Assay and kinetics of arginase

A sensitive colorimetric assay for arginase was developed. Urea produced by arginase was hydrolyzed to ammonia by urease, the ammonia was converted to indophenol, and the absorbance was measured at 570 nm. The assay is useful with low concentrations of arginase (0.5 munit or less than 1 ng rat liver arginase) and with a wide range of arginine concentrations (50 microM to 12.5 mM). Michaelis-Menten kinetics and a Km for arginine of 1.7 mM were obtained for Mn2+-activated rat liver arginase; the unactivated enzyme did not display linear behavior on double-reciprocal plots. The kinetic data for unactivated arginase indicated either negative cooperativity or two types of active sites on the arginase tetramer with different affinities for arginine. The new assay is particularly well suited for kinetic studies of activated and unactivated arginase.     

Partial purification and properties of purine nucleoside phosphorylase from rabbit erythrocytes.

1. The partial purification of purine nucleoside phosphorylase from rabbit erythrocytes is described. 2. Analytical and preparative isoelectric focusing gave a pI value for the enzyme of 4.65. 3. Gel-chromatography and sucrose-density-gradient-centrifugation techniques gave estimates of the molecular weight in the range 75000-83000. 4. Lineweaver-Burk plots of kinetic data were non-linear at high inosine concentrations. Extrapolation of the linear part of such plots yielded a Km value for inosine of about 70 micrometer for the rabbit erythrocyte and liver enzymes. 5. A Hill interaction coefficient of 0.75 was obtained, suggesting negative co-operativity with respect to the binding of inosine. 6. Treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) caused partial inactivation, and subsequent Lineweaver-Burk plots with inosine as substrate displayed complete linearity, with an increase in Km value for inosine to 200 micrometer. 7. Starch-gel electrophoresis did not reveal the presence of secondary isoenzymes; all tissue extracts examined gave electrophoretic patterns similar to those obtained with the partially purified enzyme from erythrocytes. 8. Results of hybridization studies with nucleoside phosphorylase from human foetal liver suggest that the rabbit enzyme is also a trimer.     

Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver

Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.     

A slow-tight binding inhibitor of dopamine beta-monooxygenase: a transition state analogue for the product release step

The steady-state kinetic data show that 3-hydroxy-4-phenylthiazole-2(3H)-thione (3H4PTT) is a potent tight-binding inhibitor for dopamine beta-monooxygenase (DbetaM) with a dissociation constant of 0.9 nM. Ackermann-Potter plots of the enzyme dependence of the inhibition revealed that the stoichiometry of the enzyme inhibition by 3H4PTT is 1:1. Pre-steady-state progress curves at varying inhibitor with fixed reductant and enzyme concentrations clearly show the slow binding behavior of the inhibitor. The observed kinetic behavior is consistent with the apparent direct formation of the tightly bound E x I* complex. The k(on) and k(off) for 3H4PTT which were determined under pre-steady-state conditions at variable inhibitor concentrations were found to be (1.85 +/- 0.07) x 10(6) M(-1) s(-1) and (1.9 +/- 0.6) x 10(-3) s(-1), respectively. The dissociation constant calculated from these rates was similar to that determined under steady-state conditions, confirming that 3H4PTT is a kinetically well-behaved inhibitor. The steady-state as well as pre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is competitive with respect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized form of the enzyme. The kinetic behavior and the structural properties of 3H4PTT are consistent with the proposal that the E x 3H4PTT complex may mimic the transition state for the product (protonated) release step of the enzyme. Therefore, 3H4PTT could be used as a convenient probe to examine the properties of the E x P complex of the DbetaM reaction and also as an active site titrant for the oxidized enzyme.     

Kinetic and molecular properties of citraconyl-aldolase. The reversible denaturation and hybridization of the native and modified enzymes

1. The preparation of enzymically active N-citraconyl derivatives of fructose diphosphate aldolase from rabbit muscle is described. Reaction is restricted to amino groups and the derivatives are not very heterogeneous with respect to the number of substituents. 2. Linear double-reciprocal plots of enzyme velocity against substrate concentration are found up to about 15% blocking of amino groups. With more than 15% blocking, there is a marked downward curvature in the double-reciprocal plots at high substrate concentrations. 3. Over the range 0-25% blocking of amino groups the apparent V(max.) for fructose diphosphate falls to 10% that of the native enzyme, and the apparent K(m) rises from 1 to 400mum. 4. Various pieces of evidence suggest that citraconyl-aldolase is slightly distorted in structure compared with the native enzyme. However, the kinetic properties and tetrameric structure of citraconyl-aldolase can be completely recovered after denaturation in 4m-guanidine hydrochloride. 5. After removal of the citraconyl groups in acid conditions the kinetic and molecular properties of native enzyme are restored. 6. Hybrid forms of aldolase can be constructed containing native and citraconylated subunits and the suitability of these derivatives for the study of subunit interactions in the enzyme is discussed. 7. The kinetic properties of hybridized aldolase containing native and citraconylated subunits are not exactly those predicted from the kinetic properties of the two parental forms. This result is interpreted in terms of conformational changes induced in the native and modified subunits when both are present in a hybrid molecule, evidently as a result of interactions in the tetramer.     

