Maintenance Of Enzyme Activity Research Articles

Bio-compatible n-HAPs/polymer monolithic composites templated from CO2-in-water high internal phase emulsions

According to the applications of materials in food and biology etc. fields, the open-cell macroporous polymer composite must contain a non-toxic component, and be bio-compatible, and the preparation processes should be green and facile. Herein, bio-compatible macro-porous nano-hydroxyapatite particles (n-HAPs)/polymer monolithic composites were templated from green and facile CO2-in-Water high internal phase emulsion (C/W HIPE). The bio-active n-HAPs that acted as emulsifiers were incorporated, which enable the well-dispersion of functional fillers in these monolithic composites. Green and facile C/W HIPE templating combined with radical polymerization of bio-compatible monomers involves nontoxic chemicals, and produced polymer monolith with macro-porous open-cell structure (80–120 μm in pore size, 20–40 μm in pore-throat size), and enable the n-HAPs/polymer improved flexibility and mechanical performance (above 80% compression strain, > 400 kPa of compression strength). Results of bio-compatibility tests show that these n-HAPs/polymer monoliths have non-cytotoxicity on HepG2 cells; Furthermore, the immobilization of pectinase by the composite indicates that composites have physical adsorption of pectinase, and the introduction of n-HAPs is more conducive to the immobilization of pectinase and the improvement and maintenance of enzyme activity; after reusing the immobilized pectinase 10 times, the relative catalytic activity can still be above 85%. Therefore, this kind of porous composite has promising potential in applications of bio-tissue engineering, food, and pharmaceutical.

A kinetic model of phospholipase C-γ1 linking structure-based insights to dynamics of enzyme autoinhibition and activation

Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain–pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.

Polymers as Encapsulating Agents and Delivery Vehicles of Enzymes.

Enzymes are versatile biomolecules with broad applications. Since they are biological molecules, they can be easily destabilized when placed in adverse environmental conditions, such as variations in temperature, pH, or ionic strength. In this sense, the use of protective structures, as polymeric capsules, has been an excellent approach to maintain the catalytic stability of enzymes during their application. Thus, in this review, we report the use of polymeric materials as enzyme encapsulation agents, recent technological developments related to this subject, and characterization methodologies and possible applications of the formed bioactive structures. Our search detected that the most explored methods for enzyme encapsulation are ionotropic gelation, spray drying, freeze-drying, nanoprecipitation, and electrospinning. α-chymotrypsin, lysozyme, and β-galactosidase were the most used enzymes in encapsulations, with chitosan and sodium alginate being the main polymers. Furthermore, most studies reported high encapsulation efficiency, enzyme activity maintenance, and stability improvement at pH, temperature, and storage. Therefore, the information presented here shows a direction for the development of encapsulation systems capable of stabilizing different enzymes and obtaining better performance during application.

Magnetic COFs as satisfactory support for lipase immobilization and recovery to effectively achieve the production of biodiesel by maintenance of enzyme activity

BackgroundProduction of biodiesel from renewable sources such as inedible vegetable oils by enzymatic catalysis has been a hotspot but remains a challenge on the efficient use of an enzyme. COFs (Covalent Organic Frameworks) with large surface area and porosity can be applied as ideal support to avoid aggregation of lipase and methanol. However, the naturally low density limits its application. In this work, we reported a facile synthesis of core–shell magnetic COF composite (Fe3O4@COF-OMe) to immobilize RML (Rhizomucor miehei lipase), to achieve its utilization in biodiesel production.ResultThis strategy gives extrinsic magnetic property, and the magnetic COFs is much heavier and could disperse in water medium well, facilitating the attachment with the enzyme. The resultant biocomposite exhibited an excellent capacity of RML due to its high surface area and fast response to the external magnetic field, as well as good chemical stability. The core–shell magnetic COF-OMe structure not only achieved highly efficient immobilization and recovery processes but also maintained the activity of lipase to a great extent. RML@Fe3O4@COF-OMe performed well in practical applications, while free lipase did not. The biocomposite successfully achieved the production of biodiesel from Jatropha curcas Oil with a yield of about 70% in the optimized conditions.ConclusionMagnetic COFs (Fe3O4@COF-OMe) for RML immobilization greatly improved catalytic performance in template reaction and biodiesel preparation. The magneticity makes it easily recovered and separated from the system. This first successful attempt of COFs-based immobilized enzyme broadened the prospect of biodiesel production by COFs with some inspiration.

