 14 days. Associated with the increase in LSI was an elevation of hepatic vitellogenin mRNA and zinc concentrations. The vitellogenin mRNA concentrations peaked at 7 days after treatment. The zinc concentrations increased to a peak at day 14. MT was analysed by using differential pulse polarography and a rainbow-trout MT RNA probe. The MT mRNA concentrations rose after 14 days and remained elevated at 21 and 28 days. The MT concentrations increased after 14 days and remained elevated throughout the experimental period. The concentrations of MT-bound zinc increased in association with the elevation in MT concentrations in the oestradiol-treated rainbow trout. These findings indicate that MT is involved in the regulation of zinc during the period of vitellogenin induction and that MT may function by maintaining the pool of available zinc at an appropriate concentration.

Animals↗

PubMed2002

Effects of the protein kinase C beta inhibitor LY333531 on neural and vascular function in rats with streptozotocin-induced diabetes.

Elevated protein kinase C activity has been linked to the vascular and neural complications of diabetes. The aim of the present study was to examine the involvement of the beta-isoform of protein kinase C in abnormalities of neuronal function, neural tissue perfusion and endothelium-dependent vasodilation in diabetes, by treatment with the selective inhibitor LY333531 (10 mg.kg(-1).day(-1)). Diabetes was induced in rats by streptozotocin; the duration of diabetes was 8 weeks. Nerve conduction velocity was monitored, and responses to noxious mechanical and thermal stimuli were estimated by the Randall-Sellito and Hargreaves tests respectively. Sciatic nerve and superior cervical ganglion blood flow were measured by microelectrode polarography and hydrogen clearance. Vascular responses were examined using the in vitro mesenteric bed preparation. An 8-week period of diabetes caused deficits in sciatic motor (20%) and saphenous nerve sensory (16%) conduction velocity, which were reversed by LY333531. Diabetic rats had mechanical and thermal hyperalgesia. LY333531 treatment did not affect mechanical thresholds, but corrected thermal hyperalgesia. Sciatic nerve and superior cervical ganglion blood flow were both reduced by 50% by diabetes; this was almost completely corrected by 2 weeks of LY333531 treatment. Diabetes caused a 32% reduction in vasodilation of the mesenteric vascular bed in response to acetylcholine, mediated by nitric oxide and endothelium-derived hyperpolarizing factor. When the former was abolished during nitric oxide synthase inhibition, an 80% diabetic deficit in the remaining relaxation was noted. LY333531 treatment attenuated the development of these defects by 64% and 53% respectively. Thus protein kinase C beta contributes to the neural and vascular complications of experimental diabetes; LY333531 is a candidate for further study in clinical trials of diabetic neuropathy and vasculopathy.

Animals↗

PubMed2001

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed.

Amino Acid Sequence↗

PubMed1999

DNA interactions of cisplatin tethered to the DNA minor groove binder distamycin.

Modifications of natural DNA in a cell-free medium using cisplatin tethered to the AT-specific, minor groove binder distamycin, were studied using various methods of biochemical analysis or molecular biophysics. These methods include: binding studies using differential pulse polarography and flameless atomic absorption spectrophotometry, mapping DNA adducts using a transcription assay, use of ethidium bromide as a fluorescent probe for DNA adducts of platinum, measurement of DNA unwinding by gel electrophoresis, measurement of CD spectra, an interstrand cross-linking assay using gel electrophoresis under denaturing conditions, measurement of melting curves with the aid of absorption spectrophotometry and the use of terbium ions as a fluorescent probe for distorted base pairs in DNA. The results indicate that attachment of distamycin to cisplatin changes several features of the DNA-binding mode of the parent platinum drug. Major differences comprise different conformational alterations in DNA and a considerably higher efficiency of the conjugated drug to form in DNA interstrand cross-links. Cisplatin tethered to distamycin, however, coordinates to DNA with similar base sequence preferences as the untargeted platinum drug. The results point to a unique profile of DNA binding for cisplatin-distamycin conjugates, suggesting that tethering cisplatin to minor groove oligopeptide binders may also lead to an altered biological activity profile.

Antineoplastic Agents↗

PubMed1995

[Effects of lumbar peridural anesthesia on tissue pO2 of the large intestine in man].

