Molecular and cellular mechanism of the effect of La(III) on horseradish peroxidase

Horseradish is an important economic crop. It contains horseradish peroxidase (HRP) and lots of nutrients, and has specific pungency. Lanthanum is one of the heavy metals in the environment. It can transfer through the food chain to humans. In this paper, the molecular and cellular mechanism of the toxic effects of La(III) on HRP in vivo was investigated with an optimized combination of biophysical, biochemical, and cytobiological methods. It was found that La(III) could interact with O and/or N atoms in the backbone/side chains of the HRP molecule in the cell membrane of horseradish treated with 80 μM La(III), leading to the formation of a new complex of La and HRP (La–HRP). The formation of the La–HRP complex causes the redistribution of the electron densities of atoms in the HRP molecule, especially the decrease in the electron density of the active center, Fe(III), in the heme group of the La–HRP molecule compared with the native HRP molecule in vivo. Therefore, the electron transfer and the activity of HRP in horseradish treated with 80 μM La(III) are obviously decreased compared with those of the native HRP in vivo. This is a possible molecular and cellular mechanism for the toxic effect of La(III) on HRP in vivo. It is suggested that the accumulation of La in the environment, especially the formation of the La–HRP complex in vivo, is harmful to organisms.     

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 °C. The immobilized HRP showed higher storage stability than the free HRP.     

Dynamics of the retrograde transport of horseradish peroxidase in the trigeminal nerve during burn keratitis

The dynamics of trigeminal neuron labelling with horseradish peroxidase (HRP) has been studied in rats during thermal burn keratitis. HRP application on the cornea after burn prolongs the time of its appearance in the perikarya cells of trigeminal ganglia, decreasing the number of labelled neurons. A more marked inflammation led to the decline in the number of nervous cells containing HRP. The decreased number of labelled HRP cells was recorded during the regeneration phase. This difference in the dynamics of neuron labelling in trigeminal ganglia was associated with the damaging effect of high temperature and inflammation on HRP uptake and transportation.     

Cytochemical observations on the transport of horseradish peroxidase in different segments of the nephron

Synopsis Kidney slices from rats injected with horseradish peroxidase (HRP) 5–10 min before sacrifice were fixed with formaldehyde vapour for 4 hr at 37°C and compared with tissue fixed by perfusion or by immersion. Much more of the injected protein was retained in extracellular and vascular spaces of the nephron in vapour-fixed than in perfusion-fixed or immersion-fixed tissue. The extracellular localization of HRP in the lateral intercellular spaces and in the infoldings of the basal cell membranes showed characteristic differences in different segments of the nephron. The high concentration of HRP in the lateral intercellular spaces of the collecting tubules, as well as the early location of small phagosomes containing HRP in the apical, lateral, and basal cell regions suggested that HRP was reabsorbed through the cytoplasm into the intercellular spaces or excreted in the opposite direction. The intercellular spaces in the terminal segments of the proximal tubules also showed high concentrations of HRP which suggests participation of these spaces in protein transport between the lumen and the peritubular capillaries. The extracellular concentration of HRP early after injection was found, by colorimetric assays of homogenates, to be several times higher in the papilla than in the cortex.     

The role of the mannose/N-acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate-,proteose peptone-, and Corynebacterium parvum-elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 micrograms/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate-elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10-20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N-acetylglucosamine receptor. PMA stimulates fluid-phase pinocytosis of HRP by thio macrophages but does not affect receptor-mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) selectively inhibits bulk-phase uptake by thio macrophages.     

Cytochemical examination of the compartments involved in the transcellular transport of horseradish peroxidase in rat hepatocytes

Horseradish peroxidase (HRP, 10 mg/100 g body weight) was intravenously injected into rats in order to investigate the nature of the compartments involved in the transcellular transport of the protein through hepatocytes into bile. Double cytochemistry for HRP and the marker enzymes for cytoplasmic organelles was used. HRP was shown to be taken up by hepatocytes via vesicles at the sinusoidal surface, some of which were positive for 5'-nucleotidase activity. HRP was then found in the smooth-surfaced vesicles and tubules which were negative in 5'-nucleotidase, glucose 6-phosphatase, thiamine pyrophosphatase and acid phosphatase activity, suggesting that the tubular structures are neither the endoplasmic reticulum, the Golgi apparatus nor lysosomes. Biochemical studies revealed that the lead procedures used for the double cytochemistry did not inhibit the peroxidatic activity of HRP, and conversely that HRP did not interfere with the marker enzyme activity. Such cytochemical observations seemed to be supported by the observation that administration of monensin (3.5 mg/100 g) and chloroquine (5 mg/100 g), which markedly altered the structure of the Golgi apparatus and lysosomes, respectively, slightly altered the biliary excretion of HRP but not to a significant extent.     

