Use of an enzyme thermistor in continuous measurements and enzyme reactor control.

The enzyme thermistor measures the heat produced by the action of an immobilized enzyme on a substrate present in the sample. Its application in analysis of discrete samples, e.g., in clinical chemistry, is well documented, but it has not been used so far for continuous measurements. We decribe here the application of the enzyme thermistor for continuous monitoring and control of enzyme reactors. An enzyme thermistor filled with coimmobilized glucose oxidase and catalase was used to measure the amount of glucose in the outflow from a column reactor containing immobilized lactase acting on a lactose solution pumped through the reactor. The lactose conversion was kept on a constant level, irrespective of the actual enzymatic activity in the reactor, by regulating the flow through the reactor. The experiments were carried out with aqueous solutions of lactose as well as with whey from cow's milk.     

Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system.

The possibility of using the enzyme thermistor (ET) for the direct determination of kinetic parameters (Km, Ki, Vm) of immobilized enzyme (IME) was evaluated using different preparations of invertase conjugated to bead celluloses. Two different ET columns packed with IME were operated in the mode of a differential enzyme reactor (short length, low substrate conversion). Kinetic parameters of the above IME reactor were computed by a nonlinear curve-fitting procedure. The obtained kinetic parameters were superverified by means of an independent differential reactor (DR) system. This system utilized an indirect postcolumn analytical method based on determination of glucose concentration in the stirred reservoir. Best agreement between the data acquired by direct (ET) and indirect (DR) methods was obtained if the ET column was operated at flow rates within the range of 1.0-1.5 ml min-1 using invertase-cellulose chlorotriazine conjugate. Influence of heat loss and flow nonideality is discussed. The proposed ET method offers a rapid, convenient, and general approach to determination of kinetic constants of IME preparations by omitting postcolumn analytical methods.     

Microcalorimetric methods for substrate determination in flow systems with immobilized enzymes.

The enthalpy of processes catalyzed by immobilized enzymes in the reaction cell of a LKB-flow calorimeter is used for determination of urea (0.5-5 mumol) and glucose (0.03-0.5 mumol). Accuracy is 2-5% and the time needed for one analysis is 20 min. A sensitive "enzyme thermistor" consisting of a flow through cell with an immobilized enzyme and two thermistors is described, which permits glucose determinations (0.05-1 mumol +/- 0.03 mumol) by means of temperature difference caused by reaction heat. Coupling of enzyme reactions for increasing reaction heat and consequently sensitivity in calorimetric determinations is demonstrated.     

Enzyme kinetics determined using calorimetry: a general assay for enzyme activity?

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.     

Measurement of heat evolution and correlation with oxygen consumption during microbial growth. Reprinted from Biotechnology and Bioengineering, Vol. XI, Issue 3, Pages 269-281 (1968)

A procedure for measuring the rate of heat production from a fermentation has been developed. The method is based on measuring the rate of temperature rise of the fermentation broth resulting from metabolism, when the temperature controller is turned off. The heat accumulation measured in this manner is then corrected for heat losses and gains. A sensitive thermistor is used to follow the temperature rise with time. This procedure is shown to be as accurate as previous methods but much simpler in execution. Using this technique, the rate of heat production during metabolism was found to correlate with the rate of oxygen consumption. Experiments were performed using bacteria (E. coli and B. subtilis), a yeast (C. intermedia), and a mold (A. niger). The substrates investigated included glucose, molasses, and soy bean meal. The proportionality constant for the correlation is independent of the growth rate, slightly dependent on the substrate, and possibly dependent on the type of organism growth. This correlation has considerable potential for predicting heat evolution from the metabolism of microorganisms on simple or complex substrates and providing quantitative parameters necessary for heat removal calculations.     

A comparison of the effects of diabetes induced with either alloxan or streptozotocin and of starvation on the activities in rat liver of the key enzymes of gluconeogenesis