Gene expression and activity of antioxidant enzymes in rice plants, cv. BRS AG, under saline stress.

The rice cultivar (Oryza sativa L.) BRS AG, developed by Embrapa Clima Temperado, is the first cultivar designed for purposes other than human consumption. It may be used in ethanol production and animal feed. Different abiotic stresses negatively affect plant growth. Soil salinity is responsible for a serious reduction in productivity. Therefore, the objective of this study was to evaluate the gene expression and the activity of antioxidant enzymes (SOD, CAT, APX and GR) and identify their functions in controlling ROS levels in rice plants, cultivar BRS AG, after a saline stress period. The plants were grown in vitro with two NaCl concentrations (0 and 136mM), collected at 10, 15 and 20days of cultivation. The results indicated that the activity of the enzymes evaluated promotes protection against oxidative stress. Although, there was an increase of reactive oxygen species, there was no increase in MDA levels. Regarding genes encoding isoforms of antioxidant enzymes, it was observed that OsSOD3-CU/Zn, OsSOD2-Cu/Zn, OsSOD-Cu/Zn, OsSOD4-Cu/Zn, OsSODCc1-Cu/Zn, OsSOD-Fe, OsAPX1, OsCATB and OsGR2 were the most responsive. The increase in the transcription of all genes among evaluated isoforms, except for OsAPX6, which remained stable, contributed to the increase or the maintenance of enzyme activity. Thus, it is possible to infer that the cv. BRS AG has defense mechanisms against salt stress.

Biochemical and milk-clotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium.

For a long time, proteolytic enzymes have been employed as key tools of industrial processes, especially in the dairy industry. In the present work, we used Phanerochaete chrysosporium for biochemical characterization and analysis of catalytic specificity of an aspartic peptidase. Our results revealed an aspartic peptidase with molecular mass ∼38kDa, maximal activity at pH 4.5 and 50°C, and stability above 80% in the pH range of 3-8 and temperature up to 55°C for 1h. In a milk-clotting assay, this peptidase showed maximal milk clotting activity at 60-65°C and maintenance of enzymatic activity above 80% in the presence of 20mM CaCl2. In a specificity assay, we observed stronger restriction of catalysis at the S1 subsite, with a preference for lysine, arginine, leucine, tyrosine, and phenylalanine residues. The restricted proteolysis and milk-clotting potential are attractive properties for the use in cheese production.

Zwitterionic polymer-protein conjugates reduce polymer-specific antibody response

Summary Proteins are promising therapeutics with several design challenges, such as their inherent immunogenicity due to their exogenous source or short circulation time. The common solution to such issues is the chemical conjugation of the amphiphilic polymer poly(ethylene glycol) (PEG), a process known as PEGylation. However, several studies demonstrated a decrease in protein bioactivity or an increase in the presence of specific antibodies post PEGylation, highlighting the importance of an alternative strategy. Here we compare the performance of a superhydrophilic zwitterionic polymer in protecting an immunogenic protein to PEG in (i) the maintenance of enzyme activity and stability post modification, (ii) mitigation of the antibody response elicited by the polymer and any subsequent effect on pharmacokinetics, and (iii) minimization of host response toward the underlying protein. In contrast to PEG, zwitterionic conjugation decreases host antibody response without sacrificing bioactivity or generating polymer-specific antibodies.

Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability.

BackgroundCrotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV.MethodsThe enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromatography to confirm its purity. Subsequently, its N-terminal amino acid sequence was determined by Edman degradation. The enzyme activity and stability were evaluated by a microplate colorimetric assay and the molecular mass was estimated by SDS-PAGE using periodic acid-Schiff staining and determined by mass spectrometry.ResultsThe first 39 N-terminal amino acid residues exhibited high identity with other snake venom L-amino acid oxidases. Bordonein-L is a homodimer glycoprotein of approximately 101 kDa evaluated by gel filtration. Its monomer presents around 53 kDa estimated by SDS-PAGE and 58,702 Da determined by MALDI-TOF mass spectrometry. The enzyme exhibited maximum activity at pH 7.0 and lost about 50 % of its activity after five days of storage at 4 °C. Bordonein-L’s activity was higher than the control when stored in 2.8 % mannitol or 8.5 % sucrose.ConclusionsThis research is pioneering in its isolation, characterization and enzyme stability evaluation of an LAAO from CdtV, denominated bordonein-L. These results are important because they increase the knowledge about stabilization of LAAOs, aiming to increase their shelf life. Since the maintenance of enzymatic activity after long periods of storage is essential to enable their biotechnological use as well as their functional studies.

