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1.
Anionic trypsin from Pacific saury (Cololabis saira) pyloric ceca was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. It was purified to 53.7-fold with a yield of 6.1%. The apparent molecular weight of the enzyme was about 24 kDa, as determined by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On native-PAGE, trypsin showed a single band. The purified anionic trypsin displayed optimal activity at pH 8.5 and 55°C. The enzyme was stable at neutral and alkaline pH and in the temperature range of 20–50°C. The stability was affected by the calcium ion. The activity of purified anionic trypsin was completely inhibited by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and partially inhibited by ethylenediaminetetraacetic acid (EDTA). NaCl (0–30%) decreased the activity in a concentration-dependent manner. The kinetic trypsin constants Km and Kcat were 0.19 mM and 210 s?1, respectively, while the catalytic efficiency (Kcat/Km) was 1105.26 s?1 mM?1. The N-terminal amino acid sequences of anionic trypsin, IVGGYECQAH, were found and were homologous to those of trypsin from other fish species.  相似文献   

2.
Trypsin, with molecular weight of 28 kDa from the intestine of genetically improved Nile tilapia (Oreochromis niloticus), was purified by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. Purified trypsin had maximal activity at pH 8.0 and 60°C for hydrolysis of N α-p-tosyl-L-arginine methyl ester. The enzyme was stable at temperatures up to 50°C and pH range of 6.0–11.0. Its activity was strongly inhibited by metal ions such as Pb2+ and Fe3+ and protease inhibitors including soybean trypsin inhibitor and phenylmethylsulfonyl fluoride. Also, the ion Ca2+ slightly inhibited this activity. The Michaelis-Menten constant (K m) and catalytic constant (K cat) of purified trypsin were 0.036 mM and 152 s?1, respectively. Furthermore, trypsin contained low amounts of hydrophobic and aromatic amino acids as well as β-sheet (20.2%) and β-turn (25.0%).  相似文献   

3.
The purification of trypsin from the common kilka (Clupeonella cultriventris caspia) viscera (pyloric caeca) resulted in a 28.3-fold increase and 12% yield by ammonium sulfate precipitation (30–60%), Sephadex G-75, and DEAE-cellulose chromatography. Trypsin showed a molecular weight of 23.2 kDa and appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, and zymography. The trypsin had optimal activity at pH 8.0 and 60°C for the hydrolysis of α-N-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) substrate. Trypsin was stable up to 50°C and at pH range of 7.0–10.0. Activity was significantly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) inhibitors (p < 0.05). The enzyme was relatively stable toward oxidizing agents, retaining 59.7 and 98.0% of its initial activity after 1 h incubation in the presence of 15% H2O2 and 1% sodium perborate, respectively. Trypsin was significantly activated by surfactants and Ca2+, Mg2+, and Mn2+ and inactivated by Fe2+, Zn2+, Cu2+, Al3+, Ba2+, and Co2+ (p < 0.05). Nevertheless, Na+ and K+ had no significant effect on trypsin activity (p > 0.05). The purified trypsin showed significantly higher catalytic efficiency (kcat/Km) than porcine pancreatic trypsin against BAPNA and N-α-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) substrates (p < 0.05).  相似文献   

4.
Trypsin from viscera of Indian mackerel (Rastralliger kanagurta) was purified by ammonium sulphate precipitation and chromatographic techniques such as size exclusion, ion exchange, and affinity chromatography, with a 14.4-fold increase in specific activity and 18.7% recovery. The molecular weight of the trypsin was estimated to be approximately 26 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified trypsin showed amidase-specific activity which was determined using benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for isolated trypsin activity were 9.0 and 50°C, respectively. The purified trypsin was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK). Purified trypsin showed almost 40% recovery at high NaCl concentration (30%). The N-terminal amino acid sequence of the first 10 amino acids of purified trypsin was IVGGYESQPH. The Michaelis-Menten constant (Km) and catalytic constant (Kcat) of purified trypsin were 0.430 mM and 0.77 s?1, respectively, determined using BAPNA as a substrate. Purified trypsin showed digestion of casein similar to bovine trypsin by the fluorometric method.  相似文献   