Influence of forskolin and carbachol on intestinal absorption of horseradish peroxidase in the goldfish (Carassius auratus)

The transepithelial route for mucosa-to-serosa transport of the tracer macromolecule horseradish peroxidase (HRP; MW 40 kDa) and modulation of this transport by forskolin and carbachol have been studied in vi-tro in stripped goldfish intestinal epithelium mounted in Ussing-type chambers. Uptake and transport have been investigated by measuring the HRP flux from the muco-sal to serosal sides by an enzymatic method and by visualising HRP reaction products in the mucosa with electron-microscopical techniques. Both the cholinergic agonist carbachol (which is thought to increase intracellular Ca²⁺ and activate protein kinase C activity) and forskolin (a direct activator of adenylylcyclase) affect the amount of enzymatically active HRP in the tissue. In control tissue, HRP product is found only within the epithelial cells, the transepithelial flux reaching a constant value of about 1.5 pmoles/cm² per h. Carbachol increases the amount of HRP product in the cells, but has no significant effect on the HRP flux compared with control values. Forskolin decreases the amount of HRP product in the cells; however, in the presence of forskolin, the lateral intercellular spaces become filled with HRP product. HRP is found in the lamina propria and the transepithelial protein flux increases more than 2.5-fold. In the presence of forskolin plus carbachol, the results are no different from the control. It is concluded that carbachol increases the endocytotic uptake of HRP, whereas forskolin inhibits the uptake but increases the paracellular permeability for HRP in goldfish intestine. Received: 10 July 1995 / Accepted: 4 February 1996     

Polymerization of horseradish peroxidase in the presence of inorganic adsorbents

Efficiencies of binding between horseradish peroxidase (HRP) and its polymers (HRPp) with inorganic adsorbents (precipitated and coprecipitated) were studied. In aqueous solutions, HRP efficiently adsorbed to aluminum oxide and the coprecipitated sorbent (composed of calcium orthophosphate, magnesium hydroxide, and aluminum hydroxide). HRP readily bound to zinc hydroxide but not to aluminum hydroxide in 25.0 mM bicarbonate buffer (pH 9.0). Several variants of HRP polymerization and HRPp modification with diamines in the presence of Al2O3 and Zn(OH)2 were compared. Synthesis of HRPp according to the scheme comprising HRP activation in solution followed by its polymerization in the presence of Zn(OH)2 appeared the most efficient. HRP and HRPp bound to Zn(OH)2 displayed a high catalytic activity in the presence of high H2O2 concentrations.     

Plasmodium falciparum: effect of anti-malarial drugs on the production and secretion characteristics of histidine-rich protein II

Plasmodium falciparum histidine-rich protein II (HRP2) is one of the best documented malaria proteins. However, little is known about the development of HRP2 concentrations under the influence of anti-malarial drugs. HRP2 levels were determined in cell medium mixture, cellular compartment, and in culture supernatant using a double-site sandwich ELISA specific for HRP2. Characteristic increases in the overall HRP2 levels were found during the later ring and the trophozoite stages. Throughout the later schizont development, rupture, and reinvasion, however, the HRP2 levels remained comparatively stable. When the cultures were exposed to serial dilutions of anti-malarial drugs, a distinct inhibition of HRP2 production was seen with increasing concentrations of drugs, resulting in sigmoid dose-response curves, similar to those obtained from conventional drug sensitivity assays. HRP2 therefore allows for a very accurate estimation of parasite development and its inhibition and may therefore be ideally suited for use in drug sensitivity or bioassays.     

