Μg Of Enzyme Research Articles

Optimization of Free Phytoprostane and Phytofuran Production by Enzymatic Hydrolysis of Pea Extracts Using Esterases.

Given the growing interest in phytoprostanes (PhytoPs) and phytofurans (PhytoFs) in the fields of plant physiology, biotechnology, and biological function, the present study aims to optimize a method of enzymatic hydrolysis that utilizes bacterial and yeast esterases that allow the appropriate quantification of PhytoPs and PhytoFs. To obtain the highest concentration of PhytoPs and PhytoFs, a response surface methodology/Box-Behnken design was used to optimize the hydrolysis conditions. Based on the information available in the literature on the most critical parameters that influence the activity of esterases, the three variables selected for the study were temperature (°C), time (min), and enzyme concentration (%). The optimal hydrolysis conditions retrieved differed between PhytoPs (21.5 °C, 5.7 min, and 0.61 μg of enzyme per reaction) and PhytoFs (20.0 °C, 5.0 min, and 2.17 μg of enzyme per reaction) and provided up to 25.1- and 1.7-fold higher contents relative to nonhydrolyzed extracts. The models were validated by comparing theoretical and experimental values for PhytoP and PhytoF yields (1.01 and 1.06 theoretical/experimental rates, respectively). The optimal conditions were evaluated for their relative influence on the yield of individual nonesterified PhytoPs and PhytoFs to define the limitations of the models for obtaining the highest concentration of most considered compounds. In conclusion, the models developed provided valuable alternatives to the currently applied methods using unspecific alkaline hydrolysis to obtain free nonesterified PhytoPs and PhytoFs, which give rise to more specific hydrolysis of PhytoP and PhytoF esters, reducing the degradation of free compounds by classical chemical procedures.

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of alpha1beta1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain beta1(1-385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (beta1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat beta1 distal heme pocket (Ile-145 --> Tyr and Ile-149 --> Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe(II)-O(2) complexes and is likely the first observation of a Fe(II)-O(2) complex in the full-length alpha1beta1 protein. Additionally, these data suggest that atypical sGCs stabilize O(2) binding by a hydrogen bonding network involving tyrosine and glutamine.

Use of Substrate Analogs and Mutagenesis to Study Substrate Binding and Catalysis in the Sir2 Family of NAD-dependent Protein Deacetylases

The Sir2 family of enzymes is highly conserved throughout evolution and functions in silencing, control of life span, apoptosis, and many other cellular processes. Since the discovery of the NAD-dependent deacetylase activity of Sir2 proteins, there has been a flurry of activity aiming to uncover the mode of substrate binding and catalysis. Structural and biochemical studies have led to several proposed reaction mechanisms, yet the exact catalytic steps remain unclear. Here we present in vitro studies of yeast homolog Hst2 that shed light on the mechanism of Sir2 proteins. Using acetyl-lysine substrate analogs, we demonstrate that the Hst2 reaction proceeds via an initial SN2-type mechanism with the direct formation of an ADP-ribose-acetyl-lysine intermediate. Kinetic studies further suggest that ADP-ribose inhibits the Hst2 reaction in a biologically relevant manner. Through biochemical and kinetic analyses of point mutants, we also clarify the role of several conserved core domain residues in substrate binding, stabilization of the ADP-ribose-acetyl-lysine intermediate, and catalysis. These findings bring us a few steps closer to understanding Sir2 activity and may provide a useful platform for the design of Sir2-specific inhibitors for analysis of Sir2 function and possibly therapeutic applications.

Recombinant Expression and Biochemical Characterization of the Unique Elongating β-Ketoacyl-Acyl Carrier Protein Synthase Involved in Fatty Acid Biosynthesis of Plasmodium falciparum Using Natural and Artificial Substrates

The human malaria parasite Plasmodium falciparum synthesizes fatty acids by using a type II synthase that is structurally different from the type I system found in eukaryotes. Because of this difference and the vital role of fatty acids, the enzymes involved in fatty acid biosynthesis of P. falciparum represent interesting targets for the development of new antimalarial drugs. beta-Ketoacyl-acyl carrier protein (ACP) synthase (PfFabBF), being the only elongating beta-ketoacyl-ACP synthase in P. falciparum, is a potential candidate for inhibition. In this study we present the cloning, expression, purification, and characterization of PfFabBF. Soluble protein was obtained when PfFabBF was expressed as a NusA fusion protein in Escherichia coli BL21(DE3)-CodonPlus-RIL cells under conditions of osmotic stress. The fusion protein was purified by affinity and ion exchange chromatography. Various acyl-P. falciparum acyl carrier protein (PfACP) substrates were tested for their specific activities, and their kinetic parameters were determined. Activity of PfFabBF was highest with C(4:0)- to C(10:0)-acyl-PfACPs and decreased with use of longer chain acyl-PfACPs. Consistent with the fatty acid synthesis profile found in the parasite cell, no activity could be detected with C(16:0)-PfACP, indicating that the enzyme is lacking the capability of elongating acyl chains that are longer than 14 carbon atoms. PfFabBF was found to be specific for acyl-PfACPs, and it displayed much lower activities with the corresponding acyl-CoAs. Furthermore, PfFabBF was shown to be sensitive to cerulenin and thiolactomycin, known inhibitors of beta-ketoacyl-ACP synthases. These results represent an important step toward the evaluation of P. falciparum beta-ketoacyl-ACP synthase as a novel antimalaria target.