5.
Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2′,5′-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K m values for G6-P and NADP+ were determined to be 0.026, 0.0068 mM, respectively. The V max values for G6-P and NADP+ were 2.20 and 2.27 μM min?1 mg protein?1, respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10?6 min?1 mg protein?1, respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC50 values of Zn+2, Mn+2, Al+3, Cu+2, and Cd+2 were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn+2, Al+3, Cu+2, and Cd+2 were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn+2, Al+3, and Cd+2 showed competitive inhibition, while Cu+2 inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.  相似文献   

6.
An alkaline phosphatase was purified from the gut of sea cucumber Stichopus japonicus by n-butyl alcohol extract, ammonium sulfate precipitation, ion exchange chromatography with diethylaminoethyl cellulose, gel filtration chromatography with Sephacryl S-200 and preparative electrophoresis with polyacrylamide gel electrophoresis. The native enzyme was estimated to be 166 ± 9 kDa and produced a single predominant band corresponding to active enzyme on nondenaturing electrophoresis, but showed 2 bands of 97 and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is composed of two dissimilar subunits. The enzyme displayed maximum activity at pH 11 and 40 °C, showing narrow pH stability (pH 10–12) and thermal instability at temperature higher than 30 °C. The activity of the purified alkaline phosphatase was enhanced by Mg2+, whereas inhibited by Zn2+, Ca2+ and EDTA at 1 and 10 mM, suggesting its activity is in a magnesium ion-dependent manner. The product-analog WO4 2? and product HPO4 2? showed strong inhibitory effects on the enzyme activity. Using p-nitrophenyl phosphate as substrate, the V max and K m values were 24.45 μmol/L min and 5.76 mM, respectively.  相似文献   

7.
Carboxypeptidase B (CPB) from zebra blenny (Salaria basilisca) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 28-fold increase in specific activity and 21.72% recovery. The molecular weight of the enzyme was estimated to be 34.5 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60°C, respectively, using Hippuryl-l-Arg as a substrate. The enzyme was unstable above 50°C and below pH 5.0. The enzyme was activated by Co2+ and Zn2+ and inhibited by ethylenediaminetetraacetic acid (EDTA). The N-terminal amino acid sequence of the enzyme was determined as S P S Y T K Y N T. The CPB kinetic constants, Km and kcat for Hippuryl-l-Arg, were 0.32 mM and 36.23 s?1, respectively.  相似文献   

8.
Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS–PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-l-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants K m and k cat on BAPNA were 0.13 mM and 1.56 s−1, respectively, while the catalytic efficiency k cat /K m was 12 s−1 mM−1. Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.  相似文献   

9.
Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30–70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg2+, Fe2+, Cu2+, Li1+, Mg2+, K1+, Mn2+, while Ca2+ had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K m and V max values were 0.72 ± 1.4 mM and 1.15 ± 0.06 μmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.  相似文献   

10.
Histidine decarboxylase (HDC) from Staphylococcus epidermidis TYH1, a halotolerant histamine-producing bacterium isolated from Japanese fermented fish-miso, was purified to homogeneity for the first time. The enzyme was purified 182-fold from cell-free extracts by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The N-terminal amino acid sequences of two polypeptide chains of 27–30 and 7–9 kDa were highly homologous with those of α- and β-chains of other staphylococcal HDCs. The optimum pH and temperature for the enzyme were 6.0 and 60 °C, respectively. This enzyme did not decarboxylate lysine, arginine, tyrosine, tryptophan or ornithine. The enzyme activity decreased with the addition of NaCl. At pH 4.8, the V max and K m values were 45.5 μmol histamine min?1 mg?1 and 1.10 mmol/L, respectively. Moreover, this enzyme was resistant to heat treatment (80 °C for 15 min) and was stable upon freezing at ?30 °C for 7 days. The very similar physiological properties of this enzyme and the almost identical N-terminal amino acid sequence to that of the HDC from S. capitis indicated that this enzyme may be evolutionally highly conserved in the genus Staphylococcus. The biophysical properties of staphylococcal HDC were elucidated using native purified enzyme.  相似文献   

