Quantification of enzyme activities in brain sections by microphotometry

1. Catalytic enzyme histochemistry offers the possibility to demonstrate enzyme activities quantitatively (microphotometry) in brain sections of those sites where they are localized. 2. A prerequisite for quantification are appropriate histochemical procedures for the demonstration of enzymes, which are shortly discussed. 3. For the microphotometric determination of enzymes in brain sections the scanning microphotometry is at present the technique of choice. 4. This is described in the example of an image plane scanning system. 5. Using this technique two measuring procedures can be applied for the quantification of enzyme activities, i.e. kinetic and end-point measurements. 6. Methods for the microphotometric determination of certain important oxido-reductases and further enzymes are presented. 7. It is concluded that quantitative catalytic enzyme histochemistry could be a source of results complementary to those provided by conventional biochemistry.     

Control of the flux in the arginine pathway of Neurospora crassa. Modulations of enzyme activity and concentration.

The influence of particular enzyme activities on the flux of metabolites in a pathway can be estimated by 'modulating' enzymes (i.e. changing turnover or concentration) and measuring the response in various parts of the system. By controlling the nuclear ration of two genetically different nuclear types in heterokaryons, the enzyme concentrations at four different steps in the arginine pathway were decreased over a range. This range was extended by the use of bradytrophs, mutant strains specifying enzymes with greatly diminished enzyme activities. Strains altered simultaneously at more than one step were also constructed by genetic recombination. By measuring the outputs of the pathway and the steady-state concentrations of intermediate pools, the fluxes in different parts of the pathway were calculated. This allowed the construction of flux/enzyme relationships, the slope of which is a measure of the sensitivity of a flux to the change in enzyme activity at that step. All fluxes were found to be considerably buffered for quite substantial decreases in the activities of all enzymes. Mass action plays an important part in this phenomenon, as do inhibition and repression. Because of the existence of expansion fluxes in growing systems, we find quantitatively different fluxes in different parts of the single pathway. For the same reason some enzyme modulations given decreased fluxes in one part and increased fluxes in another. The understanding of control in the pathway thus involves consideration of many mechanisms operating simultaneously and the estimation of changes in the whole system. The concept of a 'rate-limiting step' is found to be inadequate and is replaced by a quantitative measure, the Sensitivity Coefficient, which takes account of all the interactions. It is shown that control of the flux is shared among all the enzymes of the pathway. The results are discussed in terms of the theory of flux control.     

Principle and method of kinetic microphotometric enzyme activity determination in situ

An advanced apparative set-up is described for multipositional microphotometric recording of histochemical enzyme reactions in cryostat sections. It consists of a computer controlled microscope photometer with scanning stage. Measurements on the same tissue section may be performed at 12 preselected positions. These are repeatedly brought into the measuring beam in several measuring cycles. The complete measuring process, storage of measuring position coordinates, movements of the stage and statistical evaluation of the data is under computer control. By use of the gel film technique, extinction changes in tetrazolium coupled enzyme reactions can be measured continuously at initial rate conditions. Measurements are performed at identical conditions and can thus be analysed as relative enzyme activities.     

Principle and method of kinetic microphotometric enzyme activity determination in situ

Summary An advanced apparative set-up is described for multipositional microphotometric recording of histochemical enzyme reactions in cryostat sections. It consists of a computer controlled microscope photometer with scanning stage. Measurements on the same tissue section may be performed at 12 preselected positions. These are repeatedly brought into the measuring beam in several measuring cycles. The complete measuring process, storage of measuring position coordinates, movements of the stage and statistical evaluation of the data is under computer control. By use of the gel film technique, extinction changes in tetrazolium coupled enzyme reactions can be measured continuously at initial rate conditions. Measurements are performed at identical conditions and can thus be analysed as relative enzyme activities.     

NanoDrop fluorometry adopted for microassays of proteasomal enzyme activities

NanoDrop spectrophotometry and NanoDrop fluorospectrometry are used almost exclusively to determine the concentrations of nucleic acids and proteins. We propose that NanoDrop fluorospectrometry can also be applied for measuring enzyme activities using fluorogenic substrates such as the proteolytic activities of the 26S proteasome. Because the NanoDrop ND-3300 device requires only 2 μl of sample, the amount of sample extract, substrate, and cofactors used for an enzyme assay can be significantly reduced. In this report, we present exemplary microassays for proteasomal activities (chymotrypsin-, trypsin-, and PGPH [peptidyl-glutamyl peptide hydrolase]-like sites) in extracts of isolated hemocytes from a marine crab, Cancer pagurus (Crustaceae).     