AIM: The aim of this study was to investigate the effect of 15 ml bupivacaine 0.25%, given via an epidural catheter, on the tissue-PO2 of the colon wall in humans. METHODS: Nine patients were studied during elective colorectal surgery. The tissue-PO2 was measured by polarography, using a multiwire surface probe. RESULTS: There was no significant change in cardiac index and arterial oxygen delivery after the injection of bupivacaine. At the serosal side of the colon wall the tissue-PO2 increased from 34 to 51 mmHg after epidural bupivacaine, which is statistically significant. Spinal nerve block produces sympathetic denervation of the bowel with an increase of regional nutritive blood flow. CONCLUSION: It is concluded that epidural bupivacaine has a favourable effect on tissue oxygenation of the colon during colorectal surgery.

Aged↗

PubMed2002

Mitochondrial function during ischemic preconditioning.

Background. Ischemic preconditioning (IPC) protects the myocardium from ischemia reperfusion injury. The effect of IPC on the mitochondria is not well known. However, one of the mechanisms postulated in IPC (the opening of the mitochondrial K(ATP) channels) is likely to result in changes in mitochondrial function. Therefore, the purpose of this study was to determine the effect of IPC on mitochondrial function during ischemia reperfusion. Methods. Isolated rat hearts (n = 6/group) were subjected to (1) 30 minutes of equilibration, 25 minutes of ischemia, and 30 minutes of reperfusion (RP) (control group) or (2) 10 minutes of equilibration, two-5 minute episodes of IPC (each followed by 5 minutes of re-equilibration), 25 minutes of ischemia, and 30 minutes of RP (IPC group). Left ventricular rate pressure product (RPP) was measured. At end-equilibration (end-EQ) and at end-reperfusion (end-RP) mitochondria were isolated. Mitochondrial respiratory function (state 2, 3, and 4), respiratory control index (RCI), rate of oxidative phosphorylation (ADP/Delta t), and ADP:O ratio were measured by polarography with the use of NADH- or FADH-dependent substrates. Results. IPC improved recovery of RPP at end-RP (72% +/- 5% in IPC vs 30% +/- 4% in control, P <.05). Ischemia reperfusion (IR) decreased state 3, ADP/Delta t, and RCI in both groups compared with end-EQ. IPC improved state 3 (47 +/- 3 in IPC vs 37 +/- 2 ng-atoms O/min/mg protein in control), ADP/Delta t (17 +/- 1 in IPC vs 13 +/- 1 nmol/s/mg protein in control), and RCI (3.7 +/- 0.1 in IPC vs 2.1 +/- 0.2 in control) at end-RP compared with control with the use of NADH-dependent substrate (P <.05 vs control). IPC also improved state 3 (85 +/- 6 in IPC vs 71 +/- 4 ng-atoms O/min/mg protein in control), ADP/Delta t (18 +/- 2 in IPC vs 12 +/- 1 nmol/s/mg protein in control), RCI (2 +/- 0.1 in IPC vs 1.5 +/- 0.1 in control), and ADP:O ratios (1.4 +/- 0.04 in IPC vs 1.7 +/- 0.09 in control) at end-RP compared with control with the use of FADH-dependent substrate (P <.05 vs control). Conclusions. The cardioprotective effects of IPC can be attributed at least in part to the preservation of mitochondrial function during reperfusion.

Adenosine Triphosphate↗

PubMed1984

Detection of superoxide generated by endothelial cells.

Superoxide and lipid free-radical generation in cultured endothelial cells treated with menadione or nitrazepam were measured using electron paramagnetic resonance spectroscopy. Superoxide was detected both intracellularly and extracellularly. Extracellular generation of superoxide and hydrogen peroxide was also measured, either by spectrophotometric measurement of succinoylated cytochrome c reduction or by polarography. Extracellular superoxide was generated due to reduced menadione diffusing across the plasma membrane and reacting with oxygen to generate superoxide in the medium. Increased intracellular oxygen tension favored intracellular oxidation of reduced menadione, thus decreasing diffusion of reduced menadione from the cells and, hence, decreasing extracellular superoxide production. The nitro anion free radical of reduced nitrazepam, which cannot cross the plasma membrane, did not generate detectable extracellular superoxide. Our results show that intracellular superoxide can be spin-trapped using 5,5-dimethyl-1-pyrroline-1-oxide and that secondary free-radical injury to membrane lipids, due to excess production of partially reduced species of oxygen by intact cells, can be detected by spin-trapping lipid free radicals with phenyl N-tert-butylnitrone.

Animals↗

PubMed2004

Biosynthetic Ca2+/Sr2+ exchange in the photosystem II oxygen-evolving enzyme of Thermosynechococcus elongatus.

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr(2+)-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr(2+)- and Ca(2+)-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr(2+)-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD* is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ* to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr(2+)-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects.