11.
Two forms of rhodanese were purified from the liver of Clarias gariepinus Burchell, designated catfish rhodanese I (cRHD I) and rhodanese II (cRHD II), by ion-exchange chromatography on a CM-Sepharose CL-6B column and gel filtration through a Sephadex G-75 column. The apparent molecular weight obtained for cRHD I and cRHD II was 34,500 ± 707 and 36,800 ± 283 Da, respectively. The subunit molecular weight determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis was 33,200 ± 283 and 35,100 ± 141 Da for cRHD I and cRHD II, respectively. Atomic absorption spectrophotometric analysis revealed that cRHD II contained a high level of iron (Fe), which presumably was responsible for the brownish colour of the preparation. In contrast, no Fe was identified in cRHD I, and its preparation was colourless. Further characterization of cRHD II gave true Michaelis–Menten constant (K m) values of 25.40 ± 1.70 and 18.60 ± 1.68 mM for KCN and Na2S2O3, respectively, an optimum pH of 6.5 and an optimum temperature of 40°C. The Arrhenius plot of the effects of temperature on the reaction rate consisted of two linear segments with a break occurring at 40°C. The apparent activation energy values from these slopes were 7.3 and 72.9 kcal/mol. Inhibition studies on the cRHD II enzyme showed that the activity of the enzyme was not affected by Mn2+, Co2+, Sn2+, Ni2+ and NH4 +, but Zn2+ inhibited the enzyme considerably.  相似文献   

12.
Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20–70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0–11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K m and k cat were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s−1, respectively. The N-terminal sequences of the first 10 amino acids were “I V G G Y E C Q K Y” for trypsin A and “I V G G Y E A Q S Y” for trypsins B and C. These sequences showed highly homology to other fish trypsins.  相似文献   

13.
β-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from rhizostomous jellyfish mesogloea and characterized. Using two purification steps, this enzyme was purified up to 27.4-fold with a recovery rate of 46% compared with crude extract. The molecular weight of the enzyme was estimated to be about 136 kDa, composed of subunit molecular weights of 68 kDa. The enzyme activity was inhibited by SH-reagents, indicating that it contains a SH-group in its active site. The enzyme has a high affinity for pNPGlcNAc with Km value of 0.021 mM. The rate of hydrolysis of N-acetylchito-oligosaccharides tended to decrease with increasing degree of polymerization of the substrate. The parameters of k cat were 92.0 s−1 for pNPGlcNAc, 38.2 s−1 for GlcNAc2, 14.0 s−1 for GlcNAc3, 4.1 s−1 for GlcNAc4, 1.6 s−1 for GlcNAc5, 0.9 s−1 for GlcNAc6, respectively. These results suggest that this β-N-acetylhexosaminidase is an exo-fashion hydrolytic enzyme involved in chitin degradation. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

14.
Tyrosine hydroxylase (TH) which catalyses the rate – limiting step in catecholamine (CA) synthesis shows significant annual variations with activity and kinetics increasing with the progress of gonad recrudescence up to spawning and decreasing thereafter. Estradiol-17 β (E2) exerts biphasic effects on in vivo and in vitro enzyme activity and kinetics: low dosages/concentrations stimulated, and high dosages/concentrations inhibited them. Preincubations of hypothalamic enzyme preparations with low (10?9 M) or high (10?3 M) E2 for 15 min at 30 °C, followed by cAMP (1.0 mM) for 10 min at 30 °C produced differential effects: an additive effect in the low concentration group and an inhibitory response in the high concentration group. The stimulatory or inhibitory effects on TH activity could be related to changes in apparent Km and Vmax of the enzyme for substrate and cofactor. The results suggest that TH activity and kinetics are influenced by the circulating titer of E2 and the steroid interacts with the cAMP signaling pathway in the acute regulation of TH.  相似文献   

