Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32

X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-prolyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40°C. The enzyme was incubated at 40°C for 15 min with various metal ions. It was activated by Mg²⁺ (2.5 mM), Ca²⁺ (0.1 to 2.5 mM), Na⁺ (10 to 50 mM), and K⁺ (10 to 50 mM) and was inhibited by Hg²⁺ (0.1 to 2.5 mM), Cu²⁺ (0.1 to 2.5 mM), and Zn²⁺ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40°C), 1,10-phenanthroline (1.0 mM, 15 min at 40°C), phenylmethylsulfonyl fluoride (1.0 mM), N-ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40°C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40°C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Separation of carbamazepine and five metabolites,and analysis in human plasma by micellar electrokinetic capillary chromatography

A rapid and feasible method was developed for the analysis of carbamazepine and its five metabolites (10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 10,11-dihydro-10-hydroxycarbamazepine, 2-hydroxycarbamazepine and 3-hydroxycarbamazepine) in human plasma. Separation of the analytes is based on micellar electrokinetic chromatography, in untreated fused-silica capillary (48.5/40.0 cm length, 50 microm I.D.) with phosphate buffer (30 mM, pH 8.00) as background electrolyte, containing 50 mM sodium dodecylsulfate, and methanol (15%, v/v) as organic modifier. Clean up of human plasma samples was carried out by means of a solid-phase extraction procedure, which gave a high extraction yield for all six carbamazepines (>88%). The overall precision of the method gives a mean RSD of about 1.8%. The limit of quantitation for all analytes is < or = 0.30 microg ml(-1), the limit of detection < or = 0.12 microg ml(-1).     

Implantable hollow fiber bioreactor as a potential treatment for hypercholesterolemia: characterization of the catalytic unit

Previous studies have shown that the modification of low density lipoprotein (LDL) by the enzyme phospholipase A(2)(PLA(2))results in a reduction of cholesterol levels in the plasma of hypercholesterolemic rabbits, due to accelerated clearance of the modified LDL. In the current study, we established techniques and optimized the ratio of enzyme to support for the immobilization of PLA(2) on a polymeric support. Hollow fiber bioreactors made from polytetrafluoroethylene (PTFE) polymers were used to encapsulate immobilized PLA(2). This design was adopted to eliminate hemolysis of red blood cells by the enzyme. Characterization of the resulting immobilized enzyme in terms of its activity, Michaelis-Menten kinetic constants, and the variation of its activity with incubation time is presented. The enzyme activity was not significantly altered upon incubation at 37 degrees C in lipoprotein-deficient serum (LPDS), over the course of 2 months. The Michaelis-Menten kinetics constants are K(M) = 8.9 mM, V(max) = 6434.2 for the free enzyme and K(app) (M) = 16.7 mM, V(app) (max) = 619.7 for the immobilized enzyme. These data suggest that a system based on immobilized PLA(2) in conjunction with hollow fiber bioreactors (HFBs) may be a good candidate for lowering LDL levels in plasma. (c) 1995 John Wiley & Sons, Inc.     

Development of a commercial amperometric biosensor electrode for the ketone D-3-hydroxybutyrate

Representatives of the common classes of quinoid NADH redox mediator, including Meldola Blue (MB) 3, 4-methyl-1,2-benzoquinone (4-MBQ) 4, 1-methoxy phenazine methosulphate (1-MeO-PMS) 5 and 2,6-dichloroindophenol (DCIP) 6, are shown to inhibit the NAD-dependent enzyme D-3-hydroxybutyrate dehydrogenase (HBDH), severely limiting their utility in the construction of a stable biosensor electrode for the ketone body D-3-hydroxybutyrate (3-OHB). It is proposed that these mediators bind covalently to important thiol groups in the enzyme. This mode of inhibition is overcome through the use of mediators such as 1,10-phenanthroline quinone (1,10-PQ) 7, which avoid 1,4-nucleophilic addition with enzyme amino acid residues such as Cys. As a result, 1,10-PQ 7 was selected for incorporation in a biosensor electrode for 3-OHB. The resulting MediSense Optiumtrade mark beta-Ketone electrode is stable (相似文献   

Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of Glyphosate and AMPA in Three Representative Agricultural Austrian Soils with a HPLC-MS/MS Method

