Tracing of the Bile-chemotactic migration of juvenile Clonorchis sinensis in rabbits by PET-CT

Background

Adult Clonorchis sinensis live in the bile duct and cause clonorchiasis. It is known that the C. sinensis metacercariae excyst in the duodenum and migrate up to the bile duct through the common bile duct. However, no direct evidence is available on the in vivo migration of newly excysted C. sinensis juveniles (CsNEJs). Advanced imaging technologies now allow the in vivo migration and localization to be visualized. In the present study, we sought to determine how sensitively CsNEJs respond to bile and how fast they migrate to the intrahepatic bile duct using PET-CT.

Methodology/Principal Findings

CsNEJs were radiolabeled with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG). Rabbits with a gallbladder contraction response to cholecystokinin-8 (CCK-8) injection were pre-screened using cholescintigraphy. In these rabbits, gallbladders contracted by 50% in volume at an average of 11.5 min post-injection. The four rabbits examined were kept anesthetized and a catheter inserted into the mid duodenum. Gallbladder contraction was stimulated by injecting CCK-8 (20 ng/kg every minute) over the experiment. Anatomical images were acquired by CT initially and dynamic PET was then carried out for 90 min with a 3-min acquisition per frame. Twelve minutes after CCK-8 injection, about 3,000 ¹⁸F-FDG-labeled CsNEJs were inoculated into the mid duodenum through the catheter. Photon signals were detected in the liver 7–9 min after CsNEJs inoculation, and these then increased in the whole liver with stronger intensity in the central area, presenting that the CsNEJs were arriving at the intrahepatic bile ducts.

Conclusion

In the duodenum, CsNEJs immediately sense bile and migrate quickly with bile-chemotaxis to reach the intrahepatic bile ducts by way of the ampulla of Vater.     

Sequence Analysis and Molecular Characterization of Clonorchis sinensis Hexokinase,an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors of CsHK to interfere with glycolysis in C. sinensis.     

Advanced Enzymology,Expression Profile and Immune Response of Clonorchis sinensis Hexokinase Show Its Application Potential for Prevention and Control of Clonorchiasis

BackgroundApproximately 35 million people are infected with Clonorchis sinensis (C. sinensis) globally, of whom 15 million are in China. Glycolytic enzymes are recognized as crucial molecules for trematode survival and have been targeted for vaccine and drug development. Hexokinase of C. sinensis (CsHK), as the first key regulatory enzyme of the glycolytic pathway, was investigated in the current study.Conclusions/SignificanceDue to differences in putative spatial structure and enzymology between CsHK and HK from the host, its extensive distribution in adult worms, and its expression profile as a component of excretory/secretory products, together with its good immunogenicity and immunoreactivity, as a key glycolytic enzyme, CsHK shows potential as a vaccine and as a promising drug target for Clonorchiasis.     

The Hsp70 chaperone Ssa1 is essential for catabolite induced degradation of the gluconeogenic enzyme fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme of gluconeogenesis. In the yeast Saccharomyces cerevisiae, it is only expressed when cells are grown in medium with nonfermentable carbon sources. Addition of glucose to cells leads to inactivation of FBPase and degradation via the ubiquitin-proteasome system. Polyubiquitination of FBPase is carried out by the Gid complex, a multi-subunit ubiquitin ligase. Using tandem affinity purification and subsequent mass spectrometry we identified the Hsp70 chaperone Ssa1 as a novel interaction partner of FBPase. Studies with the temperature-sensitive mutant ssa1-45^ts showed that Ssa1 is essential for polyubiquitination of FBPase by the Gid complex. Moreover, we show that degradation of an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is also affected in ssa1-45^ts cells demonstrating that Ssa1 plays a general role in elimination of gluconeogenic enzymes.     

Fishborne Trematode Metacercariae in Freshwater Fish from Guangxi Zhuang Autonomous Region, China

A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.     

Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis

Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The K_m and V_max values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min^?1 mg protein^?1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.     

Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.     

