BEHAVIOUR OF HORSERADISH PEROXIDASE IN AOT REVERSED MICELLES

The activity and stability of horseradish peroxidase (HRP) solubilised in AOT reversed micelles in isooctane and decalin was studied using guaiacol (2-methoxyphenol) as the electron donor.

The activity of the enzyme in both reversed micellar systems increases with the water content until reaching a maximum value that remains fairly constant for water contents higher than 3.05% (v/v) in isooctane and 2.20% in decalin. The effect of pH on the activity profile was studied in the system AOT/isooctane. The enzyme is fully active at pH 7 and 8 for water contents higher than 3.05% (v/v) but it was completely deactivated at pH 9. The effect of surfactant concentration on HRP activity was also investigated. At low water contents a strong dependence was observed, whilst no further activity increase was observed for water content values higher than 2.7% (v/v).

The stability of HRP was found to be strongly dependent on the water content of the system with higher levels of stability obtained for higher values of water content. HRP stability is also affected by the presence of substrates. Whilst the stability increases markedly when the enzyme is incubated with guaiacol, it does not appear to be so strongly affected by the presence of hydrogen peroxide, at the concentrations studied.     

Distinct heme-substrate interactions of lactoperoxidase probed by resonance Raman spectroscopy: difference between animal and plant peroxidases

Resonance Raman scattering from cow milk lactoperoxidase (LPO) and its complexes with various electron donors and inhibitors was investigated. The Raman spectrum of LPO is strikingly close to that of hog intestinal peroxidase but distinctly dissimilar to that of horseradish peroxidase (HRP). The v10 frequency suggested the six-coordinate high-spin structure of heme for native LPO in contrast with the five-coordinate high-spin structure for HRP. For the v10 band, benzohydroxamic acid caused a frequency shift with HRP but not with LPO. Guaiacol, o-toluidine, and histidine brought about a frequency shift of the v4 mode for LPO but not for HRP. The frequency shift was restored upon removal of the substrate or inhibitor by dialysis. The down shift of the v4 frequency is considered to represent an appreciable donation of electrons from the substrate or inhibitor to the porphyrin LUMO and thus their direct interaction with the heme group. From the relative intensity of the shifted and unshifted v4 lines, the dissociation constant was determined to be Kd = 52 mM for guaiacol and Kd = 87 mM for histidine at pH 7.4. The binding of histidine was relatively retarded in the presence of sulfate anion (Kd = 150 mM for 0.53 M sulfate present), and imidazole alone yielded no frequency shift, indicating the binding of the carboxyl group of histidine to the protein cationic site on one hand and a weak charge-transfer interaction between the imidazole group and the heme group on the other.     

Effect of ionic liquid on the kinetics of peroxidase catalysis

The effect of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), on the horseradish peroxidase (HRP)-catalyzed oxidation of 2-methoxyphenol (guaiacol) with hydrogen peroxide (H2O2) was investigated. HRP maintains its high activity in the aqueous mixtures containing various concentrations of the ionic liquid and even in 90% (v/v) ionic liquid. In order to minimize the effect of solution viscosity on the kinetic constants of HRP catalysis, the enzymatic reactions in the subsequent kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. As the concentration of [BMIM][BF4] increased for the oxidation of guaiacol by HRP, the K(m) value increased with a slight decrease in the k(cat) value: The K(m) value increased from 2.8 mM in 100% (v/v) water to 22.5 mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to HRP.     

Immobilization of horseradish peroxidase on activated wool

This work is aimed to immobilize partially purified horseradish peroxidase (HRP) on wool activated by multifunctional reactive center, namely cyanuric chloride. The effect of cyanuric chloride concentration, pH and enzyme concentration on immobilization of HRP was studied. FT-IR and SEM analyses were detected for wool, activated wool and immobilized wool-HRP. The wool-HRP, prepared at 2% (w/v) cyanuric chloride and pH 5.0, retained 50% of initial activity after seven reuses. The wool-HRP showed broad optimum pH at 7.0 and 8.0, which was higher than that of the soluble HRP (pH 6.0). The soluble HRP had an optimum temperature of 30 °C, which was shifted to 40 °C for immobilized enzyme. The soluble and wool-HRP were stable up to 30 and 40 °C after incubation for 1 h, respectively. The apparent kinetic constant values (K_ms) of wool-HRP were 10 mM for guiacol and 2.5 mM for H₂O₂, which were higher than that of soluble HRP. The wool-HRP was remarkably more stable against proteolysis mediated by trypsin. The wool-HRP exhibited more resistance to heavy metal induced inhibition. The wool-HRP was more stable to the denaturation induced by urea, Triton X-100, isopropanol, butanol and dioxan. The wool-HRP was found to be the most stable under storage. In conclusion, the wool-HRP could be more suitable for several industrial and environmental purposes.     