Carbon metabolism and the sign of control coefficients in metabolic adaptations underlying K-ras transformation

Metabolic adaptations are associated with changes in enzyme activities. These adaptations are characterized by patterns of positive and negative changes in metabolic fluxes and concentrations of intermediate metabolites. Knowledge of the mechanism and parameters governing enzyme kinetics is rarely available. However, the signs-increases or decreases-of many of these changes can be predicted using the signs of metabolic control coefficients. These signs require the only knowledge of the structure of the metabolic network and a limited qualitative knowledge of the regulatory dependences, which is widely available for carbon metabolism. Here, as a case study, we identified control coefficients with fixed signs in order to predict the pattern of changes in key enzyme activities which can explain the observed changes in fluxes and concentrations underlying the metabolic adaptations in oncogenic K-ras transformation in NIH-3T3 cells. The fixed signs of control coefficients indicate that metabolic changes following the oncogenic transformation-increased glycolysis and oxidative branch of the pentose-phosphate pathway, and decreased concentration in sugar-phosphates-could be associated with increases in activity for glucose-6-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, and decrease for transketolase. These predictions were validated experimentally by measuring specific activities. We conclude that predictions based on fixed signs of control coefficients are a very robust tool for the identification of changes in enzyme activities that can explain observed metabolic adaptations in carbon metabolism.     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.     

A single and continuous spectrophotometric assay for various lipolytic enzymes, using natural, non-labelled lipid substrates

A rapid, continuous spectrophotometric assay for measuring the amount and activity of several lipolytic enzymes is described. It is based on the metachromatic properties of the cationic dye safranine, and makes use of the fact that an adequate combination of a lipolytic enzyme with one of its substrates leads to a change in the net negative charge at the lipid/water interface, which is monitored by the absorbance change of safranine. Utilizing this method, most lipolytic enzymes can be detected in very low amounts (milliunit or less) in about 1 min without employing radiolabelled lipids or synthetic lipid analogues. Over a wide range of enzyme concentrations, there is a good linearity between the initial hydrolysis rate (determination by the safranine method) and the amount of enzyme. The versatility of the assay is illustrated by examples showing how phospholipase A2, triacylglycerol hydrolase, phospholipase D or phospholipase C (either general or phosphatidylinositol-specific) activities can be detected, either separately or sequentially. Due to its high sensitivity, simplicity, and rapidity, this assay should find its main application in monitoring column effluents during the purification steps of lipolytic enzymes.     

Screening for and identification of starch-, amylopectin-, and pullulan-degrading activities in bifidobacterial strains

Forty-two bifidobacterial strains were screened for alpha-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.     

Quantitative enzyme histochemistry in the brain

Two main groups of quantitative methods are used in the brain to relate enzymatic processes to cellular structures, i.e. the methods of microchemistry and microscopic histochemistry. Microchemistry tries to quantify enzyme activities in very small brain regions by miniaturizing biochemical methods, whereas microscopic histochemistry applies staining procedures to tissue sections, preserving the structural relationship that is present in situ and giving topological information on the distribution of enzymes which is indispensable in structural heterogeneous tissue as is the brain. The present review deals preferentially with microscopic methods and, in particular, with scanning microphotometry (image plane scanning). Using this technique two measuring procedures can be applied for the quantification of enzyme activities, i.e. end-point and kinetic (continuous monitoring) measurements which are described in detail. Methods for the microphotometric demonstration of certain important dehydrogenases (isocitrate dehydrogenases, succinate dehydrogenase, NAD-linked malate dehydrogenase, glutamate dehydrogenase and glycerol 3-phosphate dehydrogenase), of cytochrome c oxidase, hexokinase and acetylcholinesterase are presented. These methods were adapted for giving optimal demonstration of enzyme activities in the rat hippocampus. The examples are given to illustrate the aptitude and possibilities of this technique in the quantification of enzymes in the complex matrix of the brain.     

Enzymatic measurement of saccharopine with saccharopine dehydrogenase

We have developed an enzymatic method for measuring saccharopine, a key intermediate in lysine metabolism. With the enzyme saccharopine dehydrogenase, saccharopine can be oxidized to lysine and alpha-ketoglutarate with the corresponding conversion of NAD to NADH. The natural equilibrium favors saccharopine formation, but using hydrazine to trap one of the products, alpha-ketoglutarate, shifts the reaction toward quantitative oxidation of saccharopine. A stable endpoint is reached in 15-20 min, and although high concentrations of alpha-ketoglutarate slow the reaction, the end product is fully recovered. Unlike previous assays this technique is specific, convenient, and capable of measuring saccharopine directly in protein-free biological fluids or extracts.     

Immunoassay of circulating enzymes: current status

This report reviews the current status of immunoassays for circulating enzymes. The necessity for measuring the protein concentration of circulating enzymes is evident in the light of the inability of conventional enzyme assays to measure the levels of released enzymes accurately, especially when these enzymes are inactivated, bound to an inhibitor, or present as proenzymes. In some cases, therefore, measurement of enzyme protein concentrations rather than their catalytic activities provide more relevant information for detection of lesions and evaluation of their extent in the organs from which the enzymes are derived. Facts to be considered in the measurement and interpretation of the values obtained by immunoassays are discussed.     