Physico-kinetic and functional features of a novel β-glucosidase isolated from milk thistle (Silybum marianum Gaertn.) flower petals

A highly abundant β-glucosidase from petals of Silybum marianum has been purified and characterized for its physico-kinetic properties. The 135 kDa enzyme was a homodimer with subunit molecular mass of 67.6 kDa. The characteristic catalytic properties of the enzyme included acidic pH optimum (5.5), meso-thermostability, and β-linked substrate specificity with preference for gluco-conjugate but a marked (>50 %) activity with D-fuco-conjugates and considerable (~16 %) activity towards D-galacto-conjugates. The enzyme showed high affinity for p-nitrophenyl glucoside (pNPG) with K_m and V_max values of 0.25 mM and 5.35 μkat.mg^?1 enzyme protein. Thus, the enzyme had a very high (292,000 M^?1.s^?1) catalytic efficiency (K_cat/K_m). Thermal catalytic optimum of enzyme was 40 °C with activation energy value 8.26 kCal.Mol^?1. The enzyme showed significant insensitivity to D-gluconic acid lactone inhibition (57 % at 5 mM) with an apparent K_i 3.8 mM. The transglucosylating ability of enzyme was noticed for glucosylation of geraniol and withaferin-A with pNPG as glucosyl donor but cellobiose did not serve as the glycosyl donor. Partial proteomics of the enzyme revealed two peptide fragment sequences, VTPSNEVH and KRSEESNF. These motifs showed significant matching/sequence conservation with some other glycohydrolases. The novelties of purified enzyme hold potential to expand a library of catalytically characteristic members of the hydrolase family from plants for use in biotransformation applications.     

Purification,kinetic studies,and homology model of Escherichia coli fructose-1,6-bisphosphatase

Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The k_cat of the E. coli FBPase was 14.6 s⁻¹ as compared to 21 s⁻¹ for the pig kidney enzyme, while the K_m of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg²⁺ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg²⁺, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg²⁺ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.     

Regulation of Vid-dependent degradation of FBPase by TCO89, a component of TOR Complex 1

A pivotal gluconeogenic enzyme in Saccharomyces cerevisuae, fructose-1, 6-bisphosphatase (FBPase) was selectively turned over in vacuole via Vid (vacuole import and degradation) dependent pathway in response to the fresh glucose after chronic glucose starvation. TCO89, a novel and unique component of Tor Complex I (TORCI), was found to physically associate with FBPase and significantly affect FBPase degradation via Vid pathway. Further investigation indicated that Δtco89 mutant strongly impaired FBPase''s importing into Vid vesicles and Vid24''s association with Vid vesicles. Inactivation of TORCI by rapamycin treatment strongly blocked FBPase degradation. Other components of TORCI were also found to physically associate with FBPase. The P1S mutation of FBPase, reported to block its degradation, was observed to impair the association of FBPase with TORCI components. These results implicated an important regulatory role of TCO89 and TORCI in this pathway.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn²⁺ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.     

Fructose-1,6-bisphosphatase from a cold-hardy insect: Control of cryoprotectant glycerol catabolism

Fructose 1,6-bisphosphatase (FBPase) from the larvae of the gall moth, Epiblema scudderiana, was purified to homogeneity with a final specific activity of 1.6 U/mg protein. The enzyme had a native molecular weight of 74.0 ± 6.5 kD and a subunit molecular weight of 37.6 ± 3.0 kD; the dimeric structure of the enzyme in this species is unusual. The pH optimum was 7.00 in imidazole buffer at 22°C and rose to 7.31 at 5°C. An Arrhenius plot of enzyme activity vs. temperature was linear with an activation energy of 91 ± 4.1 kJ/mol^?1. K_m values for FBPase decreased from 4.7 ± 0.34 μM at 22°C to 1.3 ± 0.05 μM at 5°C. No allosteric activators were identified, but the enzyme was inhibited by fructose 2,6-bisphosphate (F2,6P₂), AMP, ADP, dihydroxyacetonephosphate, glycerol, and KCI. Inhibition by AMP and F2,6P₂ increased at low temperature, and effects of these compounds may be key to preventing futile cycling of carbon at the FBPase/phosphofructokinase loci during the biosynthesis of glycerol cryoprotectant. Oppositely, glycerol clearance in the spring and reconversion into glycogen is promoted by interactions of temperature, inhibitors, and glycerol that promote FBPase activity: I₅₀ values for AMP and F2,6P₂ increase at 22°C (compared with 5°C), high glycerol levels override F2,6P₂ inhibition of the enzyme, and deinhibitors (ATP, citrate) partially reverse AMP inhibition of the enzyme. © 1995 Wiley-Liss, Inc.     