Stabilization of diluted aqueous solutions of horseradish peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55 degrees C and infinite dilution, HRP was inactivated with a rate constant of 2.86 x 10(-3) s-1. CaCl2, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the "glycerol-based" one containing 10% ethanol and 20% glycerol, or the "protein-based" one containing 0.1% Kathon CG and 0.2 g/l of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl2 were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.     

Studies on the Refolding Process of Recombinant Horseradish Peroxidase

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl₂ in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.     

High-valent intermediates in the reaction of N alpha-acetyl microperoxidase-8 with hydrogen peroxide: models for compounds 0, I and II of horseradish peroxidase.

N-acetyl microperoxidase-8 (Ac-MP-8) is a water soluble, ferric heme model for the peroxidases. The reaction of Ac-MP-8 with H2O2 in 10 mM potassium phosphate over the pH range of 7-11 gives rise sequentially to relatively stable green and red species with properties that closely mimic those of HRP compounds I and II, respectively. Low-temperature stopped-flow studies of this reaction carried out in 50% v/v methanol/10 mM potassium phosphate, pH* 9.1 at -25.8 degrees C indicate that the pseudo-first-order rate constant, kobs, that describes the formation of the green intermediate exhibits saturation kinetics as a function of [H2O2] with kmaxobs = 95 s-1 and KM = 87 mM. Rapid-scan studies carried out with [H2O2] = 200 mM at -38.0 degrees C show that a compound 0 species with a characteristic band near 340 nm is formed whose conversion to the green species is rate limiting. Thus, Ac-MP-8 has high-valent forms that are models for all three known intermediates in the peroxidase cycle of horseradish peroxidase.     

Direct Interaction between Terbium Ion and Peroxidase in Horseradish at Different pH Values

Rare earth elements (REEs) entering plant cells can directly interact with peroxidase in plants, which is the structural basis for the decrease in the activity of peroxidase. Different cellular compartments have different pH values. However, little information is available regarding the direct interaction between REEs and peroxidase in plants at different pH values. Here, we investigated the charge distribution on the surface of horseradish peroxidase (HRP) molecule as well as the interaction of terbium ion (Tb³⁺, one type of REEs) and HRP at different pH values. Using the molecular dynamics simulation, we found that when the pH value was from 4.0 to 8.0, a large amount of negative charges were intensively distributed on the surface of HRP molecule, and thus, we speculated that Tb³⁺ with positive charges might directly interact with HRP at pH 4.0–8.0. Subsequently, using ultraviolet-visible spectroscopy, we demonstrated that Tb³⁺ could directly interact with HRP in the simulated physiological solution at pH 7.0 and did not interact with HRP in other solutions at pH 5.0, pH 6.0 and pH 8.0. In conclusion, we showed that the direct interaction between Tb³⁺ and HRP molecule depended on the pH value of cellular compartments.     

Phenol removal from aqueous solutions by peroxidase extracted from horseradish

Horseradish peroxidase (HRP) is one of the most recently used enzymes in the process of enzymatic phenol removal. It has a catalytic ability over a broad range of pH, temperature and contaminant concentrations. In this study we revealed the possibility of successful use the crude peroxidase obtained from horseradish roots for the phenol removal from aqueous solutions in the presence of the low molecular polyethylene glycol (PEG 300) at room temperature (20°C) and pH 7.2. Reaction was monitored by direct measuring of the absorbance changes in a samples taken at certain time intervals from the reaction mixture. At the first time PEG 300 was shown to be a more stabilizing effect on crude HRP and provided a higher phenol removal in comparison with PEG 3350. Crude HRP used in these study demonstrated a greater resistance on phenol and hydrogen peroxide inactivation that allowed a higher phenol removal. The highest phenol removal was achieved when the concentration of PEG 300, phenol and hydrogen peroxide were 300 mg/L, 2.0 and 2.5 mM, respectively.     

