Seasonal differences in activity of perch (Perca flavescens) gill Na/K ATPase

We measured Na⁺/K⁺ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na⁺/K⁺ ATPase activity was measured at 8, 17, 25, and 37°C. V_max values for winter fish increased from 0.48±0.07 μmol P mg⁻¹ protein h⁻¹ at 8°C to 7.21±0.79 μmol P mg⁻¹ protein h⁻¹ at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg⁻¹ protein h⁻¹. The K_m for ATP and for Na⁺ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na⁺/K⁺ ATPase from summer fish was 10 309 (μmol P mg⁻¹ h⁻¹) K⁻¹. In winter fish, the K_m for ATP and Na⁺ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The K_m values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression.     

Physico-chemical and Catalytic Properties of Clostridium perfringens Hyaluronidase: An Update

Hyaluronidase (E.C. 4.2.2.1 hyaluronate lyase) or Mu toxin is one of the main components ofClostridium perfringens toxin complex. Although this enzyme has been studied for many years, data on its physico-chemical and catalytic characteristics are still quite contradictory and lack lucidity and completeness. In order to update knowledge of enzymatic properties of clostridial hyaluronidase, a chromatographically purified preparation from C. perfringens type A BP6K free of side phospholipase C (alpha toxin), neuraminidase (sialidase) and collagenase (kappa toxin) activities was obtained and characterized. The purification procedure included the following steps: processing the culture liquid with calcium phosphate gel, precipitation of the enzyme with acetone, ultrafiltration, and chromatography on Sephadex G-100 column. The purified hyaluronidase was homogenous as judged by rechromatography, SDS-PAGE and isoelectric focusing. Being a glycoprotein, the enzyme was most active at pH 5.7–6.2 (depending on the nature of the buffer used), at temperatures 37–45°C and at a relatively high ionic strength (0.15 and higher). The hyaluronidase was unstable when at pH values below 5.0 and above 9.0 as well as at temperatures below 30°C and above 50°C. The enzyme was most sensitive to Cu²⁺, Pb²⁺and Al³⁺ions, while the inhibitory effect of EDTA was moderate. Molecular mass of hyaluronidase was 96kDa as estimated by gel filtration and 48kDa when estimated by SDS-PAGE, suggesting that enzyme is composed of two subunits. The isoelectric point of the enzyme was 4.4. Substrate specificity of the enzyme was narrow (appart from hyaluronate, it slightly split chondroitin, but did not split heparin and various chondroitinsulphates). Moreover, unsplit glycosaminoglycans appeared to be competitive inhibitors with K_ivalues 5.3×10⁻², 4.9×10⁻², 4.5×10⁻²and 4.2×10⁻²mg/mL for heparin, chondroitinsulphates A, B and C, respectively. The Michaelis constant in regard to potassium hyaluronate was calculated to be (15.4±2.6)×10⁻²mg/mL.     

Membrane Permeability Characteristics of Metaphase II Mouse Oocytes at Various Temperatures in the Presence of Me2SO

In this study, the hydraulic conductivity (L_p), Me₂SO permeability ( _Me2SO), and the reflection coefficients (ς) and their activation energies were determined for Metaphase II (MII) mouse oocytes by exposing them to 1.5 M Me₂SO at temperatures of 30, 20, 10, 3, 0, and −3°C. These data were then used to calculate the intracellular concentration of Me₂SO at given temperatures. Individual oocytes were immobilized using a holding pipette in 5 μl of an isosmotic PBS solution and perfused with precooled or prewarmed 1.5 M Me₂SO solutions. Oocyte images were video recorded. The cell volume changes were calculated from the measurement of the diameter of the oocytes, assuming a spherical shape. The initial volume of the oocytes in the isoosmotic solution was considered 100%, and relative changes in the volume of the oocytes after exposure to the Me₂SO were plotted against time. Mean (means ± SEM) L_pvalues in the presence of Me₂SO ( ^Me₂SO_p) at 30, 20, 10, 3, 0, and −3°C were determined to be 1.07 ± 0.03, 0.40 ± 0.02, 0.18 ± 0.01, 7.60 × 10⁻²± 0.60 × 10⁻², 5.29 × 10⁻²± 0.40 × 10⁻², and 3.69 × 10⁻²± 0.30 × 10⁻²μm/min/atm, respectively. The _Me2SOvalues were 3.69 × 10⁻³± 0.3 × 10⁻³, 1.07 × 10⁻³± 0.1 × 10⁻³, 2.75 × 10⁻⁴± 0.15 × 10⁻⁴, 7.83 × 10⁻⁵± 0.50 × 10⁻⁵, 5.24 × 10⁻⁵± 0.50 × 10⁻⁵, and 3.69 × 10⁻⁵± 0.40 × 10⁻⁵cm/min, respectively. The ς values were 0.70 ± 0.03, 0.77 ± 0.04, 0.81 ± 0.06, 0.91 ± 0.05, 0.97 ± 0.03, and 1 ± 0.04, respectively. The estimated activation energies (E_a) for ^Me₂SO_p, _Me2SO, and ς were 16.39, 23.24, and −1.75 Kcal/mol, respectively. These data may provide the fundamental basis for the development of more optimal cryopreservation protocols for MII mouse oocytes.     