1. Measurements of the activities in rat liver of the four key enzymes involved in gluconeogenesis, i.e. pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), have been carried out, all four enzymes being measured in the same liver sample. Changes in activities resulting from starvation and diabetes have been studied. Changes in concentration (activity/unit wet weight of tissue) were compared with changes in the hepatic cellular content (activity/unit of DNA). 2. Each enzyme was found to increase in concentration during starvation for up to 3 days, but only glucose 6-phosphatase and phosphoenolpyruvate carboxykinase showed a significant rise in content. Fructose 1,6-diphosphatase appeared to decrease in content somewhat during the early stages of starvation. 3. There was a marked increase in the concentration of all four enzymes in non-starved rats made diabetic with alloxan or streptozotocin, for the most part similar responses being found for the two diabetogenic agents. On starvation, however, the enzyme contents in the diabetic animals tended to fall, often with streptozotocin-treated animals to values no greater than for the normal overnight-starved rat. Deprivation of food during the period after induction of diabetes with streptozotocin lessened the rise in enzyme activity. 4. The results are compared with other published values and factors such as substrate and activator concentrations likely to influence activity in vivo are considered. 5. Lack of correlation of change in fructose 1,6-diphosphatase with the other enzymes questions whether it should be included in any postulation of control of gluconeogenic enzymes by a single gene unit.     

Energy-Metabolising Enzymes in Brain Regions of Adult and Aging Rats

Abstract: The regional enzyme activities of glucose metabolism in the rat brain were investigated. Hexokinase (EC 2.7.1.1) and pyruvate dehydrogenase (EC 1.2.4.1), key enzymes for glucose metabolism, showed no changes in activity in all the regions studied of the aging brain as compared with the adult brain. However, the activity of d -3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) is low throughout the adult brain and, in contrast with hexokinase and pyruvate dehydrogenase, its activity decreases significantly during aging. Other enzymes that showed significant decreases during aging are aldolase (EC 4.1.2.13), lactate dehydrogenase (EC 1.1.1.27), citrate synthase (EC 4.1.3.7), and NAD⁺-linked isocitrate dehydrogenase (EC 1.1.1.41). The catabolic enzyme in cholinergic metabolism, acetylcholinesterase (EC 3.1.1.7), selected as an example of a non-energy-metabolising enzyme, also showed significant decreases in all regions of the brain in aging, although its highest activity remained in the striatum. These results are discussed with respect to the energy metabolism in various brain regions and their status with aging.     

Hydrolyzing Pathway,Substrate Specificity and Inhibition of Tannin Acyl Hydrolase of Asp. oryzae No. 7

Tannin acyl hydrolase (EC 3.1.1.20) of Asp. oryzae No. 7 hydrolyzes tannic acid to glucose and gallic acid. The intermediate hydrolyzates are 1,2,3,4,6-pentagalloyl glucose, 2,3,4,6-tetragalloyl glucose and two kinds of monogalloyl glucose.

The enzyme hydrolyzes ester compounds of gallic acid, but does not hydrolyze any other substrate analogues such as methyl-resorcyrate.

The enzyme reaction is inhibited competitively by substrate analogues which have phenolic hydroxyls with the exception that 2,6-dihydroxy benzoic acid inhibits noncompetitively. Therefore the binding site of the enzyme may be able to react with any kind of phenolic hydroxyl, although the substrate forming a true ES-complex must be an ester compound of gallic acid.     

Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.     

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bacteroides caccae

AIMS: To compare fermentation pattern in cultures of Bacteroides caccae supplied with pectin and glucose, and identify enzymes involved in metabolism of pectin. METHODS AND RESULTS: A strain KWN isolated from the rabbit caecum was used. Fermentation pattern, changes of viscosity and enzyme reactions products were determined. Cultures grown on pectin produced significantly more acetate and less formate, lactate, fumarate and succinate than cultures grown on glucose. Production of cell dry matter and protein per gram of substrate used was the same in pectin- and glucose-grown cultures. The principal enzymes that participated in the metabolism of pectin were extracellular exopectate hydrolase (EC 3.2.1.67), extracellular endopectate lyase (EC 4.2.2.2) and cell-associated 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). The latter enzyme is unique to the Entner-Doudoroff pathway. Activities of pectinolytic enzymes in cultures grown on glucose were low. Activity of KDPG aldolase was similar in pectin- and glucose-grown cells. CONCLUSIONS: Metabolites and activities of pectin-degrading enzymes differed in cultures of B. caccae KWN grown on pectin and glucose. Yields of dry matter and protein were the same on both substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on metabolism of pectin in animal strains of Bacteroides is incomplete. This study extends the knowledge on metabolism in bacteria from the rabbit caecum.     

The localization of trehalase in polyacrylamide gels with eugenol by a coupled enzyme assay

Methods for the localization of trehalase (α, α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) activity following electrophoresis in nondenaturing polyacrylamide gels have been developed, in which trehalose hydrolysis is coupled to oxidation of the peroxidase substrate eugenol (2-methoxy-4-allyl phenol) by the use of glucose oxidase (EC 1.1.3.4), and peroxidase (EC 1.11.1.7) as ancillary enzymes. The basis for this procedure stems from the fact that free radicals of eugenol which are generated during the coupled trehalase assay condense to form a white precipitate whose location in the gel may be determined by densitometric scanning and whose surface area is a linear function of the enzyme level subjected to electrophoresis.     