Removal of pentachlorophenol by immobilized horseradish peroxidase

Researches on the polymerization of aqueous pentachlorophenol (PCP) by the catalysis of horseradish peroxidase (HRP) with the existence of hydrogen peroxide (H₂O₂) were conducted. Factors, such as acidity, temperature, enzyme activity, and initial concentration of PCP and H₂O₂ that could influence the degradation were studied. Results showed that the optimum pH value for free enzyme was 5–6; relative higher temperature could accelerate the reaction greatly; PCP removal increased with an increase of enzyme concentration, and PCP (initial concentration 12.6 mg/L) removal percentage could reach nearly 70% under the highest enzyme concentration (about 0.05 u/ml) adopted in the experiment; removal percentage increased slightly with an increase of initial concentration of PCP, and when initial PCP concentrations were 13.0 and 0.7 mg/L, the removal percentages were about 73.7% and 35.7%, respectively; the molar ratio of the reaction between PCP and H₂O₂ was about 1:2.Based on the above results, researches on the removal of PCP by the immobilized HRP were conducted. The free HRP was immobilized on the polyacrylamide gel prepared by gamma-ray radiation method; then the immobilized HRP was filled into a column, and PCP was successfully removed by the immobilized HRP column. The results were compared with results using free HRP enzyme, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP, and when pH=5.15, the immobilized HRP could reduce PCP with initial concentration 13.4 mg/L to the concentration of 4.9 mg/L within 1 h, and the immobilized HRP column could be used to repeatedly.     

Preparation of photo-crosslinkable cinnamate modified hyaluronic acid for immobilization of horseradish peroxidase

In this work, a photo-responsive hydrogel membrane based on cinnamate-modified hyaluronic acid (HA-CM) was developed and safely cross-linked under UV light curing. The obtained material was effectively utilized for immobilization of horseradish peroxidase (HRP) enzyme via encapsulation and entrapment strategy with efficiency above 95%. The prepared HA-CM biopolymer was investigated before the UV curing using instrumental and spectral techniques including Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR). During the UV irradiation, the progress of the cross-linking reaction was monitored by the UV–vis light spectroscopy. In addition, when the photo-induced cross-linking had accomplished, the morphological appearance of the hydrogel membrane was recorded using a scanning electron microscope (SEM). The HRP immobilized in HA-CM membranes displayed remarkable stability against the environmental pH changes especially under alkaline media and shift the optimum pH to 8 compared to the free HRP, which exhibited the highest activity at pH 7. Also, the entrapped enzyme was able to preserve above 85% of its catalytic activity at higher temperature values where the free enzyme had deactivated by approximately 50%. Moreover, HA-CM-HRP maintained 87% of its activity after 10 sequential reuse cycles, which indicate the economic value of the employed immobilization strategy.     

Phenylboronic acid-salicylhydroxamic acid bioconjugates. 1. A novel boronic acid complex for protein immobilization

A chemical affinity system exhibiting antibody-like properties is described. The system exploits bioconjugates with appended phenylboronic acid (PBA) moieties and a support-bound phenylboronic acid complexing reagent derived from salicylhydroxamic acid (SHA) for protein immobilization on a chromatographic support. The structure of the PBA.SHA complex was characterized by 11B NMR and mass spectrometry and compared with complexes derived from model compounds. Protein modification reagents were synthesized from 3-aminophenylboronic acid and utilized to prepare bioconjugates from alkaline phosphatase (AP) and horseradish peroxidase (HRP). AP obtained from one source afforded PBA bioconjugates exhibiting significant loss of enzymatic activity, whereas AP obtained from a second source afforded PBA bioconjugates exhibiting only a modest loss of enzymatic activity. Conversely, HRP afforded PBA bioconjugates exhibiting no loss of enzymatic activity. SHA-modified Sepharose was prepared by reaction of methyl 4-[(6-aminohexanoylamino)methyl]salicylate with CNBr-activated Sepharose 4B, followed by treatment with aqueous alkaline hydroxylamine. PBA-AP and PBA-HRP conjugates were efficiently immobilized on SHA-Sepharose at pH 8.3. PBA-AP conjugates were retained after washing with acidic buffers at pH 6.7, 4.2, and 2.5, whereas PBA-HRP conjugates were retained after washing with buffer at pH 6.7, but were eluted to some extent at and below pH 4.2. The results are interpreted in terms of multivalent interactions involving boronic acid complex formation between the enzyme bioconjugates and immobilized complexing reagent.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Ionic strength and pH effect on the Fe(III)-imidazolate bond in the heme pocket of horseradish peroxidase: an EPR and UV-visible combined approach