15.
The effect of irradiance and temperature on the photosynthesis of two Japanese agarophytes, Gelidium elegans and Pterocladiella tenuis (Gelidiales), was determined using dissolved oxygen sensors and pulse amplitude modulated (PAM) fluorometry. Gross photosynthesis and dark respiration rates were determined over a range of temperatures (8–36 °C). The highest gross photosynthetic rates were 40.3 and 37.0 mg O2 g ww ?1  min?1 and occurred at 24.3 and 25.5 °C [95 % Bayesian credible interval (BCI) 20.7–28.0 and 23.4–28.3 °C], respectively. The dark respiration rate in G. elegans and P. tenuis increased with increasing temperature at a rate of 0.10 and 0.31 mg O2 g ww ?1  min?1 °C?1 , respectively. Modeling the net photosynthesis–irradiance (P–E) responses of G. elegans and P. tenuis at 20 °C revealed that the net photosynthetic rates quickly increased at irradiance levels below the estimated saturation irradiance of 88 and 83 µmol photons m?2 s?1, with a compensation irradiance of 14 and 19 µmol photons m?2 s?1, respectively. The highest value of the maximum effective quantum yield (Φ PSII) occurred at 20.1 °C (BCI 18.9–21.5 °C) and 21.3 °C (BCI 20.2–22.5 °C) for G. elegans and P. tenuis and was 0.49 and 0.45, respectively. These optimal temperatures of Φ PSII were relatively lower than those determined by the photosynthesis–temperature model of oxygen evolution. The temperature response of these species indicates that they are probably well adapted to the current range of seawater temperatures in the study site but that they are near the boundary of their tolerable limits.  相似文献   

16.
We investigated the effect of exposure to low salinity water on plasma ion regulation and survival rates in artificially wounded devil stinger Inimicus japonicus. All fishes survived in 33% seawater (SW), while survival rate in 100% SW was 5.1% at 24 h. In 100% SW, plasma Na+, K+, Mg2+, and Ca2+ concentrations significantly increased to 238?±?49.9, 9.6?±?2.4, 15.1?±?3.5 and 5.0?±?0.7 mmol/l at 6 h, respectively; the gill Na+/K+–ATPase (NKA) activity was almost stable, although only one fish survived to 24 h. In 33% SW, plasma Na+ and K+ concentrations remained at the same level, and plasma Mg2+ and Ca2+ concentrations gradually increased to 16.2?±?0.7 and 4.5?±?0.2 mmol/l until 24 h, respectively. The NKA activity significantly increased to 5.1?±?1.1 µmol ADP/mg protein per h at 6 h. A positive correlation was observed between the wound surface area against body weight and the plasma ion concentrations, although no difference was observed in the restoration rate of the wounded area between 100 and 33% SW. These results indicate that exposure of wounded fish to low salinity water improves survivability by favoring plasma ion regulation without influencing the restoration rate.  相似文献   

17.
Changes in quality parameters including pH, water activity, texture, and lipid oxidation were studied in pasteurized chum salmon (Oncorhynchus keta) ikura samples packaged using two films with different oxygen transmission rates (OTR) (40 and 62 cm3·m?2·day?1; F-40 and F-62), during 60 days storage at 4°C. No significant differences in pH and water activity (aw) were observed between ikura packaged using two different films with different OTR (P > 0.05). However, compared to the first day of study, water activity decreased significantly in ikura (P < 0.05). Ikura thiobarbituric reactive substance (TBARS) in the pouches significantly increased during the storage at 4°C (P < 0.05). Both pouches showed similar trends in TBARS until day 29, while after day 29, ikura packaged in F-62 (OTR = 62 cm3·m?2·day?1) showed a significant increase in TBARS compared to F-40 with less OTR (P < 0.05). The texture of ikura became softer compared to the first day; however, no significant difference was observed between the ikura samples in two pouches (P < 0.05). The quality changes of ikura measured during storage indicate that packaging ikura in a lower OTR film would provide greater quality retention than one with higher OTR.  相似文献   

