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.     

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: Comparison of kinetic properties among the three isozymes

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25^oC, the major and minor components of the E3 isozyme catalyzed the reaction with V_max of 1.1 and 0.8 μmol NADH min⁻¹ mg⁻¹ protein, respectively, compared to 0.067 and 0.060 μmol NADH min⁻¹ mg⁻¹ protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K_m for methylglyoxal of 8.6 μM, the lowest compared to 46 μM for E1 and 586 and 552 μM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with K_i values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 μM for E1 and E2 isozymes, respectively.     

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.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

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.     

Immobilized 4-aminophenyl 1-thio-β- -galactofuranoside as a matrix for affinity purification of an exo-β- -galactofuranosidase

An alternative and fast method for the purification of an exo-β- -galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β- -galactofuranoside affinity chromatography system and specific elution with 10 mM -galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM -galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β- -galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and K_m and V_max values of 0.311 mM and 17 μmol h⁻¹ μg⁻¹ respectively, when 4-nitrophenyl β- -galactofuranoside was employed as the substrate.     

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.     

α-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of α-cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.     

Bacterial Expression and Purification of the Arabidopsis NADPH–Cytochrome P450 Reductase ATR2

An N-terminally modified form of the Arabidopsis NADPH–cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2′,5′-ADP–agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 μmol min⁻¹ mg⁻¹ and the K_m for cytochrome c reduction was 15 ± 2 μM. The purified NADPH–cytochrome P450 reductase was able to support function of CYP79B2.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

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.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

Microbial production of d-amino acids

The production of d-aminoacylase by Alcaligenes denitrificans and Alcaligenes faecalis has been studied. The enzyme was inducibly produced and N-acetyl-d-leucine and N-acetyl-d-valine were the most effective inducers. d-methionine, d-valine, d-phenylalamine and d-leucine were produced by the enzymic hydrolysis of the appropriate N-acetyl-d-amino-acids with whole cell biomass. The hydrolysis of N-acetyl-d-methionine by A. denitrificans and N-acetyl-d-valine by A. faecalis was preferential. Maximum yields of d-methionine and d-valine were 94.3 and 84.7% at a specific product formation rate of 20.10 and 19.19 μmol min⁻¹ mg⁻¹ of wet cells at 20 mM substrate concentration and 5 mg ml⁻¹ of cell density.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

Dinuclear complexes of transition metals containing carbonate ligands. VIII. Kinetics and mechanism of carbon dioxide uptake by the tri-μ-hydroxo-bis (1,5-diamino-3-aza-pentane)cobalt(III) ion in weakly basic aqueous carbonate solution

The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO₃²⁻35 mM and at an ionic strength of 0.1 M (LiClO₄). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M⁻¹ s⁻¹ with ΔH≠ = (13.2±0.7) kcal mol⁻¹ and ΔS≠ = (−15.1 ± 0.7) cal deg⁻¹ mol⁻¹. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.     

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.     

Transport of neutral, cationic and anionic amino acids by systems B, b, XAG, and ASC in swine small intestine

Amino acid influx across the brush border membrane of the intact pig ileal epithelium was studied. It was examine whether in addition to system B, systems ASC and b^o,+ were involved in transport of bipolar amino acids. The kinetics of interactions between lysine and leucine demonstrates that system b^o,+ is present and accessible also to -glutamine. -aspartate (K_1/2 0.3 mM) and -glutamate (K_i 0.5 mM) share a high affinity transporter with a maximum rate of 1.3 μmol cm⁻² h⁻¹, while only -glutamate with a K_1/2 of 14.4 mM uses a low affinity transporter with a maximum rate of 2.7 μmol cm⁻² h⁻¹, system ASC, against which serine has a K_i of 1.6 mM. In the presence of 100 mM lysine, -glutamine (A), leucine (B), and methionine (C) fulfilled the criteria of the ABC test for transport by one and the same transporter. However, serine inhibits not only transport of -glutamate but also of glutamine (K_i 0.5 mM), and -glutamate inhibits part of the transport of glutamine. The test does, therefore, only indicate that the three bipolar amino acids have similar affinities for transport by systems B and ASC. Further study of the function of system B must be carried out under full inhibition by lysine and glutamate.     

Standard electromotive force of the H2-AgCl;Ag cell in 30, 40, and 50 mass% glycerol/water from −20 to 25 °C: pK2 and pH values for a standard “Mops” buffer in 50 mass% glycerol/water

Data are presented regarding the establishment of the pH (designated pH^*) of a standard buffer solution suitable as a pH reference in 50 mass% glycerol/water mixtures at temperatures ranging from −20 to 25 °C. The buffer material selected was the ampholyte Mops [(3-N-morpholino)-propane sulfonic acid], and the reference standard consists of equal molal amounts of Mops and its sodium salt. The assignment of pH^* values is based on measurements of the electromotive force (emf) of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦ Mops, Na Mopsate, NaCl ¦ AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Mops)^±/ah (Mopsate)⁻ + H ⁺. The standard emf of the silver-silver chloride electrode in 30, 40, and 50 mass% glycerol/water mixtures was determined from emf measurements of the cell at subzero temperatures with HCl solutions replacing the buffer-chloride mixtures.     

Purification and characterization of recombinant Pneumocystis carinii thymidylate synthase

Gatalytically active Pneumocystis carinii thymidylate synthase is expressed to the extent of about 4% of the soluble protein in Escherichia coli χ2913 harboring plasmid pUETS-1.8 (U. Edman, J. C. Edman, B. Lundgren, and D. V. Santi, Proc. Natl. Acad. Sci. USA 86, 6503–6507, 1989). Ion-exchange, affinity, hydrophobic, and reactive dye agarose chromatography steps were explored to devise a large-scale purification protocol for P. carinii thymidylate synthase. Sequential DE52, Q-Sepharose, phenyl-Sepharose, and Cibacron Blue F3GA chromatography yielded enzyme that was homogeneous by SDS-PAGE in a yield of over 50%. The sequence of the first 10 amino acid residues of the purified protein was in accord with that predicted from the DNA sequence. Isoelectric focusing gave a pI of 6.2. Kinetic analysis of the purified enzyme revealed that the the K_m values were 4.7 ± 1.3 μM for dUMP and 15.7 ± 4.3 μM for 5,10-methylenetetrahydrofolate, similar to those of many other thymidylate synthases; the κ_cat of the most active preparation was 0.8 s⁻¹. The enzyme is stable for at least 2 months when stored at −80°C in the presence of 40% glycerol, Tris-HCl, and thiol.