The effects of chloride, dihydrogenphosphate and ionic strength on the spectroscopic properties of horseradish peroxidase in aqueous solution at pH=3.0 were investigated. A red-shift (lambda=408 nm) of the Soret band was observed in the presence of 40 mM chloride; 500 mM dihydrogenphosphate or chloride brought about a blue shift of the same band (lambda=370 nm). The EPR spectrum of the native enzyme at pH 3.0 was characterized by the presence of two additional absorption bands in the region around g=6, with respect to pH 6.5. Chloride addition resulted in the loss of these features and in a lower rhombicity of the signal. A unique EPR band at g=6.0 was obtained as a result of the interaction between HRP and dihydrogenphosphate, both in the absence and presence of 40 mM Cl-. We suggest that a synergistic effect of low pH, Cl- and ionic strength is responsible for dramatic modifications of the enzyme conformation consistent with the Fe(II)-His170 bond cleavage. Dihydrogenphosphate as well as high chloride concentrations are shown to display an unspecific effect, related to ionic strength. A mechanistic explanation for the acid transition of HRP, previously observed by Smulevich et al. [Biochemistry 36 (1997) 640] and interpreted as a pure pH effect, is proposed.     

Catalytic and structural properties of surfactant-horseradish peroxidase complex in organic media

A surfactant-horseradish peroxidase (HRP) complex that is catalytically active in organic media has been successfully prepared by a method utilizing water-in-oil (W/O) emulsions. To optimize conditions for preparation of the HRP complex, the effects of some key parameters in the aqueous phase of W/O emulsions were investigated. The surfactant-HRP complex prepared with a nonionic surfactant exhibited a high catalytic activity compared to those with a cationic or anionic surfactant in anhydrous benzene. At the preparation step, the pH of the aqueous solution had a prominent effect on the enzymatic activity of the HRP complex in organic media. Several kinds of salts present in the HRP complex could be employed to enhance the catalytic performance in organic media. However, anionic ions present in the preparation process appeared to lower the catalytic activity owing to the complexation with heme iron. UV-visible absorption spectra of the HRP complex in benzene, which were prepared from a KCN solution (pH 7.0) or an alkaline solution (pH 12), were comparable with those of native HRP in aqueous solution under the same conditions. Resonance Raman spectroscopic studies also revealed that no significant change in the coordination state of the heme iron occurred even after coating the enzyme with surfactant molecules, lyophilization, and solubilization in nonaqueous media.     

A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies

An extracellular, thermostable, alkaline lipase was partially purified from a thermophilic Bacillus strain J 33. It was optimally active at pH 8.0 at 60°C, retaining 50% activity at 70°C for 30 min. It had native molecular mass of 45 kDa. The lipase was stable in 90% (v/v) hexane or benzene mixtures in water. It converted 66% oleic acid at 0.25 M with 0.4 M methanol in hexane to methyl oleate at 60°C in 16 h. Activity was stimulated by Mg2 (10 mM) but inhibited by EDTA (10 mM) and PMSF (10 mM). It was stable in Triton X-100, Tween 20 and Tween 80 (0.1% v/v). © Rapid Science Ltd. 1998     

Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode

Direct electron transfer of immobilized horseradish peroxidase on gold colloid and its application as a biosensor were investigated by using electrochemical methods. The Au colloids were associated with a cysteamine monolayer on the gold electrode surface. A pair of redox peaks attributed to the direct redox reaction of horseradish peroxidase (HRP) were observed at the HRP/Au colloid/cysteamine-modified electrode in 0.1 M phosphate buffer (pH 7.0). The surface coverage of HRP immobilized on Au colloid was about 7.6 x 10(-10) mol/cm(2). The sensor displayed an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The calibration range of H(2)O(2) was 1. 4 microM to 9.2 mM with good linear relation from 1.4 microM to 2.8 mM. A detection limit of 0.58 microM was estimated at a signal-to-noise ratio of 3. The sensor showed good reproducibility for the determination of H(2)O(2). The variation coefficients were 3. 1 and 3.9% (n = 10) at 46 microM and 2.8 mM H(2)O(2), respectively. The response showed a Michaelis-Menten behavior at higher H(2)O(2) concentrations. The K(app)(M) value for the H(2)O(2) sensor was found to be 2.3 mM.     