Calcium↗

PubMed2002

Drug-related taste disturbances.

Drug-induced change in taste is the second most common cause of taste disturbance among our patients, accounting for approximately 25% of cases. About 170 drugs have been associated with taste disturbances, either when used alone or in combinations. We studied the zinc-chelating capability of 20 drugs associated with taste disturbance, using the pH titration test, DC polarography, spectrophotometry and tests of intestinal absorption of zinc in the presence of these drugs. The results of these analyses and our review of the literature indicate that there are a number of possible mechanisms for drug-related taste disturbance and that zinc plays a key role.

Humans↗

PubMed1984

Overview of analysis of carcinogenic and/or mutagenic metals in biological and environmental samples. I. Arsenic, beryllium, cadmium, chromium and selenium.

One of the most dangerous and pernicious forms of pollution arises from the potential mobilization of a spectrum of toxic trace metals and metalloids in our environment. Among the most important elements in this regard are arsenic, beryllium, cadmium, chromium and selenium whose adverse toxic effects are now well recognized including their carcinogenicity and/or mutagenicity. These agents (and their derivatives) can be widely dispersed throughout the environment as a result of fossil fuel combustion, industrial and agricultural processes and natural processes. The trend for the immediate future appears to be of greater exposure to these metals not only as a result of generally increased usage patterns but also because of prospective enhanced use of fossil fuels for space heating and electricity generation. In order to more readily evaluate trends of human exposure as well as the toxicity, bioavailability, bioaccumulation and transport of these elements, sensitive analytical procedures are required for the determination of their various oxidation states (as well as their organic derivatives) in complex matrices such as those found in both environmental and biological samples. Hence, the principal objective of this overview is to highlight the more recent trends and state-of-the-art methodologies for the determination of arsenic, beryllium, cadmium, chromium and selenium (in their various forms) in environmental compartments such as air, water, soil and in human tissues (primarily blood, urine, and milk). Techniques to be discussed primarily include atomic absorption spectrometry, neutron activation analysis, gas chromatography, differential pulse polarography and electrochemical analysis. The importance of quality control and differentiation according to speciation will also be stressed.

Air↗

PubMed1987

Analytical chemistry of chromium species in the environment, and interpretation of results.

Valence-selective species determination of chromium in environmental and biological material presents intricacies primarily due to the complex nature of the samples and the instability of the oxidation states of chromium. In particular organic matter ubiquitously present in environmental samples introduces the risk of chromium(VI) reduction during analysis which takes place preferably in acidic solution. Also chromium(III) oxidation may occur in alkaline medium. Hence analytical methods should be adequately adapted to the reliable determination of chromium(VI) in blood, natural water, effluents, sediments and soil. It can be shown that polarography is most effectually suitable to the determination of chromium(VI) compounds. Chromium(VI) is electrochemically active over the entire pH range, so that medium pH can be selected for measuring, thus protecting samples most effectively from undergoing redox reactions during the analytical procedure. Other established methods such as spectrophotometry or valence-specific extraction are more crucial, because these generally require acidic medium. In some cases sample pre-treatment can be employed to eliminate reductants prior to final measurement. Very few methods are available to selectively measure chromium(III). Indirect determination is therefore preferred. Optimum conditions for the determination of total chromium by AAS are shortly outlined.

Chromium↗

PubMedDate not supplied

Studies on biomarkers of copper exposure and toxicity in the marine amphipod Gammarus locusta (Crustacea): I. Induction of metallothionein and lipid peroxidation.

Sublethal exposures of the marine amphipod Gammarus locusta to a concentration range of copper (Cu) in water (4 days' exposure; 3, 5 and 10 micro g Cu l(-1)) or spiked sediments (28 days' exposure; 1, 3 and 6 mg Cu kg(-1) dry weight) were performed, and the resulting bioaccumulation of Cu and effects on putative metallothionein (MT) and lipid peroxidation (LP) were investigated. A time-course exposure study (over 10 days) to a single water-borne concentration of Cu (4 micro g l(-1)) was also carried out. MT and LP were quantified, respectively, by differential pulse polarography and as thiobarbituric acid-reactive malondialdehyde equivalents. The increasing levels of Cu in water and sediment exposures resulted in enhanced uptake of the metal by G. locusta. Synthesis of putative MT occurred in response to exposure to water-borne Cu, the levels being higher (p < 0.05) over the dose range of Cu compared with controls. A positive correlation was observed between putative MT levels and the Cu body-burden concentration (p < 0.001). However, no increase in LP was observed in these animals. In contrast, in the time-course experiment, LP levels increased within 1 day of exposure, subsequently peaking at 4 days (68% greater than control, p < 0.001), before returning to control values by day 6. Higher levels of MT were also observed in this exposure, but at days 6 and 10 (55% and 38%, respectively), paralleling the decrease in LP. No increase in MT levels was recorded with exposure to Cu-contaminated sediments, whereas higher levels of LP were seen in comparison with controls (p < 0.001). Overall, the inverse relationship between putative MT induction and the occurrence of LP indicates that MT may protect against the prooxidant effects of Cu. It is concluded that MT and LP offer potential for application as biomarkers in G. locusta.