Quantitation of fluid phase uptake in paramecium. 1. Kinetics in the cells blocked in phagocytic activity

Uptake of horseradish peroxidase (HRP) was quantified in the unicellular eukaryote Paramecium aurelia. About 80% of the total HRP accumulated within 20 min entered the cells via a fluid phase pathway as demonstrated measuring the HRP internalization in the presence of yeast mannan. The rate of HRP accumulation was concentration-dependent and was found to be linear over the range of 50–500 μg HRP per ml of the extracellular medium. During the first 10 min of exposure to HRP, the mannan-uninhibitable uptake was found to reach 1.2–1.65 ng HRP per mg protein (depending on the concentration of the marker in the medium), which corresponds to 0.68 fl per cell/min. Accumulation of HRP reached a plateau within the next 10–15 min and its intracellular uptake was 2–2.55 ng HRP per mg protein. When the phagocytic activity of the cells was blocked with 1-propranolol, the amount of cell-accumulated HRP was 1.8–2.1-fold higher than in the control and the rate of the marker uptake within the first 10 min of incubation reached 1.114 fl per cell/min.     

Permeability of the mouse gallbladder to blood-borne horseradish peroxidase

Summary Horseradish peroxidase (HRP) was administered intravenously to mice by bolus injection. The subsequent uptake and fate of the HRP by the lateral and basal cell surfaces of resting and stimulated gallbladder epithelial cells was followed by light and electron microscopy. At 10 min after injection, HRP was visible in the lamina propria of the gallbladder and within 20 min of injection, HRP had permeated the basement membrane and had entered the lateral intercellular space, extending as far as the apical tight junction. Over the following 30 min, there was evidence of vesicular epithelial HRP uptake and 1 h after injection, HRP was visible in epithelial secretory granules within the lumen of the gallbladder and apical transport vesicles. These data provide evidence of a blood-to-bile transport pathway which could represent an important route of entry to bile by various blood-borne macromolecules.     

Competition between ligands of glycosyltransferases and horseradish peroxidase for binding sites on intracellular and plasma membranes of HeLa cells. Application of a micro-method for the semi-quantitation of surface-bound HRP

A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37 degrees C. The incubation medium contained HRP, lysozyme and Ca2+ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37 degrees C in HEPES buffer containing 20 mM Ca2+. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37 degrees C in the absence of Ca2+, and was measured by spectrophotometry. The addition of 20 mM Ca2+ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca2+. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Binding of Bispecific Monoclonal Antibodies to the Solid Phase-Adsorbed Antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-human IgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (K _a) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than K _afor Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90–95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5–10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-hIgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90-95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5-10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

Uptake of horseradish peroxidase from CSF into the choroid plexus of the rat,with special reference to transepithelial transport

Summary Protein uptake from cerebral ventricles into the epithelium of the choroid plexus, and transport across the epithelium were studied ultrastructurally in rats. Horseradish peroxidase (HRP, MW 40,000) was used as protein tracer. Steady-state ventriculo-cisternal perfusion with subatmospheric pressure (-10cm of water) in the ventricular system was applied. HRP dissolved in artificial CSF was perfused from the lateral ventricles to cisterna magna for various times, and ventriculo-cisternal perfusion, vascular perfusion or immersion fixation with a formaldehyde-glutaraldehyde solution was performed.Coated micropinocytic vesicles containing HRP were seen both connected with the apical, lateral and basal epithelial surface and within the cells. Heavily HRP-labeled vesicles were often fused with the lining membrane of slightly labeled or unlabeled intercellular spaces. Since the apical tight junctions of the epithelium never appeared open or never contained HRP in the spaces between the fusion points, and since the intercellular spaces between adjacent epithelial cells below the junctions only infrequently contained tracer after 5 min, by increasing amounts after 15–60 min of HRP perfusion, a vesicular transport of HRP from the apical epithelial surface to the intercellular spaces, bypassing the tight junctions, is suggested.In addition to the transepithelial transport, micropinocytic vesicles also transported HRP to the lysosomal apparatus of the epithelial cells. With increasing length of exposure to HRP, a sequence of HRP-labeled structures could be evaluated, from slightly labeled apical vacuoles and multivesicular bodies to very heavily labeled dense bodies.     

Efferent neurons of the auditory center in the midbrain of the frog Rana ridibunda

Individual cells which produce projections from the torus semicircularis in the frog have been visualized after injection of horseradish peroxidase (HRP) to various thalamic and isthmal areas. Labeled toral cells were observed if HRP had been injected to the posterodorsal areas of the thalamus or to the isthmal areas where lateral lemniscus fibers and cells of the premature lateral lemniscal nucleus are situated. Medium and large size cells in the rostrolateral torus semicircularis were mainly labeled. Thalamic injections of the HRP produced more labeled cells in the lateral part of the magnocellular nucleus, whereas isthmal injections produced labeled cells mainly in the lateral part of the laminar nucleus. A few HRP containing cells were observed in the principal nucleus of the torus. Specificity of the neuronal organisation of the auditory pathway in amphibians is discussed.     

