An alternative 7-ethoxyresorufin o-deethylase activity assay: A continuous visible spectrophotometric method for measurement of cytochrome P-450 monooxygenase activity

A procedure to directly measure the cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase activity with a visible spectrophotometer is described and compared to the standard fluorometric method. The two assays yielded identical results with both β-naphthoflavone-treated mammalian (rat) and fish (scup, Stenotomus chrysops) liver microsomes. The assay takes advantage of a clean distinction in visible absorption spectra obtained for highly purified 7-ethoxyresorufin (substrate) and resorufin (enzymatic product). The purification and characterization of resorufin, the enzymatic product, are detailed, and its extinction coefficient (ε₅₇₂ = 73 mm^?1 cm^?1) provides for an accurate quantitation of enzyme activity. The large visible extinction coefficient of the product chromophore provides a high sensitivity for low-activity samples. The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays. The utility of the visible assay is further demonstrated by the simple determinations of the coupling ratio for 7-ethoxyresorufin oxidation in scup liver microsomes and the K_I for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction.     

Enhanced chemiluminescence in the measurement of proteins and haptens: Evaluation of choriogonadotropin (hCG) and free thyroxin

We evaluated the Amerlite system (Amersham, Bucks, UK) for hCG and FT4. The within-run imprecision (CV%) for hCG was 4.05 at 19.6 U/I (n = 10), 6.28 at 43.45 U/I (n = 10) and 4.62 at 298.57 U/I (n = 10). The between-run imprecision (five replicates for ten days) was 4.8%, 15% and 11%, respectively. The system was linear up to 200 U/I. A good correlation between Amerlite hCG and an IRMA assay (Becton Dickinson, r = 0.91), Delfia (Pharmacia, r = 0.91) and an automated ELISA assay on ES 600 (Boehringer, r = 0.92) was observed on 70 samples. Within-run imprecision for FT4 was 3.8% at 0.7 ng/dl (n = 10), 3.3% at 1 ng/dl (n = 10) and 4.32% at 5.15 ng/dl (n = 10), and between-run was 5.95%, 4.4% and 8.2%, respectively. The comparison with a commercial direct RIA (Becton Dickinson) showed good correlation (r = 0.90, n = 100 samples). The diagnostic value of the association of thyrotropin and FT4, in comparison with the traditional thyroid tests (T3, T4, thyrotropin, FT4, FT3) has been assessed in various thyroid diseases.     

Immunological comparison of the starch branching enzymes from potato tubers and maize kernels

Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M_r = 79,000) shares a high degree of similarity with maize branching enzyme I (M_r = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.     

Enzymatic synthesis of [U-14C] ribose-labeled inosine.

A very potent anticholinesterase compound, 7-(diethoxyphosphinyloxy)-N-methylquinolinium fluorosulfate, has been used to determine the normality of acetylcholinesterase solutions. The inhibitor reacts rapidly and completely with acetylcholinesterase. The bimolecular rate constant is 2.5 × 10⁸m^?1 min^?1 and the equilibrium constant is about 10⁶. The reaction produces an inactive diethylphosphoryl enzyme in which the active serine is phosphorylated. The reaction produces the highly fluorescent 1-methyl-7-hydroxyquinolinium dipolar ion as a leaving group. The inhibited enzyme is quite stable and hydrolyzes to produce active enzyme only at the rate of 0.04%/min. The inhibitor was used in two ways for measuring the normality of acetylcholinesterase solutions: (1) The very fast reaction of the inhibitor with cholinesterase makes it convenient to determine the normality of enzyme solutions by measuring the decrease in enzyme activity caused by the addition of an accurately known quantity of the inhibitor. (2) The highly fluorescent nature of the leaving group makes it possible to measure the low concentration that is produced by the reaction of excess inhibitor with the enzyme. The two methods yielded activities per site of 6.9 × 10⁵ min^?1 and 7.3 × 10⁵ min^?1 using enzyme normalities of 1–2 × 10^?8m and 1–5 × 10^?m, respectively, using a commercial 11 S enzyme preparation from electric eel and acetylthiocholine as the enzyme substrate.     

Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone.

A method for enzyme immunoassay of thyroid-stimulating hormone (TSH) is described, TSH was conjugated with horseradish peroxidase according to periodate oxidation method. Separation of the bound and free was obtained by double-antibody solid-phase technique using Sepharose 4B-anti-rabbit immunogiobulin G (IgG)-geat IgG. The fluorescence reaction using tyramine and hydrogen peroxide as substrates was used for the determination of enzyme activity in order to increase the sensitivity of enzyme immunoassay. The standard curve for serum TSH was satisfactory to recognize TSH concentrations as 0.06 μU/tube. TSH values obtained by this method correlated well with those obtained by radioimmunoassay (r, 0.96). The coefficients of variation were 1.8 to 5.3% (within assay) and 5.1 to 10.5% (between assay). The method is about equal to radioimmunoassay with respect to sensitivity. Since it requires minimal equipment and is less expensive than radioimmunoassay, it is possible to perform routine assays even in laboratories with limited facilities.     