We developed a novel method to quantify adsorbed glyphosate and AMPA in soils based on an extraction utilizing Na-tetraborate, an SPE clean-up step, and subsequent LC-MS detection. Reversed phase-based separation of glyphosate and AMPA was realized after FMOC-derivatization. The quantification involved external calibration and 1,2–¹³C, ¹⁵N- labeled glyphosate as well as ¹³C, ¹⁵N labeled AMPA as internal standards. The optimum recovery for extraction was obtained with 40 mM Na-tetraborate. The method was applied in three representative soils (Kirchberg, Phyra, and Pixendorf, Austria) where glyphosate was applied by standard agricultural practices. The recovery for glyphosate extracted with 40 mM Na-tetraborate buffer was 93.5% (RSD <2%) for glyphosate at Kirchberg-cambisol; 95.7% (RSD < 2%) at Pixendorf- chernozem and 79.1% (RSD <7%) at Phyra-stagnosol. The corresponding values for AMPA were 92.4% (RSD <2%) at Kirchberg, 98.1% (RSD <2%) at Pixendorf and 69.9% (RSD <4%) at Phyra. The limits of detection for glyphosate were 6.8 μg kg^?1(RSD <10%) at Kirchberg, 4.3 μg kg^?1 (RSD <10%) at Pixendorf, and 46.5 μg kg^?1 (RSD <7%) at Phyra. The limits of detection for AMPA were 26.7 μg kg^?1 (RSD <10%) at Kirchberg, 25.2 μg kg^?1 (RSD <10%) at Pixendorf, and 120.3 μg kg^?1 (RSD <9%) at Phyra. Accordingly, the limits of quantification were 22.7 μg kg^?1(RSD <5%) for glyphosate, and 88.9 μg kg^?1 (RSD <2%) for AMPA at Kirchberg and respectively 14.4 μg kg^?1 (RSD <6%) and 84 μg kg^?1 (RSD <5%) at Pixendorf and 13.8 μg kg^?1 (RSD <6%) and 87.2 μg kg^?1 (RSD <8%) at Phyra. Both substances in the soils were lower than the LOQ before applying the herbicide Roundup. The influence of higher contents of iron oxides, clay, and acidic pH, resulting in a more pronounced adsorption of glyphosate and AMPA in the soils of Phyra and Kirchberg, is demonstrated.     

Use of Chitosan Membrane from the Carapace of the Soldier Crab <Emphasis Type="Italic">Mictyris brevidactylus</Emphasis> for Biosensor Construction

Glucose oxidase (EC 1.3.4.3) was immobilized on chitosan membrane (<0.1 mm in thickness) prepared from the carapace of the soldier crab Mictyris brevidactylus. A glucose electrode was constructed by covering a platinum electrode (2.0 mm in diameter) with the enzyme membrane. The enzyme electrode sensed glucose amperometrically (1.0 µA/mM, with linear range up to 0.5 mM, r = 0.999) when positively imposed with 0.6 V against an Ag/AgCl reference electrode. The glucose biosensor was sensitive (<0.1 µM, S/N > 3), reproducible (CV for 55 µM glucose <3%, n = 5), reagentless, and durable for months.     

Effects of some drugs on the activity of glucose 6-phosphate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro

Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) were investigated. The enzyme was purified 2488-fold in a yield of 76.8% using ammonium sulfate precipitation and 2′,5′-ADP Sepharose 4B affinity gel at 4°C. The drugs pental sodium, MgSO₄, vancomycin, metamizol, marcaine, and prilocaine all exhibited inhibitory effects on the enzyme. While MgSO₄ (K_i = 12.119 mM), vancomycin (K_i = 1.466 mM) and metamizol (K_i = 0.392 mM) showed competitive inhibition, pental sodium (K_i = 0.748 mM) and marcaine (K_i = 0.0446 mM) displayed noncompetitive inhibition.     

Kinetic study of a cellobiase purified from Neocallimastix frontalis EB188

A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.     

Regulation of glucokinase activity in liver of hibernating ground squirrel Spermophilus undulatus

The kinetic properties of glucokinase (GLK) from the liver of active and hibernating ground squirrels Spermophilus undulatus have been studied. Entrance of ground squirrels into hibernation from their active state is accompanied by a sharp decrease in blood glucose (Glc) level (from 14 to 2.9 mM) and with a significant (7-fold) decrease of GLK activity in the liver cytoplasm. Preparations of native GLK practically devoid of other molecular forms of hexokinase were obtained from the liver of active and hibernating ground squirrels. The dependence of GLK activity upon Glc concentration for the enzyme from active ground squirrel liver showed a pronounced sigmoid character (Hill coefficient, h = 1.70 and S _0.5 = 6.23 mM; the experiments were conducted at 25°C in the presence of enzyme stabilizers, K⁺ and DTT). The same dependence of enzyme activity on Glc concentration was found for GLK from rat liver. However, on decreasing the temperature to 2°C (simulation of hibernation conditions), this dependency became almost hyperbolic (h = 1.16) and GLK affinity for substrate was reduced (S _0.5 = 23 mM). These parameters for hibernating ground squirrels (body temperature 5°C) at 25°C were found to be practically equal to the corresponding values obtained for GLK from the liver of active animals (h = 1.60, S _0.5 = 9.0 mM, respectively); at 2°C sigmoid character was less expressed and affinity for Glc was drastically decreased (h = 1.20, S _0.5 = 45 mM). The calculations of GLK activity in the liver of hibernating ground squirrels based on enzyme kinetic characteristics and seasonal changes in blood Glc concentrations have shown that GLK activity in the liver of hibernating ground squirrels is decreased about 5500-fold.     

