Postnatal ontogeny of kinetics of porcine jejunal brush border membrane-bound alkaline phosphatase,aminopeptidase N and sucrase activities

Our objectives were to determine postnatal changes in the maximal enzyme activity (V(max)) and enzyme affinity (K(m)) of jejunal mucosal membrane-bound alkaline phosphatase, aminopeptidase N and sucrase using a porcine model which may more closely resemble the human intestine. Jejunal brush border membrane was prepared by Mg(2+)-precipitation and differential centrifugation from pigs of suckling (8 days), weaning (28 days), post-weaning (35 days) and adult (70 days) stages. p-Nitrophenyl phosphate (0-8 mM), L-alanine-p-nitroanilide hydrochloride (0-28 mM) and sucrose (0-100 mM) were used in alkaline phosphatase, aminopeptidase N and sucrase kinetic measurements. V(max) of alkaline phosphatase was the lowest in the adult (4.27 micromol.mg(-1) protein.min(-1)), intermediate in the suckling (9.75 micromol.mg(-l) protein.min(-l)) and the highest in the weaning and post-weaning stage (12.83 and 10.40 micromol.mg(-l) protein.min(-l)). K(m) of alkaline phosphatase was high in the suckling and weaning stages (5.14 and 9.93 mM) and low in the adult (0.66 mM). V(max) of aminopeptidase N was low in the suckling (7.04 micromol.mg protein(-1).min(-1)) and high in the post-weaning stage (13.36 micromol.mg(-l) protein.min(-l)). K(m) of aminopeptidase N was the highest in the two weaning stages (2.96 and 3.39 mM), intermediate in the adult (2.33 mM) and the lowest in the suckling stage (1.66 mM). V(max) of sucrase increased from the suckling to the adult (0.48-1.30 micromol.mg(-l) protein.min(-l)). K(m) of sucrase ranged from 11.19 to 16.57 mM. There are dramatic postnatal developmental changes in both the maximal enzyme activity and enzyme affinity of jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase in the pig.     

Direct effect of temperature on the catalytic activity of nitric oxide synthases types I, II, and III.

The effect of temperature (between 5.0 and 45.0 degrees C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH 7.5. The value of V(max) for NOS-I activity increases from 1.8 x 10(1) pmol min(-1) mg(-1), at 5.0 degrees C, to 1.8 x 10(2) pmol min(-1) mg(-1), at 45.0 degrees C; on the other hand, the value of K(m) (=4.0 x 10(-6) M) is temperature independent. Again, the value of V(max) for NOS-II activity increases from 8.0 pmol min(-1) mg(-1), at 7.0 degrees C, to 5.4 x 10(1) pmol min(-1) mg(-1), at 40.0 degrees C, the value of K(m) (=1.8 x 10(-5) M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of DeltaH(V(max)) (=4.2 x 10(1) kJ mol(-1)), DeltaH(K(m)) (=0 kJ mol(-1)), and DeltaH((V(max))(/K(m))()) (=4.2 x 10(1) kJ mol(-1)). On the contrary, the value of K(m) for NOS-III activity decreases from 3.8 x 10(-5) M, at 10.0 degrees C, to 1.6 x 10(-5) M, at 40.0 degrees C, the value of V(max) (=6.8 x 10(1) pmol min(-1) mg(-1)) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of K(m) being temperature independent. However, when l-arginine level is higher than 2 x 10(-4) M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of K(m). As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.     

Purification and characterization of a dimethoate-degrading enzyme of Aspergillus niger ZHY256, isolated from sewage

A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50 degrees C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu(2+). The Michaelis constant (K(m)) and V(max) for dimethoate were 1.25 mM and 292 micromol min(-1) mg of protein(-1), respectively.     

Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology

The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K(m),V(max), K(cat), activation energy (E(a)) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K(m)) for casein decreased with corresponding increase in V(max), as reaction temperature was raised from 45-60 degrees C. The protease exhibited K(m) of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60 degrees C, respectively, whereas V(max) values at these temperatures were 74.07, 99.01, 116.28, and 120.48 microg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45 degrees C until 60-65 degrees C followed by thermal inactivation. Likewise, the energy of activation (E(a)) was more between 45-55 degrees C (9747 cal/mol) compared to E(a) between 50-60 degrees C (4162 cal/mol).     

Characterization of beta-xylosidase enzyme from a Pichia stipitis mutant

beta-Xylosidase production was maximal for the mutant Pichia stipitis NP54376 grown on xylan as the sole carbon source. beta-Xylosidase was purified from culture supernatant by (NH(4))(2)SO(4) precipitation and a hydrophobic interaction chromatography on phenyl sepharose. Optima of pH and temperature were 5.0 and 50 degrees C, respectively. The enzyme was inhibited by 2-mercaptoethanol (100%) and Fe(3+) (80%), and moderately affected by Cu(2+), Ag(+), NH(4)(+) and Mg(2+) and SDS. The purified xylosidase hydrolyzed xylobiose and xylo-oligosaccharides and it did not exhibit activity against cellulose, starch, maltose and cellobiose. 2.5 g l(-1) glucose repressed beta-xylosidase activity in the NP54376 strain. The K(m) and V(max) values on p-nitrophenyl-beta-xylopyranoside were 1.6 mM and 186 micromol p-nitrophenyl min(-1)mg(-1) protein, respectively. Analysis of the hydrolysis products by HPLC indicated that the major hydrolysis product is xylobiose in all the carbon sources tested.     

Phosphorylation of the yeast choline kinase by protein kinase C. Identification of Ser25 and Ser30 as major sites of phosphorylation

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C.     

Partial purification and characterization of almond seed lipase

A lipase was partially purified from the almond (Amygdalus communis L.) seed by ammonium sulfate fractionation and dialysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with K(m) and V(max) values of 25 mM and 113.63 micromol min(-1) mg(-1) for tributyrin as substrate. All triglycerides were efficiently hydrolyzed by the enzyme. The partially purified almond seed lipase (ASL) was stable in the pH range of 6-9.5, with an optimum pH of 8.5. The enzyme was stable between 20 and 90 degrees C, beyond which it lost activity progressively, and exhibited an optimum temperature for the hydrolysis of soy bean oil at 65 degrees C. Based on the temperature activity data, the activation energy for the hydrolysis of soy bean oil was calculated as -5473.6 cal/mol. Soy bean oil served as good substrate for the enzyme and hydrolytic activity was enhanced by Ca(2+), Fe(2+), Mn(2+), Co(2+), and Ba(2+), but strongly inhibited by Mg(2+), Cu(2+), and Ni(2+). The detergents, sodiumdeoxicholate and Triton X-100 strongly stimulated enzyme activity while CTAB, DTAB, and SDS were inhibitors. Triton X-405 had no effect on lipase activity. The partially purified enzyme retained its activity for more than 6 months at -20 degrees C, beyond which it lost activity progressively.     

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains

Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.     

Acute upregulation of blood-brain barrier glucose transporter activity in seizures