Sulphydryl groups of (Na+ +K+)-ATPase from rectal glands of Squalus acanthias: Titrations and classification

1. (Na⁺ +K⁺)-ATPase from rectal gland of Squlus acanthias contains 34 SH groups per mol (M_r 265000). 15 are located on the α subunit (M_r 106 000) and two on the β subunit (M_r 40 000). The β subunit also contains one disulphide bridge. 2. The reaction of (Na⁺ +K⁺)-ATPase with N-ethylmaleimide shows the existence of at least three classes of SH groups. Class I contains two SH groups on each α subunit and one on each β subunit. Reaction of these groups with N-methylmaleimide in the presence of 40% glycerol or sucrose does not alter the enzyme activity. Class II contains four SH groups on each α subunit, and the reaction of these groups with 0.1 mM N-ethylmaleimide in the presence of 150 mM K⁺ leads to an enzyme species with about 16% activity. The remaining enzyme activity can be completely abolished by reaction with 5–10 nM N-ethylmaleimide, indicating a third class of SH groups (Class III). This pattern of inactivation is different from that of the kidney enzyme, where only one class of SH groups essential to activity is observed. 3. It is also shown that N-ethylmaleimide and DTNB inactivate by reacting with the same Class II SH groups. 4. Spin-labelling of the (Na⁺ +K⁺)-ATPase with a maleimide derivative shows that Class II groups are mostly buried in the membrane, whereas Class I groups are more exposed. It is also shown that spin label bound to the Class I groups can monitor the difference between the Na⁺- and K⁺-forms of the enzyme.     

HPLC method for the determination of phytochelatin synthase activity specific for soft metal ion chelators

Phytochelatins (PCs) are nonprotein peptides with the general structure (γ-Glu-Cys)_n-Gly (PC_n), where n is greater than or equal to 2. They are synthesized through a reaction catalyzed by phytochelatin synthase (PCS) in the presence of metal cations and using the tripeptide glutathione (γ-Glu-Cys-Gly) and/or previously synthesized PC_n as the substrate. Here, a highly sensitive assay for PCS activity was devised, in which the dequenching of Cu(I)-bathocuproinedisulfonate complexes was used in the detection system of a reversed-phase high-performance liquid chromatograph. Using recombinant PCS from the higher plant Arabidopsis thaliana (rAtPCS1), this assay system was capable of determining PCS activity based on an amount of the enzyme preparation that was 100-fold less than that required for the 5,5′-dithiobis(2-nitrobenzoic acid) assay method. Although adsorption of the enzyme onto the reaction vessel hindered accurate activity determination, the inclusion of bovine serum albumin successfully resolved this issue. This method is a powerful tool for investigating PCS enzyme mechanisms with respect to the roles of metal ions.     

Exploration of the binding sites of acetylcholinesterase with protein-modifying reagents

The effects of eight protein-modifying reagents upon bovine erythrocyte acetylcholinesterase have been studied with three substrates: acetylthiocholine, p-nitrophenyl acetate, and indophenyl acetate. It was shown that a variety of interferences can occur unless the excess modifier and its product are removed chromatographically. After such removal, five of the agents were shown to have modified the enzyme; each acted in one of four ways. Class I was activation of indophenyl acetate hydrolysis with inhibition of acetylthiocholine hydrolysis. Class II was nonselective inhibition of all substrates. Class III was relatively selective inhibition of acetylthiocholine hydrolysis. Class IV was selective activation of indophenyl acetate hydrolysis.     

Plasma total antioxidant capacity is associated with dietary intake and plasma level of antioxidants in postmenopausal women

Increased plasma total antioxidant capacity (TAC) has been associated with a high consumption of fruits and vegetables. However, limited information is available on whether plasma TAC reflects the dietary intake of antioxidants and the levels of individual antioxidants in plasma. By using three different assays, the study aimed to determine if plasma TAC can effectively predict dietary intake of antioxidants and plasma antioxidant status. Forty overweight and apparently healthy postmenopausal women were recruited. Seven-day food records and 12-h fasting blood samples were collected for dietary and plasma antioxidant assessments. Plasma TAC was determined by vitamin C equivalent antioxidant capacity (VCEAC), ferric-reducing ability of plasma (FRAP) and oxygen radical absorbance capacity (ORAC) assays. TAC values determined by VCEAC were highly correlated with FRAP (r=0.79, P<.01) and moderately correlated with ORAC (r=0.34, P<.05). Pearson correlation analyses showed that plasma TAC values by VCEAC and ORAC had positive correlation with plasma uric acid (r=0.56 for VCEAC; r=0.49 for ORAC) and total phenolics (r=0.63 for VCEAC; r=0.36 for ORAC). However, TAC measured by FRAP was correlated only with uric acid (r=0.69). After multivariate adjustment, plasma TAC determined by VCEAC was positively associated with dietary intakes of γ-tocopherol (P<.001), β-carotene (P<.05), anthocyanidins (P<.05), flavones (P<.05), proanthocyanidins (P<.01) and TAC (P<.05), as well as with plasma total phenolics (P<.05), α-tocopherol (P<.001), β-cryptoxanthin (P<.05) and uric acid (P<.05). The findings indicate that plasma TAC measured by VCEAC reflects both dietary and plasma antioxidants and represents more closely the plasma antioxidant levels than ORAC and FRAP.     