TRH microdialysis into the RTN of the conscious rat increases breathing, metabolism, and temperature.

Thyrotropin-releasing hormone (TRH) injected into the retrotrapezoid nucleus (RTN) of anesthetized rats produces a large, prolonged stimulation of ventilatory output (C. L. Cream, A. Li, and E. E. Nattie. J. Appl. Physiol. 83: 792-799, 1997). Here we inject or dialyze TRH into the RTN of conscious rats. In 6 of 17 injections (200 nl, 3.1 +/- 1.7 mM), ventilation (VE) increased 31% by 10 min, with recovery by 60 min. With dialysis, each animal of one group (n = 5) received, in random order, 10 mM TRH, 10 mM TRHOH (a metabolite of TRH), and artificial cerebrospinal fluid (aCSF); each animal of a second group (n = 5) received aCSF and 1 mM TRH. TRHOH and aCSF had no sustained effects. TRH (1 mM) increased VE (32%, P < 0.02, by 10 min, with recovery by 60 min), O(2) consumption (VO(2); 19%, P < 0. 03), and body (rectal) temperature (T(re); 0.5 degrees C, P < 0.09). TRH (10 mM) increased VE (78%, P < 0.01, by 10 min, with no recovery at 60 min), VO(2) (48%, P < 0.01), and T(re) (1.0 degrees C, P < 0. 01). TRH also induced arousal. The tissue volume affected in dialysis, estimated by spread of dialyzed fluorescein (332.3 mol wt, mol wt of TRH = 362.4), was 1,580 +/- 256 nl for 10 mM (n = 5) and 590 +/- 128 nl for 1 mM (n = 5). We conclude that 1) the RTN is involved in the integration of VE, VO(2), T(re), and arousal and 2) TRH may establish the responsiveness of RTN neurons.     

The effect of galactose metabolic disorders on rat brain Na+,K+-ATPase activity

To evaluate the effect of galactose metabolic disorders on the brain Na+,K+-ATPase in suckling rats. Separate preincubations of various concentrations (1-16 mM) of the compounds galactose-1-phosphate (Gal-1-P) and galactitol (galtol) with whole brain homogenates at 37 degrees C for 1 h resulted in a dose dependent inhibition of the enzyme whereas the pure enzyme (from porcine cerebral cortex) was stimulated. Glucose-1-phosphate (Glu-1-P) or galactose (Gal) stimulated both rat brain Na+,K+-ATPase and pure enzyme. A mixture of Gal-1-P (2 mM), galtol (2 mM) and Gal (4 mM), concentrations commonly found in untreated patients with classical galactosemia, caused a 35% (p < 0.001) rat brain enzyme inhibition. Additionally, incubation of a mixture of galtol (2 mM) and Gal (1 mM), which is usually observed in galactokinase deficient patients, resulted in a 25% (p < 0.001) brain enzyme inactivation. It is suggested that: a) The indirect inhibition of the brain Na+,K+-ATPase by Gal-1-P should be due to the presence of the epimer Gal and phosphate and that the pure enzyme direct activation by Gal-1-P and Glu-1-P to the presence of phosphate only. b) The observed brain Na+,K+-ATPase inhibitions in the presence of toxic concentrations of Gal-1-P and/or galtol could modulate the neural excitability, the metabolic energy production and the catecholaminergic and serotoninergic system.     

Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus luteus) seeds

Three distinct enzymes hydrolyzing either ApppA or AppppA, or both, were separated and purified from yellow lupin seed extracts. Two of the enzymes were purified to homogeneity. These enzymes differ greatly in their catalytic and physical properties. One hydrolase, with a native molecular weight of 41,000, exhibits broad pH (from 5-8) optimum for activity, requires Mg2+ for activity, is inhibited by zinc ions (I0.5 = 25 microM) and hydrolyses ApppA (V = 1), ApppC (V = 0.38), ApppG (V = 0.2), and ribose(5')pppA (V = 0.2). The enzyme exhibits much lower activity with AppppA (V = 0.1), and ApppppA, AppppppA, ppppA, and ATP are hydrolyzed 25- to 100-fold slower then ApppA. ADP was always one of the products of the reactions catalyzed by the enzyme. AppA, NAD, NADP, FAD, cAMP, and p-nitrophenyl-thymidine 5'-phosphate were not hydrolyzed by the enzyme. The enzyme is diadenosine 5',5"'-P1, P3-triphosphatase. The second hydrolase, composed of one polypeptide chain of a molecular weight 18,000-18,500, exhibits optimal activity in the pH range from 7.5-9, requires Mg2+ for activity, is inhibited by calcium ions (I0.5 for calcium depends on the concentration of Mg2+ and is 35-180 microM in the presence of 0.5-10 mM Mg2+, respectively), and hydrolyzes AppppA (V = 1, Km = 1 microM), ApppppA (V = 0.42, Km = 1.8 microM), AppppppA (V = 0.34), AppppU (V = 0.73), AppppC (V = 0.67), AppppG (V = 0.27), and ppppA. ATP was always one of the products of the reactions catalyzed by the enzyme. Dinucleoside di- and triphosphates, ATP, cAMP, and p-nitrophenylthymidine 5'-phosphate were not hydrolyzed by the enzyme. This enzyme is diadenosine 5',5"'-P1,P4-tetraphosphatase (EC 3.6.1.17). The third hydrolase, composed of one polypeptide chain of a molecular weight of 56,000, exhibits maximal activity at pH 9-10.5, does not require Mg2+ ions for activity, is inhibited neither by divalent cations (Mg2+, Ca2+, Zn2+, Co2+, Mn2+, or Ni2+) nor by EDTA, and uses as substrates all compounds which are substrates for the diadenosine 5',5"'-P1,P3-triphosphatase and diadenosine 5',5"'-P1,P4-tetraphosphatase. In addition, the enzyme hydrolyzes p-nitrophenyl-thymidine 5'-phosphate, p-nitrophenylthymidine 3'-phosphate, bis-p-nitrophenylphosphate, ADP, AppA, NAD, NADP, and FAD, but not cAMP. With the exception of p-nitrophenylphosphate derivatives all other substrates of the enzyme yield AMP as one of the products of hydrolysis. This enzyme has a specificity similar to that of phosphodiesterases (EC 3.1.4.1) from other sources. With the lupin phosphodiesterase, ApppA (V = 1, Km = 2.2 microM) and AppppA (V = 1, Km = 2.0 microM) are better substrates than NAD (V = 0.8, Km = 9.6 microM), AppA (V = 0.4), ApppppA (V = 0.6), and AppppppA (V = 0.34).     

A one-step enzyme immunoassay for human manganese superoxide dismutase with monoclonal antibodies

A one-step enzyme immunoassay for the determination of manganese superoxide dismutase in serum has been developed with two kinds of monoclonal antibodies. Proposed method had high sensitivity (assay range, 0.4-200 ng/ml), good recovery (recovery percentage, 102.9-106.2%) and reproducibility (intraassay, C.V. = 1.87-3.66%; interassay, C.V. = 3.03-10.4%). From these results, it is possible to apply this method to routine clinical analysis and biochemical research with various purposes.     

Rat osseous plate alkaline phosphatase: Effect of neutral protease digestion on the hydrolysis of pyrophosphate and nitrohenylphospate

Collagenase treatment, commonly used to prepare alkaline phosphatase-rich matrix vesicles from epiphyseal cartilage growth plates, seems to affect the integrity of this membrane-bound enzyme. Alkaline phosphatase-rich rat osseous plates were incubated with 1000 U/mL collagenase for 3 h, at 37°C and after purification on Sepharose 4B, kinetic studies were performed using nitrophenylphosphate and pyrophosphate as substrates.The optimum apparent pH for the hydrolysis of p-nitrophenylphosphate and pyrophosphate increased from 9.4 to 10.25 and from 8.0 to 9.0, respectively, as a consequence of collagenase treatment. In the absence of Mg²⁺ ions, the enzyme hydrolyzed PNPP with K_M = 322.5 ± 15.3 M and V = 965.2 ± 45.8 U/mg, while in the presence of 2 mM Mg²⁺ ions, V increased 66%. Cobalt (K_0.5 = 5.3 ± 0.3 M) and manganese (K_0.5 = 0.72 ± 0.03 M) ions stimulated the PNPPase activity of the collagenase-treated enzyme, but with a lower apparent affinity when compared with that of not-treated enzyme. In the absence of Mg²⁺ ions pyrophosphate was hydrolyzed according to Michaelis-Menten kinetics (K_M = 105.1 ± 6.3 M and V = 64.9 ± 3.9 U/mg), but site-site interactions (n_H = 1.2) were observed in the presence of 2 mM Mg²⁺ ions (V = 110.8 ± 5.5 U/mg; K_0.5 = 42.7 ± 2.0 M).To our knowledge this is the first report showing significant alterations on phosphohydrolytic activity and metal binding properties of bone alkaline phosphatase due to associated neutral proteases in collagenase preparations often used for the isolation of matrix vesicles.     

Purification and characterization of PrbA,a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens)

The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 +/- 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K(m) and V(max) values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K(m) and V(max) values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 degrees C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), with K(i) values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine.     

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.