Comparison of in vitro methods for carboxylesterase activity determination in immortalized cells representative of the intestine, liver and kidney

Herein we compare the fluorimetric determination of total and specific carboxylesterase activity in immortalized human derived living cells and in cell lysates. The cell lines chosen are representative of metabolism occurring in the intestine (Caco-2 and HT-29), kidney (HEK-293T) and liver (Hep G2). Caco-2 and HT-29, as cells prone to differentiation, were tested along the differentiation time. For evaluation of both methods when distinguishing activity of different carboxylesterases, HEK-293T transfected with the human carboxylestarase-2 (hCES2) were also tested.Application to Caco-2 or HT-29 cells demonstrated higher activity detected in cell lysates than in cell monolayers. The difference is most striking when comparing the methods at different stages of Caco-2 and HT-29 cell maturation, highlighting substrate accessibility as a limiting step in the in vivo hydrolysis rates (possibly limited by plasma and Endoplasmic Reticulum membrane permeability) with increasing relevance as the cells differentiate. Application to Hep G2 or to hCES2 transfected and non-transfected HEK-293T cells, demonstrated a tendency for higher sensitivity in living cell suspensions than that obtained with the cell lysates which indicates the importance of cell environment in the maintenance of enzyme activity. However, quantification of hCES2 activity relative to total esterase, or to total carboxylesterase activity, was not significantly different in any case.The results herein presented help to clarify which method is best suited for evaluation of carboxylesterase activity in vitro depending on the final goal of the study.

Radio-synthesized polyacrylamide hydrogels for proteins release

The use of hydrogels for biomedical purposes has been extensively investigated. Pharmaceutical proteins correspond to highly active substances which may be applied for distinct purposes. This work concerns the development of radio-synthesized hydrogel for protein release, using papain and bovine serum albumin as model proteins. The polymer was solubilized (1% w/v) in water and lyophilized. The proteins were incorporated into the lyophilized polymer and the hydrogels were produced by simultaneous crosslinking and sterilization using γ-radiation under frozen conditions. The produced systems were characterized in terms of swelling degree, gel fraction, crosslinking density and evaluated according to protein release, bioactivity and cytotoxicity. The hydrogels developed presented different properties as a function of polymer concentration and the optimized results were found for the samples containing 4–5% (w/v) polyacrylamide. Protein release was controlled by the electrostatic affinity of acrylic moieties and proteins. This selection was based on the release of the proteins during the experiment period (up to 50h), maintenance of enzyme activity and the nanostructure developed. The system was suitable for protein loading and release and according to the cytotoxic assay it was also adequate for biomedical purposes, however this method was not able to generate a matrix with controlled pore sizes.

Sustained Delivery of Chondroitinase ABC from HydrogelSystem.

In the injured spinal cord, chondroitin sulfate proteoglycans (CSPGs) are the principal responsible of axon growth inhibition and they contribute to regenerative failure, promoting glial scar formation. Chondroitinase ABC (chABC) is known for being able to digest proteoglycans, thus degrading glial scar and favoring axonal regrowth. However, its classic administration is invasive, infection-prone and clinically problematic. An agarose-carbomer (AC1) hydrogel, already used in SCI repair strategies, was here investigated as a delivery system capable of an effective chABC administration: the material ability to include chABC within its pores and the possibility to be injected into the target tissue were firstly proved. Subsequently, release kinetic and the maintenance of enzymatic activity were positively assessed: AC1 hydrogel was thus confirmed to be a feasible tool for chABC delivery and a promising device for spinal cord injury topic repair strategies.