Brain extraction of (18)F-labeled 2-fluoro-2-deoxy-D-glucose (FDG) was significantly higher in pentylene tetrazole (PTZ)-treated rats (32 +/- 4%) than controls (25 +/- 4%). The FDG permeability-surface area product (PS) was also significantly higher with PTZ treatment (0.36 +/- 0.05 ml. min(-1). g(-1)) than in controls (0.20 +/- 0.06 ml. min(-1). g(-1)). Cerebral blood flow rates were also elevated by 50% in seizures. The internal carotid artery perfusion technique indicated mean [(14)C]glucose clearance (and extraction) was increased with PTZ treatment, and seizures increased the PS by 37 +/- 16% (P < 0.05) in cortical regions. Because kinetic analyses suggested the glucose transporter half-saturation constant (K(m)) was unchanged by PTZ, we derived estimates of 1) treated and 2) control maximal transporter velocities (V(max)) and 3) a single K(m). In cortex, the glucose transporter V(max) was 42 +/- 11% higher (P < 0.05) in PTZ-treated animals (2.46 +/- 0.34 micromol. min(-1). g(-1)) than in control animals (1.74 +/- 0.26 micromol. min(-1). g(-1)), and the K(m) = 9.5 +/- 1.6 mM. Blood-brain barrier (BBB) V(max) was 31 +/- 10% greater (P < 0.05) in PTZ-treated (2.36 +/- 0. 30 micromol. min(-1). g(-1)) than control subcortex (1.80 +/- 0.25 micromol. min(-1). g(-1)). We conclude acute upregulation of BBB glucose transport occurs within 3 min of an initial seizure. Transporter V(max) and BBB glucose permeability increase by 30-40%.     

Purification and partial characterization of polygalacturonase from Streptomyces lydicus

Polygalacturonase produced by Streptomyces lydicus was purified to homogeneity by ultrafiltration and a combination of ion exchange and gel filtration chromatographic procedures. The purified enzyme was an exo-polygalacturonase with a molecular weight of 43 kDa. It was optimally active at 50 degrees C and pH 6.0. The enzyme was stable from pH 4.0 to 7.0 and at or below 45 degrees C for 90 min. K(m) value for polygalacturonic acid was 1.63 mg/mL and the corresponding V(max) was 677.8 microM min(-1) mg(-1). The inhibition constant (K(i)) for gluconic acid d-lactone was 20.75 mM. Purified enzyme had been inhibited by N-bromosuccinimide, while l-tryptophan could induce enzyme activity, indicating the involvement of tryptophan at the active site.     

Cloning, expression, purification, and characterization of a novel epoxide hydrolase from Aspergillus niger SQ-6

A novel epoxide hydrolase from Aspergillus niger SQ-6 has now been cloned by inverse PCR. Its gene shows eight exons including a non-coding exon at its 5'-terminal (GenBank Accession No. AY966486). Phylogenetic analysis using deduced amino acid sequence (395 aa) confirms it as an epoxide hydrolase and shares 58.3% identity with that of A. niger LCP521 (GenBank Accession No. AF238460). The predicted catalytic triad is composed of Asp(191), His(369) and Glu(343). Active recombinant epoxide hydrolase has been successfully expressed in Escherichia coli as protein fusions with a poly-His tail. Scale-up fermentation can yield 2.5g/L of recombinant protein. The electrophoretic pure recombinant protein, which shows similar characterization as natural enzyme purified from A. niger SQ-6, can be easily purified by Ni(2+)-chelated affinity and gel-filtration chromatography. Optimal pH and temperature for purified enzyme are pH 7.5 and 37 degrees C, respectively. The K(m), k(cat) and maximal velocity (V(max)) for p-nitrostyrene oxide are determined to be 1.02mM, 172s(-1) and 231micromol min(-1)mg(-1), respectively. The enzyme can be inhibited by oxidant (H(2)O(2)), solvent (Tetrahydrofuran) and several metal ions including Hg(2+), Fe(2+) and Co(2+). This (R)-stereospecific epoxide hydrolase exhibits high enantioselectivity (enantiomeric excess value, 99%) for the less hindered carbon atom of epoxide. It may be an industrial biocatalyst for the preparation of enantiopure epoxides or vicinal diols.     

Purification and properties of Bacillus subtilis SA-22 endo-1, 4-beta-D-mannanase.

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.     

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.     