Response surface design for the optimization of enzymatic detection of mercury in aqueous solution using immobilized urease from vegetable waste

Soluble and alginate immobilized urease was utilized for detection and quantitation of mercury in aqueous samples. Urease from the seeds of pumpkin, being a vegetable waste, was extracted and purified to apparent homogeneity (sp. activity 353 U/mg protein; A₂₈₀/A₂₆₀ = 1.12) by heat treatment at 48 ± 0.1 °C and gel filtration through Sephadex G-200. Homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization, no leaching of enzyme was found over a period of 15 days at 4 °C. Urease catalyzed urea hydrolysis by soluble and immobilized enzyme revealed a clear dependence on the concentration of Hg²⁺. Inhibition caused by Hg²⁺ was non-competitive (K_i = 1.2 × 10⁻¹ μM for soluble and 1.46 × 10⁻¹ μM for alginate immobilized urease.). Time-dependent inhibition both in presence and in absence of Hg²⁺ ion revealed a biphasic inhibition in activity. For optimization of this process response surface methodology (RSM) was utilized where two-level-two-full factorial (2²) central composite design (CCD) has been employed. The regression equation and analysis of variance (ANOVA) were obtained using MINITAB^® 15 software. Predicted values thus obtained were closed to experimental value indicating suitability of the model. 3D response surface plot, iso-response contour plot and process optimization curve were helpful to predict the results by performing only limited set of experiments.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Purification and Characterization of the NAD-Dependent Glutamate Dehydrogenase in the Ectomycorrhizal FungusLaccaria bicolor(Maire) Orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) fromLaccaria bicolorwas purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE–Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at −75°C, 4 days at 4°C, and 1 h at 50°C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at −75°C. NAD-GDH activity was stimulated by Ca²⁺and Mg²⁺but strongly inhibited by Cu²⁺and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 μM, 89 μM, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and itsK_mvalue increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid.     

Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus

Fructose-1,6-bisphosphate (FBP) aldolase, is a glycolytic enzyme that catalyzes the reversible condensation reaction of FBP to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase gene from Aquifex aeolicus was subcloned, overexpressed in E. coli and purified to 95% homogeneity. The purified enzyme was activated by high concentrations of NH₄⁺ and low concentrations of Co²⁺. The native molecular weight of the purified FBP aldolase was identified as 67 kDa (dimer) by gel filtration chromatography. The enzyme exhibits optimum pH at 6.5 and temperature at 90 °C. Based on the kinetic characterizations, the apparent K_m was calculated to be 4.4 ± 0.07 mM, while V_max was found to be 100 ± 0.02 μM min⁻¹ mg protein⁻¹. The recombinant protein showed extreme heat stability; no activity loss was observed even at 100 °C for 2 h. In addition, the thermophilic enzyme also showed higher stability against several organic solvents viz. acetonitrile, 1,4-dioxane, and methanol. With higher stability against both heat and organic solvents than any other class II aldolase, the A. aeolicus FBP aldolase is an attractive enzyme for use as a biocatalyst for industrial applications.     

Purification and partial characterization of exopolygalacturonase I from Penicillium frequentans

A polygalacturonase with a molecular mass of 74 kDa, an isoelectric point around pH 4.2 and pH – and temperature optima of 3.9 and 50°C, respectively, was purified from a culture fluid of Penicillium frequentans. The enzyme was characterized as an exo-α-1,4-polygalacturonase (exo-PG I). K_m and V_max for sodium polypectate hydrolysis were 0.68 g/l and 596.8 U × mg⁻¹, respectively. The enzyme, a glycoprotein with a carbohydrate content of 81%, is probably the main pectinase of Penicillium frequentans responsible for cleaving monomer units from the non-reducing end of pectin.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Purification and kinetic characterization of γ-aminobutyraldehyde dehydrogenase from rat liver

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD⁺-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K_m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD⁺ is affected by the aldehyde present at the active site: K_m for NAD⁺ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD⁺ activates the enzyme (K_act 500 μM) while NADH competes for the regulatory site (K_in 70 μM). In the presence of high NAD⁺ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (K_act 10 mM). The data show that the enzyme is probably an E₃ aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.     