Triosephosphate isomerases and aldolases from light- and dark-grown Euglena gracilis

Euglena gracilis synthesizes two distinct types of triosephosphate isomerase which can be resolved by isoelectric focusing. The more acidic Type A isomerase (pI = 4.4) predominates when cells are grown photoautotrophically and is localized in the chloroplasts. The Type B isoenzyme exhibits a more basic isoelectric pH (pI = 4.8), predominates under heterotrophic growth conditions and is of cytoplasmic origin. The two isoenzymes exhibit similar molecular weights (56,000–60,000) and catalytic properties but can be distinguished by their pH activity profiles. The situation parallels that of fructose diphosphate aldolase where a chloroplastic Class I enzyme (pI = 4.6, M_r 120,000) found in autotrophically grown cells can be resolved from the cytoplasmic Class II (pI = 5.7, M_r 88,000) enzyme which predominates under heterotrophic conditions. Inhibition of chloroplastic 70S ribosomal synthesis by chloramphenicol blocks the formation of the Type A triosephosphate isomerase and the Class I aldolase.     

Final blood lactate concentration after incremental test and aerobic performance

The goal of this study was to test the hypothesis that, in groups of highly trained endurance athletes (first and junior national teams), the final blood lactate concentration at maximum aerobic performance decreased as their training status increased. This study was performed with 20 physically active volunteers and 45 highly trained middle- and long-distance endurance athletes (speed skaters, triathletes, and cross-country skiers). Significant negative correlations (r = ?0.59 to ?0.87) between the final blood lactate concentration after incremental tests until exhaustion and aerobic performance (anaerobic threshold (AT)) were found only for the groups of highly trained endurance athletes, but not for the group of physically active subjects. It was shown for highly trained speed skaters that the final lactate concentration in their blood decreased and the oxygen consumption at AT increased with an increase in the volume of type I muscle fibers in the working muscle (r = ?0.84 and r = 0.7, respectively).     

The catalytic mechanism of Pseudomonas cytochrome c peroxidase

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with H₂O₂ was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.     

Pitfalls in the study of steady state kinetics of enzymes: spurious inhibition patterns due to stray light errors

When reaction velocity measurements of enzyme reactions are carried out with single beam, single monochromator spectrophotometers, stray light in the spectrophotometer can produce systematic errors in the apparent velocities when highly absorbing solutions (optical density >2.0) are used. These errors can give rise to spurious “inhibition” patterns of the steady state kinetics. Because of a suspected error of this kind, this laboratory has recently reinvestigated the kinetics of glucose 6-phosphate dehydrogenase from Escherichia coli and found that the reported noncompetitive inhibition of the enzyme by DPNH is explained more readily by an unnoticed effect of stray light on the apparent reaction velocity than by a true enzyme inhibition. Methods for estimating and correcting such errors in spectrophotometers are presented in detail.     

Isolation of 3-methylcrotonyl-coenzyme A carboxylase from bovine kidney

3-Methylcrotonyl-CoA carboxylase (MCase), an enzyme of the leucine oxidation pathway, was highly purified from bovine kidney. The native enzyme has an approximate molecular weight of 835,000 as measured from exclusion limits by polyacrylamide gel electrophoresis at pH 7.3. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated two subunits, identified as a biotin-free subunit (A subunit; M_r = 61,000) and a biotin-containing subunit (B subunit; M_r = 73,500). The biotin content of the enzyme was 1 mol/ 157,000 g protein, consistent with an AB protomeric structure for the enzyme. The isoelectric point of the enzyme was found to be 5.4. Maximal MCase activity was found at pH 8 and 38 °C in the presence of Mg²⁺ and an activating monovalent cation such as K⁺. Kinetic constants (K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 75 μm; ATP, 82 μm; HCO₃^?, 1.8 mm. Certain acyl-CoA derivatives, including crotonyl-CoA, (2Z)-3-ethylcrotonyl-CoA, and acetoacetyl-CoA, were also substrates for the enzyme. Some data on inhibition of the enzyme by acyl-CoA derivatives, and sulfhydryl- and arginyl-reagents, are presented.     

Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata

A radioimmunoassay technique has been developed for the measurement of digoxigenin glycosides in crude extracts from both fresh and dried leaf material of Digitalis lanata, The antibody, obtained by immunizing rabbits against a conjugate of digoxin with human serum albumin, had a high affinity (K_a = 0.8 × 10¹⁰ l/mol) for digoxin and permitted detection of as little as 60 fmol digoxin (45 pg) per 0.1 ml of sample. The antiserum was highly specific for digoxigenin and its glycosides, with only diginatin showing a substantial cross reactivity (3?0%). The use of [³H]-labelled and [¹²⁵I]-labelled digoxin as tracer and of dextran-coated charcoal or ammonium sulfate for separation did not change the specificity of the assay nor the properties of the standard curve. This method has been found to correlate with the usual fluorimetric determination of digoxin, but is more sensitive by a factor of 10⁴. A correlation analysis of 8 and 30 different D. lanata plants (leaf discs and drugs analysed with both methods) gave correlation coefficients of r = 0.989 and r = 0.907 respectively. The analysis of a single leaf disc, 3 mm in diameter (obtained from a fresh leaf), gave an exact measure of the digoxin content found in the dried leaf drug (r = 0.973). With a semi-automated technique, about 2000 quantitative analyses per week can be performed by one person, thus providing the potential to screen plants for use in breeding or tissue culture work. The distribution of digoxigenin equivalents in single seeds, seedlings and plants of different ages has also been investigated.     

Organization of the Rudimentary Wing Locus in DROSOPHILA MELANOGASTER. I. the Isolation and Partial Characterization of Mutants Induced with Ethyl Methanesulfonate, Icr-170 and X Rays

New rudimentary (r) mutants have been isolated following mutagenesis with ethyl methanesulfonate (r^LE), ICR–170 (r^LI) and X rays (r^LX). From wing phenotype measurements on homoallelic females, it has been shown that the r^LE mutant series includes several leaky alleles, as well as alleles that produce moderate and strong r phenotypes. All of the tested r^LI alleles yielded strong r phenotypes in homoallelic females, whereas the r^LX series was found to include both moderate and strong alleles. Based on allele complementation for the wing phenotype, it was found that all three mutant series include both complementing and noncomplementing alleles, but the relative frequencies of these two types of alleles differ considerably among the three series. Complementing alleles comprise most of the r^LE mutant series (19 of 25) and almost one-half of the r^LX series (five of 12), while only one of 16 r^LI mutants is a complementing allele. Data from enzyme assays of mutants mostly support the direct correlation of genetic complementation units with the activities of the first three enzymes in the de novo pyrimidine biosynthetic pathway. All of these findings are discussed in light of evidence that these three enzymes are contained within a trienzyme complex in animals. We conclude that the available genetic evidence supports the contention that the trienzyme complex is encoded by a single mRNA.     

Agreement of measured and calculated muscle activity during highly dynamic movements modelled with a spherical knee joint

The inclusion of muscle forces into the analysis of joint contact forces has improved their accuracy. But it has not been validated if such force and activity calculations are valid during highly dynamic multidirectional movements. The purpose of this study was to validate calculated muscle activation of a lower extremity model with a spherical knee joint for running, sprinting and 90°-cutting. Kinematics, kinetics and lower limb muscle activation of ten participants were investigated in a 3D motion capture setup including EMG. A lower extremity rigid body model was used to calculate the activation of these muscles with an inverse dynamics approach and a cubic cost function. Correlation coefficients were calculated to compare measured and calculated activation. The results showed good correlation of the modelled and calculated data with a few exceptions. The highest average correlations were found during walking (r = 0.81) and the lowest during cutting (r = 0.57). Tibialis anterior had the lowest average correlation (r = 0.33) over all movements while gastrocnemius medius had the highest correlation (r = 0.9). The implementation of a spherical knee joint increased the agreement between measured and modelled activation compared to studies using a hinge joint knee. Although some stabilizing muscles showed low correlations during dynamic movements, the investigated model calculates muscle activity sufficiently.     

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

An efficient method for the synthesis and purification of trans-[14C]geranylgeranyl pyrophosphate.

A new and highly sensitive enzyme immunoassay of cortisol was established using horseradish peroxidase as the label. Separation of free and bound cortisol was effected by insolubilized anti-cortisol antibody which was prepared by coupling the purified immunoglobulin G of antiserum with Sepharose 4B. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. Comparison of assay results obtained by radioimmunoassay and this enzyme immunoassay showed excellent agreement of results in all cases (r = 0.913). The detection limit of cortisol was about 10 pg per assay tube. This enzyme immunoassay is applicable to the routine determination of plasma cortisol.