Demonstration of afferent connections of the forebrain in Emys orbicularis turtles by the peroxidase method

The transport of horseradish peroxidase (HRP) out of the injection site in the dorsal ventricular ridge was studied in turtles Emys orbicularis. Labeled cells in the forebrain were observed in the paleostriatum among fibers of the lateral forebrain bundle. In the thalamus most of cells containing the granular HRP reaction product were located in the n. rotundus, n. reuniens and perirotundal nuclei (n. dorso-medialis anterior, n. magnocellularis thalami, n (centralis) lateralis, n. dorso-medialis). Fewer labeled cells were revealed in the n. anterior and n. ventralis. The density of labeled cells in the majority of all thalamic nuclei increased if the HRP was extended from the dorsal ventricular ridge into the neostriatum and the pallial thickening with adjacent general cortex. HRP positive cells in the pretectal area, nuclei of the posterior commissura and mesencephalic ventro-lateral tegmentum were observed only in cases when the enzyme was diffused from the injection site into the neostriatum, while the HRP retrograde transport to n. geniculatus lateralis, pars dorsalis was revealed only when HRP was extended into the pallial thickening and adjacent general cortex. Ascending connections of the paleostriatum, thalamic nuclei and mesencephalic tegmentum with telencephalic structures, mainly with the dorsal ventricular ridge, were discussed.     

Axoplasmic transport of horseradish peroxidase in single neurons of the dorsal root ganglion studied in vitro by microinjection

Summary The dependence of anterograde axoplasmic transport on cytoskeletal components was investigated using microinjection of horseradish peroxidase (HRP) into the somata of chick dorsal root ganglion cells in vitro. Microinjected HRP was transported anterogradely in the neurites and their branches; this transport was disturbed by colchicine in a drug-dependent and time-dependent manner. Cytochalasin B, a drug that depolymerizes actin, did not inhibit the transport of HRP, despite the formation of local swellings in neurites. The microinjection of polyclonal antibodies directed against tubulin and monoclonal antibodies (mAbs) against 200-kDa neurofilaments disturbed the axoplasmic transport of co-injected HRP, which then exhibited an irregular and discontinuous distribution in the axonal branches. The transport of HRP became discontinuous after the injection of anti-tubulin antibodies and led to the formation of globular deposits of HRP. Polyclonal antibodies against actin and mAbs to 160-kDa and 68-kDa neurofilaments seemed to have no effect on the axoplasmic transport of co-injected HRP. Microinjection of antibodies against tubulin induced formation of perinuclear bundles consisting of cytoskeletal components. The transport of HRP thus appears to be regulated by an intact microtubular system and cross-linker components (200-kDa neurofilaments) of the cytoskeleton. Actin and most intermediate filament proteins do not seem to play an essential role in the transport of HRP.     

Endocytotic pathways at the ruffled borders of rat maturation ameloblasts

Summary Using horseradish peroxidase (HRP) as a soluble protein tracer, electron microscopic studies were carried out in order to analyze endocytosis in the ruffle-ended ameloblasts of rat incisors. Accumulated HRP was initially incorporated from the ruffled border into the cytoplasm by means of pinocytotic vacuoles (pinosomes) and pinocytotic coated vesicles. The majority of the HRP was taken up by the large number of pinosomes, which then formed large endocytotic vacuoles by fusing either with each other or with preexisting endocytotic vacuoles. As time passed HRP accumulated, not in the pinosomes and ruffled border but in the endocytotic vacuoles and multivesicular bodies. Frequent connections between HRP-labeled coated vesicles and these cytoplasmic bodies indicate that these vesicles serve as an HRP carrier. These findings strongly suggest that ruffle-ended ameloblasts actively absorb soluble proteins from the enamel matrix during enamel maturation.     

Interaction of aromatic donor molecules with horseradish peroxidase: identification of the binding site and role of heme iron in the binding and activity

The interaction of aromatic substrates with horseradish peroxidase (HRP) was studied. Chemical modification of HRP was performed using diethylpyrocarbonate (DEPC) and for the first time the amino acid involved in binding with these substrates has been identified. The kinetic parameters for this interaction have been calculated and the role of heme iron in the oxidation of aromatic substrates by HRP has been discussed.