Amphipoda↗

PubMed2002

Olive oil feeding up-regulates uncoupling protein genes in rat brown adipose tissue and skeletal muscle.

BACKGROUND: Some nutrients, such as carotenoids, retinoic acid, and certain types of fatty acids, increase thermogenic capacity. OBJECTIVE: The influence of 4 dietary lipid sources (olive oil, sunflower oil, palm oil, and beef tallow) on the content of uncoupling proteins 1, 2, and 3 (UCP1, UCP2, and UCP3) and their messenger RNA (mRNA) expression in several tissues of rats was compared. DESIGN: Wistar rats were randomly divided into 4 groups and fed ad libitum diets containing 40% of energy as fat. UCP1, UCP2, and UCP3 mRNA and protein were assessed by Northern blot and Western blot, respectively. Oxygen consumption in tissues was measured by polarography. Total-body oxygen consumption was assessed in an open-circuit chamber system. Circulating fuels (fatty acids and glucose) and hormones (triiodothyronine, thyroxine, corticosterone, and insulin) were measured. RESULTS: Olive oil feeding induced the highest UCP1, UCP2, and UCP3 mRNA expression in interscapular brown adipose tissue. An analogous effect was observed in gastrocnemius muscle UCP3 mRNA. No significant differences were observed in perirenal white adipose tissue UCP2 mRNA. Changes in mRNAs were not accompanied by close changes in the protein content of UCPs and were not associated with changes in adipose tissue oxygen consumption. Nevertheless, total-body oxygen consumption was higher in rats fed olive oil than in those fed the other 3 diets. No significant differences were found in body and tissue weights or in serum indexes. CONCLUSION: Olive oil induced an up-regulating effect on UCP mRNA that was probably not mediated by systemic metabolic changes, but rather related to a local effect on interscapular brown adipose tissue and skeletal muscle.

Adipose Tissue↗

PubMed1975

Analysis of fumigants and fumigant residues.

The terms fumigant and fumigant residue are defined. Interrelationships between physically and chemically bound residues, storage environments, nature of the substrate and other influencing factors are outlined. Analytical methods include polarography by DME and RPE, titrimetry, spectrophotometry, and GC with microthermal conductivity, hydrogen flame ionization, electron capture, microcoulometric, thermionic, and flame photometric detector systems, with backup by enzymatic, radiometric, NAA and X-ray flourescence methods. Various aspects are illustrated with different fumigants used commercially. Supplementary methods to extend the power and usefulness of analytical methods in fumigant research are indicated.

Bromides↗

PubMed1993

Membrane-Associated and Soluble Lipoxygenase Isoforms in Tomato Pericarp (Characterization and Involvement in Membrane Alterations).

Membrane-associated and soluble lipoxygenases from green tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) fruit have been identified. Microsomal lipoxygenase was localized partly in the plasma membrane and tonoplast fractions. The possibilities of glycosyl-phosphatidylinositol or transmembrane polypeptide anchors in the membrane were ruled out by differential solubilization and temperature-induced phase separation in Triton X-114. High performance liquid chromatography of reaction products combined with polarography showed that tomato lipoxygenase is capable of specific oxygenation of fatty acids esterified in phospholipids. This possibility of direct action on membrane phospholipids strengthened the hypothesis of a role for lipoxygenase in plant senescence and membrane turnover. Membrane-associated lipoxygenase is polymorphic, with two forms differing by their isoelectric points (pls) (around 4.2 and 5.1). The pl of the soluble lipoxygenase corresponds to the minor microsomal enzyme, with a pl of 5.1. The charge-differing isoforms were separated and analyzed by western blotting using anti-soybean lipoxygenase antibodies. A single polypeptide with an apparent molecular weight of 92,000 was identified in each case for the soluble and microsomal enzymes. It is suggested that a charge modification of the soluble lipoxygenase allows its association with the membrane.

Journal Article↗