Theme D: The Refinement of Animal Procedures

This study investigated the physiological reactions of companion dogs (Canis familiaris) used in animal-assisted activities and animal-assisted therapy by measuring salivary cortisol concentrations. The dog caregivers (owners) collected saliva samples (a) at 3 control days without therapeutic work, (b) directly before and after each therapeutic session during 3 consecutive months, and (c) again at 3 control days without therapeutic work. The study used an enzyme immunoassay to analyze the samples. Cortisol concentrations were significantly higher during therapy days than on control days. Dogs working during the first half of the day produced higher cortisol concentrations after therapeutic sessions than before, whereas dogs working in the afternoon produced lower cortisol concentrations. Cortisol concentrations were higher in short sessions than in long ones and increased relative to the number of therapeutic sessions done during the sampling period. The results indicate that therapeutic work was physiologically arousing for the dogs in this study. Whether these physiological responses are indicative of potentially negative stress or of positive excitement remains an open question.     

Application of high performance anion exchange-pulsed amperometric detection to measure the activity of key sucrose metabolising enzymes in sugarcane

A novel method using an HPAE-PAD system, which is routinely applied to detect carbohydrates at low levels (ng per sample injection), has been applied to the measurement of key sucrose metabolising enzyme activities in partially purified extracts of sugarcane tissues. Extraction and assay procedures tailored for the HPAE-PAD system enabled the accurate measurement of enzyme activities in more mature internodes than had previously been possible using enzyme coupled assay methodology. A major advantage of the HPAE-PAD method is the capability to monitor a broad range of sugars in each assay and provides an overarching perspective of the mix of competing enzymes that may be operating simultaneously in crude extracts. The technique has been successfully applied to measuring the activity of key sucrose metabolising enzymes in sugarcane stem tissue that is generally low in protein and high in endogenous sugars, primarily sucrose.     

Physiological arousal for companion dogs working with their owners in animal-assisted activities and animal-assisted therapy

This study investigated the physiological reactions of companion dogs (Canis familiaris) used in animal-assisted activities and animal-assisted therapy by measuring salivary cortisol concentrations. The dog caregivers (owners) collected saliva samples (a) at 3 control days without therapeutic work, (b) directly before and after each therapeutic session during 3 consecutive months, and (c) again at 3 control days without therapeutic work. The study used an enzyme immunoassay to analyze the samples. Cortisol concentrations were significantly higher during therapy days than on control days. Dogs working during the first half of the day produced higher cortisol concentrations after therapeutic sessions than before, whereas dogs working in the afternoon produced lower cortisol concentrations. Cortisol concentrations were higher in short sessions than in long ones and increased relative to the number of therapeutic sessions done during the sampling period. The results indicate that therapeutic work was physiologically arousing for the dogs in this study. Whether these physiological responses are indicative of potentially negative stress or of positive excitement remains an open question.     

Evidence that type II 5'-deiodinase is not a selenoprotein.

Brain type II 5'-iodothyronine deiodinase and liver type I 5'-iodothyronine deiodinase activities are decreased in rats fed a Se(2+)-deficient diet suggesting that both enzymes are Se(2+)-dependent proteins. Since serum thyroxine (T4) concentrations are twice normal in the Se(2+)-deficient animals, it is unclear whether the Se2+ deficiency or the increased circulating T4 account for the decrease in the brain enzyme. In order to separate these two possibilities, the effects of Se2+ on 5'-deiodinase in glial cells (type II) and LLC-PK1 cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+ for 3-5 days or in medium containing 75Se2+ for 24 h. Deiodinase isozymes were determined by measuring catalytic activity and by quantification of the BrAc[125I]T4 affinity-labeled substrate binding subunits. Se2+ deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused a concentration-dependent increase in glutathione peroxidase activity in both cell types, as well as in the type I enzyme, but had no effect on the type II enzyme. LLC-PK1 cells contained multiple 75Se(2+)-labeled proteins including the 27-kDa substrate binding subunit of the type I 5'-deiodinase. Glial cells contained seven 75Se(2+)-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type II substrate binding subunit. these data show that, unlike the type I enzyme, the type II enzyme does not contain a selenocysteine or selenomethionine, further emphasizing the differences between these two isozymes.     

Measurement of relative antioxidant activity of compounds: a methodological note

The relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS.+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS.+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS.+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS.+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.     

A HISTOCHEMICAL ENZYME KINETIC SYSTEM APPLIED TO THE TRYPSIN-LIKE AMIDASE AND ESTERASE ACTIVITY IN HUMAN MAST CELLS

A method for the determination of enzyme kinetic constants V_m, K_m, and K_i in a histochemical system has been devised. As a substitute for the reciprocal of the reaction velocity, the times necessary to reach a fixed amount of end product (the initial visible color) in a tissue site at various substrate concentrations are plotted, according to the method of Lineweaver and Burk, against the reciprocal of the substrate concentrations. The technique as applied to trypsin-like esterase and amidase activities in human mast cells indicates that a single enzyme or closely related enzymes in this site are responsible for the hydrolysis of both the amide and ester substrates and that typical trypsin substrates act as competitive inhibitors of their hydrolysis. Parallel biochemical studies were performed to evaluate the effect of certain aspects of the experimental histochemical method on a purified homospecific enzyme. The relative kinetic constants derived by the histochemical method afford a further means of characterizing enzymic activity in a histochemical system.     

A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.     

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.