Cytochemical observations on mannose-specific binding sites for horseradish peroxidase in liver sinusoidal cells

Paraformaldehyde-fixed, frozen sections of the liver of rats were processed for the detection of mannose-specific binding sites of horseradish peroxidase (HRP) by a method reported previously, with some modifications resulting in a more intense binding reaction. Before staining for peroxidase activity, the sections were held in buffered solutions of physiological saline at different temperatures and pH's, and in the presence or absence of added Ca2+, mannose or galactose. The gradual decrease and final disappearance of the binding reaction were observed. The release of HRP from the binding sites as determined by the disappearance of the cytochemical reaction was 50-100 times faster at 22 degrees C than at 4 degrees C and was 5-10 times faster at 37 degrees C than at 22 degrees C. The release was approximately twice as fast at pH 7.0 than at pH 9.0 and 20-30 times faster at pH 6.0 than at pH 7.0. The release of HRP was 10-15 times faster in the absence of 1 mM Ca2+ in the buffer solution and was approximately 100 times faster in the presence of 0.1 M D-mannose as compared to 0.1 M D-galactose. Pretreatment of the sections with trypsin abolished the binding reaction whereas neuraminidase, phospholipases A2 and C, and chondroitinase ABC were without effect. An acidic isoenzyme of HRP, Sigma type VIII, was bound more intensely and more widely to liver sinusoidal cells than another acidic isoenzyme, Sigma type VII, a basic isoenzyme, Sigma type IX, and the routinely used preparation, Sigma type VI. The effect of the temperature on the binding reaction was re-examined with an improved procedure. In contradistinction to the previous finding, strong binding of HRP after 2-4 h incubation at 4 degrees C was observed.     

Horseradish peroxidase catalyzed degradation of industrially important dyes.

Horseradish peroxidase (HRP) is known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols. Here, for the first time we have shown HRP to be effective in degrading and precipitating industrially important azo dyes. For Remazol blue, the enzyme activity was found to be far better at pH 2.5 than at neutral pH. In addition, Remazol blue acts as a strong competitive inhibitor of HRP at neutral pH. Horseradish peroxidase shows broad substrate specificity toward a variety of azo dyes. Kinetic constants (K(m)(app) and V(max)(app)) for two different dyes have been determined. In addition to providing a systematic analysis of the potential of HRP in degradation of dyes, this study opens up a new area on exploration of commercial dyes as inhibitors of enzymes. 2001 John Wiley & Sons, Inc.     

Receptor-mediated uptake of horseradish peroxidase in innervated and denervated skeletal muscle

The in vitro uptake of [3H]inulin and horseradish peroxidase (HRP) has been studied in innervated and 6 days denervated extensor digitorum longus muscle of the mouse. Both markers were taken up at a higher rate in denervated muscle. The increase in uptake after denervation was, however, larger for HRP than for [3H]inulin. After 2 h incubation at 37 degrees C, pH 7.3, in the presence of equimolar concentrations of HRP and [3H]inulin (approx. 2.1 microM), the uptake of HRP was approx. 8 times as great as the uptake of [3H]inulin in the same innervated muscles. In denervated muscle the HRP uptake was approx. 19 times as great as the [3H]inulin uptake in the same muscles. Various possible explanations of these differences in uptake have been considered and tested experimentally. [3H]Inulin uptake in skeletal muscle has previously been shown to obey bulk kinetics. The present investigation shows the HRP uptake to obey saturation kinetics. The HRP uptake shows dependency on divalent cations and is reduced if incubation is carried out at pH 6.4. The uptake of HRP, when used at a low, non-saturating concentration (10 micrograms/ml approx. 0.25 microM), is inhibited greater than or equal to 60% by yeast mannan (0.1 mg/ml), ribonuclease B (0.1 mg/ml, approx. 7.4 microM), mannose (30 mM), monodansylcadaverine (1 mM), chloroquine (100 microM), trifluoperazine (25 microM) or maleic acid (2 mM). It is concluded that HRP is taken up in innervated and denervated skeletal muscle by a process of receptor-mediated endocytosis and that this uptake is under neurotrophic control.