Characterization of a keratinase produced by Bacillus sp. P7 isolated from an Amazonian environment

The Amazonian bacterium Bacillus sp. P7 efficiently degraded feather keratin during submerged cultivations, producing extracellular keratinolytic enzymes. Keratinase produced during growth on feather meal broth was partially purified by ammonium sulphate precipitation, gel filtration, and ion-exchange chromatography, resulting in a purification factor of 29.8-fold and a yield of 27%. Zymography revealed two proteolytic bands, mainly inhibited by phenylmethylsulfonyl fluoride (PMSF). Partially purified keratinase had optimal activity at 55 °C and pH 9.0, was stimulated by Ca 2+ and Mg 2+, and was inhibited by Hg 2+, Cu 2+ and Zn 2+. Organic solvents 2-mercaptoethanol and Triton X-100 slightly affected the enzyme activity, whereas SDS stimulated it. PMSF and ethylenediaminetetraacetic acid (EDTA) inhibited proteolytic activity, which suggests its serine-protease feature, with the requirement of metal ions for maximum activity and/or stability. Alkaline keratinase might be employed in detergent formulations, in leather processing, and in other processes involving protein hydrolysis. The maintenance of enzyme activity in the presence of reducing agent (2-mercaptoethanol) makes this partially purified keratinase interesting for application in the breakdown of recalcitrant keratin wastes.

Cutinase inhibition by means of insecticidal organophosphates and carbamates

Due to the extensive use of pesticides in agriculture there is an increasing demand for fast performable methods for residue analysis at official food control. This report is concerned with the development of a spectrophotometric enzyme assay in 96-well plate format, with the aim to enable simultaneous screening of multiple samples for organophosphorus and carbamate insecticides by means of their inhibitory effect on cutinase from Fusarium solani pisi. Reaction time of the assay is 30 min. The influence of various solvents on cutinase activity was studied in order to optimize the loss of enzyme activity under organic solvent conditions with respect to pesticide solubility. Methanol concentrations of up to 2.5% in the inhibition batch exhibit negligible influence on cutinase activity. Aqueous mixtures of 10% methanol and 40% diethylene glycol are advantageously bridging restricted pesticide solubility with maintenance of enzyme activity. The detection limit of paraoxon, a potent cutinase inhibitor with an inhibitory rate constant of 1.6×104 L/(mol min), was determined to 0.04 mg/L. For chlorpyrifos, the most often detected pesticide on fruit and vegetables, the detection limit was 2.6 mg/L with an inhibition constant of 2.0×103 L/(mol min). Working principle and efficiency of the assay are discussed in detail in terms of enzyme kinetics.

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.

Active Kinase Proteome Screening Reveals Novel Signal Complexity in Cardiomyopathy

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions.

Sol–gel derived urease-based optical biosensor for the rapid determination of heavy metals

A urease optical biosensor for the determination of heavy metals based on sol–gel immobilization technique was developed. A fluorescent dye, FITC-dextran, was encapsulated and parameters including optical properties of the probe, relative enzyme activity, initial pH value and the buffer concentration for substrate preparation were investigated. In sol–gel immobilization, 1 mM Tris–HCl at pH 7.1 provided a sufficient buffer capacity for metal ion analysis as well as the enzyme activity maintenance. Also, two analytical procedures, incubated and un-incubated systems, were compared to understand the sensitivity and applicability to heavy metal analysis. The developed optical biosensor showed high reproducibility and the relative standard deviation (R.S.D.) of 5.1% ( n=10) was obtained. Also, eight measurements can be completed automatically within 36 min. The biosensor has high sensitivity to Cu(II) and Cd(II) and an analytical range of 10–230 μM with a detection limit of 10 μM was achieved. Moreover, biological and environmental samples were examined to evaluate the applicability of the developed biosensor. A 19–82% of inhibition was observed when 20–45 μM metal ions were amended into tested samples, revealing that the developed system has the potential for the determination of heavy metals in real samples.

Capillary isoelectric focusing: the problem of protein solubility

A whole family of protein solubilizers, compatible with native structure and maintenance of enzyme activity, is reported for preventing protein precipitation and aggregation at the p I value under conditions of very low ionic strength, as typical of isoelectric focusing methodologies. In addition to mild solubilizers proposed in the past, such as glycols (glycerol, ethylene and propylene glycols) non-detergent sulphobetaines, in concentrations up to 1 M, are found to be quite effective in a number of cass. Other common zwitterions, such as taurine and a few of the Good's buffers (e.g., Bicine, CAPS) are also quite useful in acidic pH gradients and up to pH 8. Addition of sugars, notably saccharose, sorbitol and, to a lesser extent, sorbose (20% in capillary IEF and in the 30 to 40% concentration range in gel-slab IPGs), greatly improved protein solubility in the proximity of the p I. The improvement was dramatic if these sugars were mixed with 0.2 M taurine. In the case of hydrophobic peptide antibiotics, mixtures of 6 M urea and 25% trifluoroethanol were found to markedly improve solubility. All these additives, unlike non-ionic or zwitterionic surfactant, have the advantage of remaining monomeric, i.e., of being unable to form micelles, even at concentrations > 1 M. Thus, their elimination from the protein zone can be easily accomplished by gel filtration or by centrifugation through dialysis membranes. Using these additives, capillary IEF of proteins should be now applicable to a number of difficult cases, such as the separation of mildly hydrophobic macroions.