Crystallographic studies of the mechanism of xylose isomerase

The mechanism of xylose isomerase (EC 5.3.1.5) has been studied with X-ray crystallography. Four refined crystal structures are reported at 3-A resolution: native enzyme, enzyme + glucose, enzyme + glucose + Mg2+, and enzyme + glucose + Mn2+. One of these structures (E.G.Mg) was determined in a crystal mounted in a flow cell. The other structures were equilibrium experiments carried out by soaking crystals in substrate containing solution. These structures and other studies suggest that, contrary to expectation, xylose isomerase may not use the generally expected base-catalyzed enolization mechanism. A mechanism involving a hydride shift is consistent with the structures presented here and warrants further investigation. Additional evidence in support of a hydride shift comes from comparing xylose isomerase with triosephosphate isomerase which is known to catalyze an analogous reaction via an enediol intermediate. Evidence is presented that suggests that aldose-ketose isomerases can be divided into two groups. Phospho sugar isomerases generally do not require a metal ion for activity and show exchange of substrate protons with solvent. In contrast, simple sugar isomerases all require a metal ion and show very low solvent exchange. These observations are rationalized on the basis of the need for stereospecific sugar binding.     

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rumen bacterium Lachnospira multiparus

AIMS: Lachnospira multiparus belongs to the main rumen pectinolytic bacteria. Its carbohydrate metabolism was studied in growth experiments on laboratory fermenters, and using assays of activities of enzymes involved in pectin fermentation. METHODS AND RESULTS: The type strain of this species and two substrates were used. Lachnospira multiparus ATCC 19207 grew on pectin and glucose at a similar rate and had no preference for one or the other substrate. Pectin-grown cultures, however, produced significantly more acetate and less formate, lactate, ethanol, hydrogen, cell dry matter and protein than corresponding cultures grown on glucose. Extracellular exopectate lyase (EC 4.2.2.9) was the principal enzyme degrading the pectin macromolecule. Cell extracts possessed 2-keto-3- deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) and fructosediphosphate aldolase (EC 4.1.2.13) activity. The former enzyme catalyses the final reaction in the Entner-Doudoroff pathway; the latter is the key enzyme of glycolysis and the pentose phosphate pathway. CONCLUSION: These results are consistent with the assumption that acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway. Phosphogluconate was not metabolized by cell extracts of the strain studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This suggests that the conventional Entner-Doudoroff pathway of glucose utilization does not operate in this bacterium, presumably because of the lack of 6-phosphogluconate dehydrase (EC 4.2.1.12) activity.     

Purification and properties of glucose 6-phosphate dehydrogenase from Aspergillus aculeatus

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Aspergillus aculeatus, a filamentous fungus previously isolated from infected tongue of a patient. The enzyme, apparently homogeneous, had a specific activity of 220 units mg(-1), a molecular weight of 105,000 +/- 5,000 Dal by gel filtration and subunit size of 52,000 +/- 1,100 Dal by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The substrate specificity was extremely strict, with glucose 6-phosphate (G6P) being oxidized by nicotinamide adenine dinucleotide phosphate (NADP) only. At assay pH of 7.5, the enzyme had K(m) values of 6 microM and 75 microM for NADP and G6P respectively. The k(cat) was 83 s(-1). Steady-state kinetics at pH 7.5 produced converging linear Lineweaver-Burk plots as expected for ternary-complex mechanism. The patterns of product and dead-end inhibition suggested that the enzyme can bind NADP and G6P separately to form a binary complex, indicating a random-order mechanism. The enzyme was irreversibly inactivated by heat in a linear fashion, with G6P providing a degree of protection. Phosphoenolpyruvate (PEP), adenosinetriphosphate (ATP), and fructose 6-phosphate (F6P), in decreasing order, are effective inhibitors. Zinc and Cobalt ions were effective inhibitors although cobalt ion was more potent; the two divalent metals were competitive inhibitors with respect to G6P, with Ki values of 6.6 microM and 4.7 microM respectively. It is proposed that inhibition by divalent metal ions, at low NADPH /NADP ratio, is another means of controlling pentosephosphate pathway.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Biophysical investigations on the active site of brain hexokinase