Purification and properties of malic enzyme from Pseudomonas diminuta IFO-13182

A malic enzyme from a cell-free extract of Pseudomonas diminuta IFO-13182 was purified to electrophoretic homogeneity by DEAE-Sepharose, Sephacryl, and Blue-Sepharose chromatographies. The purified enzyme required either NAD⁺ or NADP⁺ as a coenzyme. From the results of coenzyme specificity, the enzyme should be classified as l-malate: NAD⁺ oxidoreductase (decarboxylating) [EC 1.1.1.39]. The purified enzyme was most active at pH 7.5 and 50°C and was stable in the pH range from 7.0 to 9.0. The isoelectric point was pH 4.3. Its molecular weight was 680,000 by COSMOSIL 5-Diol high performance liquid gel filtration on chromatography and 65,000 by SDS polyacrylamide gel electrophoresis. This indicates that the enzyme consisted of 10 subunits. The malic enzyme activity with NADP⁺ was about twice that measured with NAD⁺.     

Prostaglandin-E2-9-ketoreductase in rabbit kidney

The 100,000 xg supernatant of rabbit kidney contains a prostaglandin-E₂-9-ketoreductase which has an obligatory requirement for NADPH. This enzyme is localised in the renal cortex and is able to quantitatively convert PGE₂ to PGF_2α. A broad pH profile was evident with an optimum at pH 7·5. Kinetic studies indicated a K_m of 3·2 × 10⁻⁴M PGE₂. The isoelectric point was at pH 5·65 and the molecular weight, as estimated by gel filtration, was 21,800. These values differ from those obtained with enzyme from monkey brain tissue and suggest a tissue specificity of PGE₂-9-ketoreductase. By combining isoelectric focussing techniques with sephadex filtration considerable purification of the renal enzyme was achieved.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO₃⁻, nmol cm^− 2 s^− 1 − 1,76 ± 0,11_control × 1,29 ± 0,08_{REN 10 μM}; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO₃⁻, nmol cm^− 2 s⁻ 1 − 0.80 ± 0.07_control × 0.60 ± 0.06_{REN 1 μM}; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na⁺/H⁺exchanger and H⁺-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.     

Purification and characterization of malate dehydrogenase from the phototrophic bacterium,Rhodopseudomonas capsulata

Malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37) has been purified about 480-fold from crude extract of the facultative phototrophic bacterium, Rhodopseudomonas capsulata by only two purification steps, involving Red-Sepharose affinity chromatography. The enzyme has a molecular mass of about 80 kDa and consists of two subunits with identical molecular mass (35 kDa). The enzyme is susceptible to heat inactivation and loses its activity completely upon incubation at 40°C for 10 min. Addition of NAD⁺, NADH and oxaloacetate, but not l-malate, to the enzyme solution stabilized the enzyme. The enzyme catalyzes exclusively the oxidation of l-malate, and the reduction of oxaloacetate and ketomalonate in the presence of NAD⁺ and NADH, respectively, as the coenzyme. The pH optima are around 9.5 for the l-malate oxidation, and 7.75–8.5 and 4.3–7.0 for the reduction of oxaloacetate and ketomalonate, respectively. The K_m values were determined to be 2.1 mM for l-malate, 48 μM for NAD⁺, 85 μM for oxaloacetate, 25 μM for NADH and 2.2 mM for ketomalonate. Initial velocity and product inhibition patterns of the enzyme reactions indicate a random binding of the substrates, NAD⁺ and l-malate, to the enzyme and a sequential release of the products: NADH is the last product released from the enzyme in the l-malate oxidation.     

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent K_m of 60 μM and V_max of 20 μmol·min⁻¹·mg⁻¹, corresponding values with mixed histones were 12 μM and 1.2 μmol·min⁻¹·mg⁻¹. With both protein substrates the apparent K_m for ATP was 11 μM. Concentrations of Mg²⁺ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated ³²P_i from [γ-³²P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.