Characterization of a New Pantothenate Kinase Isoform from Helicobacter pylori

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Two well characterized isoforms of the enzyme are known: a prokaryotic PanK that predominates in eubacteria and a eukaryotic isoform that has primarily been characterized from mammalian and plant sources. Curiously, the genomes of certain pathogenic bacteria, including Helicobacter pylori and Pseudomonas aeruginosa, do not contain a PanK similar to either isoform, although these organisms possess all the other biosynthetic machinery required for CoA production. In this study we cloned, overexpressed and characterized an enzyme from Bacillus subtilis and its homologue from H. pylori and show that they catalyze the ATP-dependent phosphorylation of pantothenate. These enzymes do not share sequence homology with any known PanK, and unlike the bacterial and eukaryotic PanK isoforms their activity is not regulated by either CoA or acetyl-CoA. They also do not accept the pantothenic acid antimetabolite N-pentylpantothenamide as a substrate or are inhibited by it. Taken together, these results point to the identification of a third distinct isoform of PanK that accounts for the only known activity of the enzyme in pathogens such as H. pylori and P. aeruginosa.

Purification of lipase from Cunninghamella verticillata by stepwise precipitation and optimized conditions for crystallization

Lipase from the oil-mill waste isolate Cunninghamella verticillata was purified by stepwise precipitation using acetone, as a sequel to our earlier conventional column chromatographic method [Gopinath et al. (2002)World Journal of Microbiology and Biotechnology 18, 449–458]. The yield of purified lipase was approx. 4-fold higher than by the previous method and the purified lipase was obtained with 70–80% acetone saturations. The enzyme was resolved as a single band with homogeneity both by native and by SDS–PAGE. The optimum condition for the lipase to crystallize was 5 μg of enzyme in 0.05 M sodium phosphate buffer (pH 6.5) with 5 mM FeCl2 and 10% 2-methyl 2,4-pentanediol (MPD).

Crystal Structure of Mouse Carnitine Octanoyltransferase and Molecular Determinants of Substrate Selectivity*[boxs

Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, medium- or long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, we report here the crystal structures at up to 2.0-A resolution of mouse carnitine octanoyltransferase alone and in complex with the substrate octanoylcarnitine. The structures reveal significant differences in the acyl group binding pocket between carnitine octanoyltransferase and carnitine acetyltransferase. Amino acid substitutions and structural changes produce a larger hydrophobic pocket that binds the octanoyl group in an extended conformation. Mutation of a single residue (Gly-553) in this pocket can change the substrate preference between short- and medium-chain acyl groups. The side chains of Cys-323 and Met-335 at the bottom of this pocket assume dual conformations in the substrate complex, and mutagenesis studies suggest that the Met-335 residue is important for catalysis.

Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105 000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH 2-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl- l-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K m value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7±0.1 mM, whereas the V max value decreased from 200 to 50 nmol/min per μg of enzyme, depending on the peptidic chain lengths. The V max value of the synthetic substrate (250 nmol/min per μg of enzyme) was 25–500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.

Phosphatidylinositol 4,5-Bisphosphate Binding to the Pleckstrin Homology Domain of Phospholipase C-δ1 Enhances Enzyme Activity

The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some co-complexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-delta1 (PLCdelta1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in a dose-dependent manner with an EC50 = 1 microM (0.3 mol%), up to 9-fold higher when 5 microM (1.5 mol%) of PIP2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 microM of PI with a molar ratio of PI:PS:PC = 1:5:5). Stimulation was specific for PIP2, since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited by inositol 1,4, 5-triphosphate (IP3) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLCdelta1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP2-mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP2. Analysis of PIP2-stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (Ks) of PLCdelta1 for the PI/PS/PC vesicles was reduced from 558 microM to 53 microM, and the interfacial Michaelis constant (Km) was reduced from 0.21 to 0.06 by PIP2. The maximum rate of PI hydrolysis (Vmax) was not affected by PIP2. These results demonstrate that a major function of the PH domain of PLCdelta1 is to modulate enzyme activity. Further, our results identify PIP2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP2.