18.
An acetylcholinesterase was purified from the gut of sea cucumber Stichopus japonicus by anion exchange chromatography followed by gel filtration chromatography. The enzyme was purified 35.49-fold with a total yield of 7.73 %. The molecular mass of purified acetylcholinesterase was 68 kDa as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme displayed maximum activity at pH 7.5 and 35 °C with acetylthiocholine iodide as substrate. The enzyme activity appeared to be stable over pH 6.0–8.0 and up to 40 °C. It displayed an apparent Michaelis–Menten behavior in the concentration range from 0.1 to 0.8 mM with K m values of 0.62 mM for acetylthiocholine iodide and 2.53 mM for butyrylthiocholine iodide. More than 95 % of acetylcholinesterase activity was inhibited by 1 mM eserine or 1,5-bis(4-allyldimethylammonium phenyl)-pentan-3-one dibromide (BW284C51), but only 19.1 % of the activity was inhibited by tetraisopropylpyrophosphoramide (iso-OMPA) at the same concentration. On the basis of the substrate and inhibitor specificities, the purified enzyme appeared to be a true acetylcholinesterase. Nevertheless, the purified acetylcholinesterase exhibited insensitivity to substrate inhibition phenomenon. Its biochemical properties were compared with those reported for different species.  相似文献   

19.
In this study, CA I and II isoenzymes were purified from Van Lake fish gills by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography and to determine the effects of some metals on the enzyme activities. For purified CA I isoenzyme, yield, specific activity, and purification fold were obtained as 42.07%, 4948.12 EU/mg protein, and 116.61 and for CA II isoenzyme, 7%, 1798.56 EU/mg protein, and 42.38 respectively. Activity of CA was determined by measuring “CO2-hydratase activity”. Purity control was checked by SDS-PAGE. In vitro inhibitory effect of Cu2+, Ag+, Cd2+, Ni2+ metal ions, and arsenic (V) oxide were also examined for both isozymes activities. Whereas Cu2+, Ag+, Cd2+, and Ni2+ ions showed inhibitory effects on both isozymes, arsenic (V) oxide showed activation effect. IC50 values were calculated by drawing activity %-[I] graphs for metal ions exhibiting inhibitory effects. IC50 values were determined as 3.39, 6.38, 13.52, and 206 μM for CA I isozyme and 6.16, 20.29, 46, and 223 μM for CA II isozyme respectively.  相似文献   

20.
This paper presents a comparative study of the roles of Cl? and HCO3 ? in the functioning of the GABAAR-associated Cl?/HCO3 ?-ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl?(15–30 mmol l?1), whereas the enzyme from MBNs was not significantly affected by Cl?. By contrast, HCO3 ?(15–30 mmol l?1) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl?(15 mmol l?1)/HCO3 ?(27 mmol l?1) and these activities were inhibited in the presence of picrotoxin (100 μmol l?1), bumetanide (150 μmol l?1), and DIDS (1000 μmol l?1). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~?56 kDa that binds [3H]muscimol and [3H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-32P and Mg2+, as well as Cl?/HCO3 ? sensitive dephosphorylation, is also associated with this 56 kDa peptide. Both preparations also showed one subunit with molecular mass 56 kDa that was immunoreactive with GABAAR β3 subunit. The use of a fluorescent dye for Cl? demonstrated that HCO3 ?(27 mmol l?1) causes a twofold increase in Cl? influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO3 ? had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl? and HCO3 ? in the regulation of the Cl?/HCO3 ?-ATPases functioning in neurons with different specializations.  相似文献   

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