Silver nanoparticle (AgNPs) doped gum acacia-gelatin-silica nanohybrid: an effective support for diastase immobilization

An effective carrier matrix for diastase alpha amylase immobilization has been fabricated by gum acacia-gelatin dual templated polymerization of tetramethoxysilane. Silver nanoparticle (AgNp) doping to this hybrid could significantly enhance the shelf life of the impregnated enzyme while retaining its full bio-catalytic activity. The doped nanohybrid has been characterized as a thermally stable porous material which also showed multipeak photoluminescence under UV excitation. The immobilized diastase alpha amylase has been used to optimize the conditions for soluble starch hydrolysis in comparison to the free enzyme. The optimum pH for both immobilized and free enzyme hydrolysis was found to be same (pH=5), indicating that the immobilization made no major change in enzyme conformation. The immobilized enzyme showed good performance in wide temperature range (from 303 to 323 K), 323 K being the optimum value. The kinetic parameters for the immobilized, (K(m)=10.30 mg/mL, V(max)=4.36 μmol mL(-1)min(-1)) and free enzyme (K(m)=8.85 mg/mL, V(max)=2.81 μmol mL(-1)min(-1)) indicated that the immobilization improved the overall stability and catalytic property of the enzyme. The immobilized enzyme remained usable for repeated cycles and did not lose its activity even after 30 days storage at 40°C, while identically synthesized and stored silver undoped hybrid lost its ~31% activity in 48 h. Present study revealed the hybrids to be potentially useful for biomedical and optical applications.     

Serine racemase homologue of Saccharomyces cerevisiae has L-threo-3-hydroxyaspartate dehydratase activity

The NH(2)-terminal amino acid sequence of L-threo-3-hydroxyaspartate dehydratase from Pseudomonas sp. T62 showed significant similarity to that of the SRY1 gene product of Saccharomyces cerevisiae (serine racemase in yeast). SRY1 was cloned and expressed in Escherichia coli, and the gene product was purified and partially characterized. The SRY1 gene product exhibited dehydratase activity specific for L-threo-3-hydroxyaspartate (K(m)=3.9 mM, V(max)=110 micromol min(-1) (mg protein)(-1)) but not for D-threo- or DL-erythro-3-hydroxyaspartate. The purified enzyme showed no detectable serine racemase activity. The activity of the enzyme was inhibited by hydroxylamine and EDTA, and was activated by Mg(2+), Ca(2+), and Mn(2+), suggesting that pyridoxal-5'-phosphate and divalent cations participate in the enzyme reaction. Gene disruption and overexpression indicated that SRY1 is responsible for the 3-hydroxyaspartate resistance of S. cerevisiae. To our knowledge, this is the first report of 3-hydroxyaspartate dehydratase activity in eukaryotic cells.     

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.     

Human neuropathy target esterase catalyzes hydrolysis of membrane lipids

A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues in Drosophila and yeast and is detected in vitro by assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727-1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A(2) (iPLA(2)) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA(2) inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (V(max) approximately 0.01 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of (14)C-labeled products from two differentially labeled (14)C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (V(max) approximately 20 micromol/min/mg and K(m) approximately 0.05 mm for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (V(max) approximately 1 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di- or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking.     

Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.     

Purification and characterization of a novel enzyme, L-threo-3-hydroxyaspartate dehydratase, from Pseudomonas sp. T62

L-threo-3-Hydroxyaspartate dehydratase (L-threo-3-hydroxyaspartate hydro-lyase), which exhibited specificity for L-threo-3-hydroxyaspartate (K(m)=0.74 mM, V(max)=37.5 micromol min(-1) (mg protein)(-1)) but not for D-threo or D, L-erythro-3-hydroxyaspartate, was purified from a cell-free extract of Pseudomonas sp. T62. The activity of the enzyme was inhibited by hydroxylamine and EDTA, which suggests that pyridoxal 5'-phosphate and divalent cations participate in the enzyme reaction. The NH(2)-terminal amino acid sequence showed significant similarity to the Saccharomyces cerevisiae YKL218c gene product, a hypothetical threonine dehydratase. However, the purified enzyme showed no threonine dehydratase activity.     

Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.