A new assay procedure to study the induction of β-ketoacyl-ACP synthase I and II, and the complete purification of β-ketoacyl-ACP synthase I from developing seeds of oilseed rape ( Brassica napus)

We have developed a new radiochemical assay for β-ketoacyl-acyl carrier protein synthase, which is rapid, sensitive and does not require the presynthesis of acyl-acyl carrier protein derivatives as substrates. The assay has allowed us to study the induction of β-ketoacyl-acyl carrier protein synthase I and II activities during development of seeds of oilseed rape ( Brassica napus). Both enzymes are coordinately induced prior to lipid accumulation in these seeds. We have also used the new assay to purify to homogeneity β-ketoacyl-acyl carrier protein synthase I from the developing seeds. The enzyme required an approx. 12000-fold purification from the cell-free crude extract, with 8 μg of enzyme being obtained from 300 g of seed. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis purified β-ketoacyl-acyl carrier protein synthase I migrated as a single polypeptide giving an M r value of 43000. Gel-filtration chromatography gave an apparent native M r value of 86 700, suggesting that the enzyme is a homodimer. On electrophoresis in non-denaturing gels, two polypeptide bands were detectable, and both were associated with β-ketoacyl-acyl carrier protein synthase I activity. On gel filtration of a partially purified preparation, β-ketoacyl-acyl carrier protein synthase I and II could be partially resolved, with synthase II having an apparent native M r value of 87 400. The two activities could be completely resolved on an FPLC Mono Q ion-exchange column.

Human prostatic acid phosphatase: purification, characterization, and optimization of conditions for radioimmunoassay

Prostatic acid phosphatase was isolated from benign hypertrophic prostate tissue by ammonium sulfate precipitation and affinity chromatography procedures. The purified enzyme was characterized by two-dimensional gel electrophoresis and shown to have a cluster of protein spots with an apparent molecular weight of 48 000 at pI 5.9 to 6.3 in 9 mol/l urea. The specific activity of the purified enzyme was 723 and 659 U/mg protein with α-naphthyl phosphate at 30°C and para-nitrophenyl phosphate at 37°C respectively. An antibody to the purified enzyme was raised in rabbits and used in a radioimmunoassay (RIA). The use of a phosphate buffer, pH 6.6, and iodination of prostatic acid phosphatase (PAP) by the Bolton-Hunter procedure improved the precision of the assay when compared to RIA's using a phosphate buffer, pH 7.0 or 7.3, or PAP iodinated by a chloramine-T procedure. The former RIA displaced 50% of the tracer at 2 μg of enzyme per liter of serum. The between-run coefficient of variation for 11 assays ranged from 3.9–7.7% with serum at 1.3 to 5.6 μg PAP/l.

A sensitive method for detecting proteolytic enzymes released from their supports

A very sensitive radioisotope method, using 125I-labelled serum albumin as substrate, is proposed for the detection of very low enzymatic activities released from insoluble matrix to eluate, storage media and washings after an immobilization procedure. In the case of papain, proteolytic activities corresponding to about 0.1μg of enzyme per ml can be detected. In contrast, simple immunochemical techniques did not give any increase in sensitivity in papain determination compared with the conventional modified Anson method.

Quantitative determination of myeloperoxidase using tetramethylbenzidine as substrate

Tetramethylbenzidine, a noncarcinogenic, nonmutagenic derivative of benzidine, has been used as a substrate to assay myeloperoxidase. The assay is sensitive to 0.1 μg of enzyme and can be used to quantitate myeloperoxidase over a pH range of 4.4 to 7.4.

Development of a peroxidase-coupled fluorometric assay for lysyl oxidase

Lysyl oxidase catalyzes the oxidation of peptidyl lysine in elastin and collagen and also acts upon nonpeptidyl amines, although the enzyme becomes slowly inactivated while processing nonpeptidyl substrates. In spite of this complexity, it has been possible to devise a continuously monitored peroxidase-coupled fluorometric assay for the oxidation of simple amines by lysyl oxidase. In the present study, optimal assay conditions have been explored and found to include assay temperatures of 50 to 60°C, the presence of urea in the assay, and the use of diaminopentane as substrate. Although the assay is subject to interference by contaminating macromolecules in enzyme fractions, a linear assay response to enzyme concentration is obtained with highly purified lysyl oxidase with a limiting sensitivity of 0.3 μg of enzyme per assay.

A Computer-Automated Reactor System for Immobilized and Soluble Enzymes

ABSTRACT A versatile reactor system has been constructed for investigation of soluble and immobilized enzymes. The system has been fully automated using a laboratory minicomputer which permits not only computer-controlled experimentation but also detailed numerical and graphical examination of the data. The system requires relatively small amounts of enzyme. Typically, this has been 10 μg of enzyme for each discrete soluble experiment or 150 μg of enzyme for the construction of an immobilized bed which can be reused in many discrete experiments.

