UDPgalactose 4-epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-epimerase activity.

UDPgalactose 4-epimerase (epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast epimerase has an N-terminal domain (residues 1-377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-epimerase, and a C-terminal domain (residues 378-699), which shows extensive identity to either the bacterial or human aldose 1-epimerase (mutarotase). The S. cerevisiae epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE-Sephacel and Resource-Q columns. Purified epimerase preparations showed mutarotase activity and could convert either alpha-d-glucose or alpha-d-galactose to their beta-anomers. Induction of cells with galactose led to simultaneous enhancement of both epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5'-UMP and l-arabinose, epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-epimerase is a bifunctional enzyme with aldose 1-epimerase activity. The active sites for these two enzymatic activities are located in different regions of the epimerase holoenzyme.     

A coupled optical enzyme assay for phosphopentomutase

Published assays for phosphopentomutase activity are based on acid lability differences between ribose 1-phosphate and ribose 5-phosphate. The present work describes a new method in which the isomerization of ribose 5-phosphate to ribose 1-phosphate is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: ribose 1-phosphate + adenine ? phosphate + adenosine (adenosine phosphorylase); adenosine + H₂O → inosine + NH₃ (adenosine deaminase). The method has been used to show some properties of Escherichia coli phosphopentomutase.     

A coupled spectrophotometric enzyme assay for methyltransferases

Adenosine deaminase (EC 3.5.4.4), purified from Aspergillus oryzae, is active in deaminating S-adenosylhomocysteine and its related thioethers, whereas the related sulfonium compound, S-adenosylmethionine, is not deaminated. By taking advantage of the different reactivity of the two compounds, a coupled optical enzyme assay for methyl transfer reactions has been developed. The amount of Ado-Hcy formed is calculated from the decrease in optical density at 265 nm, after addition of an excess of adenosine deaminase. The validity of the method has been tested with three purified enzymes, i.e., homocysteine methyltransferase, histamine methylase, and acetylserotonin methyltransferase. Some kinetic constants of these enzymes have been obtained. The procedure is highly accurate, reproducible, and relatively simple compared to the conventional radio-chemical methods currently in use.     

A coupled enzyme assay for isopenicillin N synthetase

The development of a coupled enzyme assay for the determination of isopenicillin N synthetase activity in purified extracts from Cephalosporium acremonium was described. Isopenicillin N formed from its precursor, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), by the synthetase was hydrolyzed by beta-lactamase I to the corresponding penicilloic acid. Automatic titration of the acid with standard sodium hydroxide delivered by a pH-stat gave a continuous plot of product formed vs time. This assay has been used in kinetic studies and to determine the effects of pH, ionic strength, and temperature on the enzyme's activity.     

A coupled enzyme assay for measurement of sialidase activity.

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.     

Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase.

The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-galM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S. thermophilus cells matched the mRNA levels.     

Entamoeba histolytica: molecular characterization of an aldose 1-epimerase (mutarotase)

In cells, the alpha-anomers of aldoses are the preferred metabolizable substrates, while beta-anomers of aldoses play their role in glycan structure. In the cytoplasm, alpha- and beta-anomers of aldoses interconvert through the enzyme termed aldose 1-epimerase or mutarotase (EC 5.1.3.3). We have identified a mutarotase gene in Entamoeba histolytica, the causative agent of non-bacterial dysentery in humans. Cloning and characterization of this gene in two strains of the parasite (HM-1:IMSS and Rahman) that differ in their pathogenicity, revealed that the sequence is identical in both strains. A recombinant E. histolytica mutarotase was produced as well as specific antibodies that recognized a 38 kDa protein in trophozoite lysates of both strains. Mutarotase activity was observed with the recombinant protein as well as in lysates of both HM-1:IMSS and Rahman, the former exhibiting a slightly higher mutarotase activity. Finally, we have shown by complementation that overexpression of the E. histolytica mutarotase in a mutarotase defective Escherichia coli strain restores the ability of these bacteria to grow in minimal medium with phenyl-beta-galactopyranoside as the sole carbon source.     

Fluorimetric coupled enzyme assay for lysoplasmalogenase activity in liver.

Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.     

Colorimetric coupled enzyme assay for gamma-glutamyltransferase activity using glutathione as substrate

A colorimetric coupled enzyme assay for the determination of gamma-glutamyltransferase (GGT) activity using glutathione as substrate is described. The cysteine released from glutathione upon sequential action of GGT and leucine aminopeptidase is spectrophotometrically detected through its reaction with ninhydrin at 100 degrees C in acidic conditions. The method was applied to the determination of the activity of both bovine kidney and human serum GGT. In the described assay conditions with final GGT concentrations ranging from 0.18 to 4 mU/ml, a linear relationship between produced cysteine and incubation times up to 90 min was observed. When a standard chromogenic assay for GGT using L-gamma-glutamyl-3-carboxy-4-nitroanilide as substrate and the proposed assay were applied on the same serum sample a linear relationship between the two method was observed. Since the use of GSH as substrate, the proposed method can be usefully adopted for enzymological studies on GGT-related enzymes, a class of enzymes which is still waiting to be characterized.     

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP⁺ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.     

A single-isotope enzyme assay for histamine

A simple, rapid, and sensitive radioenzymic method for histamine is described. The method utilizes a specific histamine enzyme, histamine N-methyltransferase, isolated from guinea pig brains and high specific activity tritiated S-adenosylmethionine. The sensitivity of the method as described, under best conditions, is 0.1 ng. All reagents are stable, readily prepared, and/or commercially available.     

A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity

Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available omega(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.     

A general coupled spectrophotometric assay for decarboxylases

A rapid, continuous spectrophotometric method has been developed for the assay of decarboxylases. The assay uses a coupled enzyme system in which liberated CO2 is reacted with phosphoenolpyruvate and phosphoenolpyruvate carboxylase to form oxaloacetate, which in turn is reduced by malate dehydrogenase to L-malate concomitantly with the oxidation of NADH to NAD. The resultant decrease in absorbance at 340 nm accurately reflects the activity of the decarboxylase. The method is capable of detecting the liberation of as little as 1 nmol of CO2/min and was tested in assays of lysine decarboxylase, orotidine-5'-phosphate decarboxylase, and 4'-phosphopantothenoyl-L-cysteine decarboxylase.     

A continuous coupled enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD)

Bacterial malonyl-CoA:acyl carrier protein transacylase catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of acyl carrier protein. Malonyl-ACP, the product of this enzymatic reaction, is the key building block for de novo fatty acid biosynthesis. Here, we describe a continuous enzyme assay based on the coupling of the malonyl-CoA:acyl carrier protein transacylase reaction to alpha-ketoglutarate dehydrogenase (KDH). KDH-dependent consumption of the coenzyme A generated by malonyl-CoA:acyl carrier protein transacylase is accompanied by a reduction of nicotinamide adenine dinucleotide, oxidized (NAD(+)) to nicotinamide adenine dinucleotide, reduced. The rate of NAD(+) reduction is continuously monitored as a change in fluorescence using a microtiter plate reader. We show that this coupled enzyme assay is amenable to routine chemical compound screening.     

A fluorometric assay for angiotensin-converting enzyme activity

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.