Soil enzyme activities after 1500 years of terrace agriculture in the Colca Valley, Peru

Long-term productivity and conservation of soils is critical for sustaining agricultural ecosystems. Long-term sites can provide important information about the effects of soil management practices on soil properties but there are relatively few such sites available worldwide. The Colca Valley of Peru provided a unique opportunity to study the effects of 1500 years of cultivation on Mollisols. The specific objective of the work reported was to determine the effects of cultivation at this site on soil enzyme activity as an index of soil biology and biochemistry. The study compared three key soil enzyme activities (phosphatase, β-glucosidase, and amidase) in presently cultivated and abandoned agricultural terraces, and matched uncultivated soils. Results showed that levels of organic matter, nitrogen and phosphorus were greater in agricultural than uncultivated soils. Unlike temperate regions where monoculture, intensive tillage and/or inorganic fertilizer practices have depressed soil enzyme activities, cultivated Colca soils have maintained similar or higher activities than the uncultivated/native soils. Maintenance of enzyme activities over hundreds of years in agricultural soils is partly attributed to traditional management practices including rotations with legumes, additions of animal manures, and minimum tillage.

An investigation into the significance of the N-linked oligosaccharides of Leishmania gp63

Leishmania major promastigotes, when grown in the presence of tunicamycin (TM), produced a plasma membrane-bound, proteolytically active gp63 with a lower molecular weight than the native glycoprotein. However, this lower molecular weight form of gp63 continued to be recognized by concanavalin A (Con A), suggesting that inhibition of N-linked glycosylation was not complete. Metabolic labeling of gp63, using [ 35S]methionine, demonstrated that in the range of 5–10 μg ml − TM, only the lower molecular weight form was synthesized, suggesting that inhibition was complete and that lectin binding was likely due to the GPI anchored sugars. Removal of the oligosaccharides from L. major and L. mexicana amazonensis promastigotes using endoglycosidase F, caused the gp63 molecular weight to decrease to the same value observed in the presence of TM, once again without affecting the proteolytic activity. However, this deglycosylated enzyme continued to bind Con A until subsequently treated with periodate. The latter oxidation reaction resulted in complete loss of Con A binding without inhibiting the protease activity or the substrate specificity of gp63. Further investigations revealed that both glycosylated and deglycosylated gp63 were resistant to proteolytic digestion by either autolysis or cathepsin D. These findings indicate that the N-linked oligosaccharides of gp63 are not essential for folding, transport, maintenance of enzyme activity or resistance to proteolysis.

Expression of human aspartyl-tRNA synthetase in COS cells.

Mammalian aspartyl-tRNA synthetase (DRS) occurs in a multi-enzyme complex of aminoacyl-tRNA synthetases, while DRS exists as free soluble enzymes in bacteria and yeast. The properties of human DRS transient expressed in COS cells were examined. After transfection of COS cells with the recombinant plasmids pSVL-63 that contained hDRS cDNA coding and non-coding sequences, and pSV-hDRS where the non-coding sequences were deleted, DRS in the transfected COS cells significantly increased compared to mock transfected cells. COS cells transfected with pSV-hDRS delta 32 that contained N-terminal 32 residue-coding sequence deleted hDRS cDNA showed no increase in DRS activity. Northern blot analysis showed that concentrations of corresponding mRNAs of hDRS and hDRS delta 32 were greatly enhanced in transfected cells. The increases in the level of the transcripts were much higher than those of the corresponding proteins. Gel filtration analysis showed that hDRS in pSV-hDRS transfected cells expressed as a low molecular weight form of hDRS and pSV-hDRS delta 32 transfected cells did not. Epitope tagging and indirect immunofluorescence microscopy was used to localize hDRS. Both hDRSmyc and hDRS delta 32myc were localized in the cytoplasm and showed diffused patterns. These results showed that hDRS has little tendency to aggregate in vivo and suggested that the N-terminal extension in hDRS was not involved in the expression and sub-cellular localization of hDRS, but may play a role in the maintenance of enzymatic activity of hDRS in COS cells.