6] Immunological techniques for studying β-lactamases

This chapter presents the immunological techniques for studying β-lactamases. In principle, β-lactamases are no different from other enzymes with respect to their action with antisera, and many of the techniques are capable of much wider application than that has occurred in the past. The reason for describing them, however, is that they have a particular relevance to β-lactamases because many of the minor variants of this enzyme that are found in naturally occurring strains have been characterized by these immunological methods. Once antisera to a β-lactamase have been obtained, they can be used to study the enzymes in a number of ways. These include precipitation analyses both in solution and by gel diffusion. However, perhaps the most effective is by neutralization of enzyme activity because this can be studied with as little as 0.1 μg of enzyme in impure preparations.

Méthodes simples et rapides de détection et de l'essai de l'activité de l'ACP d' Escherichia coli utilisées au cours de sa purification

Summary E. coli ACP has been brought to homogeneity by using polyacrylamide gel electrophoresis as test to follow the purification steps. This test can replace the biological assays, namely the malonyl-CoA-CO 2 exchange reaction and the stimulation of fatty acid synthesis, used throughout the preparation of ACP in classical methods described previously. It presents the advantage of simplicity, economy and a greater fiability owing to the mere fact that the product is visualized. Using this test we observed that ACP is eluted, during the chromatography on DEAE-cellulose as a sharp peak, between 0.35 and 0.5 M NaCl concentrations, easily recognizable by spectrophotometry at 225 nm (fig. 1). The central fractions of this peak consists, in most preparations and eventually after reduction by DTT, of electrophoretically pure ACP. Other fractions of the peak contain more than 80 p. cent ACP. The biological assay, demonstrating the activity of ACP, does not accompagny, in our experiments, its purification steps but is delayed till the end of the preparation. The easy oxidation of the product, a disadvantage for biological assays, constitutes in our method an advantage, allowing, if needed, the localization of ACP and its oxidized companion, since the treatment by DTT reduces them to a single electrophoretic band (compare figure 2 a and b ). This reduction, however, is postponed in our experiments until the last step just before the biological assay. We use the malony-CoA-ACP-transacylase activity of a fraction of Mycobacterium smegmatis homogenates precipitable between 65 and 95 p. cent saturation of Ammonium sulfate, in 0,1 M phosphate buffer, to test the activity of ACP. Figure 4 shows the variation of the amount of malonyl-ACP formed, expressed as the acid precipitable radioactivity, as a function of the concentration of ACP expressed as mμmoles in 0.2 ml of the incubation medium in the presence of 1 μg of enzyme containing protein fraction of M. smegmatis . Figure 5 shows the variation of the amount of malonyl-ACP formed, expressed in the same way as in figure 4, as a function of the concentration of enzyme expressed as μg protein introduced in 0.2 ml of incubation medium in the presence of a saturating amount of ACP (6 mμmoles).

5′-Adenosine Monophosphate Aminohydrolase: EVIDENCE FOR ANOMALOUS SUBSTRATE SATURATION KINETICS AT LOW ENZYME CONCENTRATIONS

Abstract Rabbit muscle 5'-AMP aminohydrolase was shown to be susceptible to a protein concentration-dependent, dilution-induced inactivation at protein concentrations used in normal spectrophotometric assays; i.e. 0.5 µg per ml. Inactivation was less evident at saturating concentrations of substrate and at pH 6.3. These properties accounted for the AMP concentration versus initial velocity profiles which at pH 7.0 and 0.5 µg of enzyme per ml were sigmoidal but hyperbolic at 50 µg per ml; at pH 6.3 substrate saturation versus initial velocity profiles were hyperbolic at both protein concentrations. Kinetic parameters for deamination of AMP determined by stopped flow spectrophotometry at 50 µg per ml where dilution inactivation was not observed are reported as a function of pH. The potassium-activated enzyme exhibits normal Michaelis-Menten kinetics in the pH range 6.15 to 7.2; Vmax = 1730 ± 260 µmoles min-1 mg-1 at 30° and is invariant over the same pH range; Km increases slightly at the higher pH.

A sensitive fluorometric assay for amino acid oxidases

An assay for amino acid oxidases is described which is capable of measuring 0.005 μg of enzyme or the oxidation of 0.5 nmole of amino acid. It involves the coupled oxidation of scopoletin by hydrogen peroxide catalyzed by peroxidase and the fluorometric determination of the scopoletin remaining. By the use of this method, it is shown that glycine is a substrate of l-amino acid oxidase.