Replacement of Mg (II), the natural activator of brain hexokinase (EC 2.7.1.1) by paramagnetic Mn (II) without affecting the physiological properties of the enzyme, has rendered brain hexokinase accessible to investigations by magnetic resonance methods. Based on such studies, a site on the enzyme, where Mn (II) binds directly with high affinity has been identified and characterized in detail. Use ofβ,γ-bidentate Cr (III) ATP as an exchange-inert analogue for Mn (II) ATP has shown that Mn (II) binding directly to the enzyme has no catalytic role but another Mn (II) ion binding simultaneously and independently to the enzyme through the nucleotide bridge participates in enzyme function. However, using this direct binding Mn (II) ion and a covalently bound spin label as paramagnetic probes a beginning has been made in mapping the ligand binding sites of the enzyme. Ultra-violet difference spectroscopy has revealed the presence of at least two glucose 6-phosphate locations on the enzyme one of which presumably is the high affinity regulatory site modulated by substrate glucose. Elution behaviour of the enzyme on a phosphocellulose column suggests that glucose induces a specific phosphate site on the enzyme to which the phosphate bearing regulatory ligands of the enzyme may bind.     

Determination of the Deoxyglucose and Glucose Phosphorylation Ratio and the Lumped Constant in Rat Brain and a Transplantable Rat Glioma

Mitochondrially bound hexokinase (ATP-D-hexose-6-phosphotransferase; EC 2.7.1.1) was dissociatively extracted from normal rat brains and intracerebral and subcutaneous implants of the 36B-10 glioma. At least 70% of the total hexokinase enzyme activity in normal and glioma tissue was associated with the mitochondrial fraction. Purification of the crude tissue extracts by ion-exchange and affinity chromatography followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a successive purification of the enzyme to homogeneity with a molecular size of 98 kilodaltons. Enzyme kinetics with glucose or 2-deoxyglucose (2-DG) as the substrate were measured spectrophotometrically by coupling the appropriate reactions to either NADPH or NAD+ formation. The Km of hexokinase with glucose as the substrate in the intracerebral glioma (0.138 mM) and subcutaneous glioma (0.183 mM) tissues was 2.1-2.7-fold higher than that observed in normal brain tissue (0.067 mM) (p less than 0.001). No significant differences were observed in the Km for hexokinase with 2-DG as the substrate in the glioma and normal brain tissue. The phosphorylation ratio for normal brain was 0.320 and was increased in the intracerebral glioma to 0.694 and in the subcutaneous glioma to 0.519. The ratios of deoxyglucose and glucose volumes of distribution in normal brain and intracerebral glioma tissues were 1.70 and 1.85, respectively. The lumped constants calculated directly from the phosphorylation ratios and the volumes of distribution of deoxyglucose and glucose were 0.517 in normal brain and 1.168 in intracerebral glioma. Our results indicate the lumped constant is increased 2.26-fold in intracerebral glioma compared with normal brain.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Factors involved in changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of acetyl-CoA carboxylase (EC 6.4.1.2), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), extramitochondrial aconitate hydratase (EC 4.2.1.3) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) have been measured in the livers of developing rats from late foetal life to maturity. 2. The effect of altering the weaning time on some enzymes associated with lipogenesis has been studied. Weaning rats at 15 days of age instead of 21 days results in an immediate increase in the activity of ;malic' enzyme (EC 1.1.1.40) whereas the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and ATP citrate lyase (EC 4.1.3.8) did not increase until 4-5 days and acetyl-CoA carboxylase 2-3 days after early weaning. Weaning rats on to an artificial-milk diet led to complete repression of the rise in activity of hepatic enzymes associated with lipogenesis normally found on weaning, except for ;malic' enzyme, which increased in activity after 20 days of age. 3. The effect of intraperitoneal injections of glucagon, cortisol, growth hormone and thyroxine on the same hepatic enzymes has been investigated. Only thyroxine had any effect on enzyme activities and caused a 20-fold increase in ;malic' enzyme activity and a twofold increase in ATP citrate lyase activity. 4. The activities of hepatic glucose 6-phosphate dehydrogenase and ;malic' enzyme are higher in adult female than in adult male rats and it has been shown that this sex difference in enzyme activities is due to both male and female sex hormones. 5. Hepatic malate, citrate, pyruvate, glucose 6-phosphate and phosphoenolpyruvate concentrations have been measured throughout development. 6. The results are discussed in relation to the dietary and hormonal control of hepatic enzyme activities during development.