Functional properties of a manganese-activated exo-polygalacturonase produced by a thermotolerant fungus Aspergillus niveus

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40 °C, presenting protein levels almost twofold higher than at 25 °C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7 % carbohydrate, molecular mass of 102.6 kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50 °C and 4.0–6.5, respectively. The enzyme was stable at pH 3.0 to 9.0 for 24 h. The DEAE-cellulose derivative was about sixfold more stable at 60 °C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232 % activated by Mn²⁺. The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K _m, V _max, K _cat, and K _cat/K _m values were 6.7 mg/ml, 230 U/mg, 393.3/s, and 58.7 mg/ml/s, respectively. The N-terminal amino acid sequence presented 80 % identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.     

Comparative physico-kinetic properties of a homogenous purified β-glucosidase from Withania somnifera leaf

β-glucosidase from Withania somnifera (Solanaceae) leaf has been purified to homogeneity and characterized for its physico-kinetic properties. The enzyme purification was achieved through a sequence of gel filtration and ion-exchange column chromatography, and PAGE revealed the homogeneity purification status of the enzyme. The properties of the enzyme included an acidic pH optima (4.8), alkaline pI (8.7), meso-thermostabity, monomeric structure with subunit molecular weight of about 50 kDa, high affinity for substrate (K _m) for pNPG (0.19 mM) and high (105,263 M^?1 s^?1) catalytic efficiency (K _cat/K _m). The mesostable enzyme had a stringent substrate specificity restricted only to β-linked gluco-conjugate. The enzyme is optimally active at 40 °C with 12.4 kcal Mol^?1 activation energy, and was highly sensitive to d-gluconic acid lactone inhibition (94 % at 1 mM) with an apparent K _i 0.21 mM. The enzyme could catalyze transglucosylation of geraniol with pNPG as glucosyl donor, but not with cellobiose. Some of the physico-kinetic properties were noted to be novel when comprehensively compared with its counterparts from plant, animal and microbial counterparts. Nevertheless, the catalytic and other features of the enzyme were relatively closer to Oryza sativa among plants and Talaromyces thermophillus among fungi. Significance of building-up of a library of novel plant β-glucosidases for structural investigation to understand naturally evolved mechanistics of catalysis has been indicated.     

Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca

Glucose isomerase (GIase) catalyzes the isomerization of d-glucose to d-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5–10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K _m and K _cat values of the enzyme are 197 mM and 1,688 min^?1, respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

Thermostable alkaline protease from Thermomyces lanuginosus: optimization,purification and characterization

A serine alkaline protease (EC.3.4.21) was isolated, purified and characterized from culture filtrate of the thermophilic fungus Thermomyces lanuginosus Tsiklinsky. Fructose (1.5 %) and gelatin (0.5 %) proved to be the best carbon and nitrogen sources, giving a maximum enzyme yield of 9.2 U/mL. Dates waste was utilized as a sole organic source to improve enzyme productivity, and the yield was calculated to be 11.56 U/mL. This yield was expressed also as 231.2 U/g of assimilated waste. The alkaline protease produced was precipitated by iso-propanol and further purified by gel filtration through Sephadex G-₁₀₀ and ion exchange column chromatography on diethyl amino ethyl (DEAE)-cellulose with a yield of 30.12 % and 13.87-fold purification. The enzyme acted optimally at pH 9 and 60 °C and had good stability at alkaline pH and high temperatures. The enzyme possessed a high degree of thermostability and retained full activity even at the end of 1 h of incubation at 60 °C. Michaelis–Menten constant (K _m), maximal reaction velocity (V _max) and turnover number (K _cat) of the purified enzyme on gelatin as a substrate were calculated to be 4.0 mg/mL, 18.5 U/mL and 1.8 s^?1, respectively. The best enzyme activators were K⁺, Ca²⁺ and Mn², respectively, while phenylmethylsulfonyl fluoride (PMSF) was the strongest inhibitory agent, thus suggesting that the enzyme is a serine type protease. The enzyme is a glycoprotein with molecular mass of 33 kDa as determined by SDS-PAGE. It retained full activity after 15 min incubation at 60 °C in the presence of the detergent Ariel, thus indicating its suitability for application in the detergent industry.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.