Possible involvement of manganese in the catalytic mechanism of bovine liver arginase.

1. Bovine liver arginase followed Michaelis-Menten kinetics in the pH range of 4.5-9.0. The variation of vi with pH implied that a basic group (pKa 8.7) functions at the catalytic site. 2. Treatment of the enzyme with N-ethylmaleimide showed that there are no critical sulfhydryl groups on the enzyme. 3. The less selective reagent, 3-bromopyruvate, caused biphasic inactivation which was unaffected by the presence of ornithine. 4. The data pointed against critical involvement of active site amino acid side chains in the catalytic sequence in arginase. 5. The observed pH-rate profile may reflect ionization of metal-bound water.     

Cooperative effects of CTP on calf liver CTP synthetase.

In all previous kinetics studies of calf liver CTP synthetase, simple Michaelis-Menten hyperbolic plots were obtained. In this study it was shown that calf liver CTP synthetase could generate sigmoidal kinetic plots as a function of the substrate UTP when in the presence of the product of the reaction, CTP. The Hill number was estimated to be 2.8. The enzyme did not generate sigmoidal plots as a function of the other substrates (L-glutamine and ATP) either in the presence or absence of CTP. Thus, CTP apparently induced changes in the liver enzyme which altered the binding of UTP to the enzyme by acting at a site distinct from the UTP binding site (allosteric site). This concept was further strengthened by the fact that 3-deazaUTP, a known competitive inhibitor of the liver enzyme, did not induce sigmoidal kinetic plots. It was also shown that CTP had no effect upon the dimerization of the enzyme, thus ruling out monomer to dimer transitions as a potential mechanism for the observed sigmoidal kinetics.     

Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme.

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 X 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.     

Cerebral-cortex hexokinases. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis

1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP(2-). Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP(2-) was the substrate varied. The K(i) for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and betagamma-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP(2-) was varied. AMP inhibition was competitive when MgATP(2-) was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.     

Production and characterization of monoclonal antibodies to rat liver arginase

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.     

Substrate inhibition of rat liver and kidney arginase with fluoride

Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis-Menten kinetics. Lineweaver-Burk and Eadie-Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase.     

The effect of bulk hydrogen ion concentration upon the apparent kinetic parameters of purified pig liver catechol O-methyltransferase

In order to investigate the pH dependence of catechol O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6), kinetic parameters have been determined for the highly purified enzyme from pig liver over the pH range 6.75-8.20 using the substrates S-adenosylmethionine (AdoMet) and 3,4-dihydroxyphenylacetic acid (DOPAC). The Km for AdoMet was found to be invariant with pH while the Km for DOPAC decreased sharply with increasing pH. The group responsible for the latter has a pK of approx. 7.1. The logarithmic (Dixon) plot of Km against pH for both substrates and that of Vmax/Km against pH for DOPAC mirror the kinetic behaviour revealed by linear plots. However, for other parameters, linear graphs indicate peaks too narrow to be explicable by a simple kinetic mechanism, whereas logarithmic plots of these parameters produce graphs apparently not reflecting this behaviour. We conclude that these results are not the products of random error or artefactual data analysis but are too complex to be explicable by a simple model of kinetic behaviour. Possible explanations (adherence of catechol O-methyltransferase to a higher-order mechanism or a dual mode of substrate binding) are advanced.     

Glutamate:4,5-dioxovaleric acid transaminase from Euglena gracilis. Kinetic studies

The kinetic properties of the enzyme L-glutamate:4,5-dioxovaleric acid aminotransferase (Glu:DOVA transaminase) from Euglena gracilis have been studied. 5-Aminolevulinic acid formation was linear with time for at least 45 min at 37 degrees C and L-glutamate was the most effective amino-group donor. Lineweaver-Burk double-reciprocal plots suggested a ping-pong reaction mechanism, with Km values for L-glutamate and DOVA of 1.92 mM and 0.48 mM respectively. Competitive parabolic substrate inhibition by DOVA at concentrations greater than 3.5-4.5 mM was observed. Glyoxylate (4-10 mM) was found to be a competitive inhibitor with respect to DOVA, whereas at low concentrations (0-4 mM) noncompetitive plots were obtained. An analysis of the possible enzyme forms involved, was carried out. In more crude preparations most of the enzyme is found to be in the form of an enzyme-glutamate complex.     

A rapid equilibrium random sequential bi-bi mechanism for human placental glutathione S-transferase

Double-reciprocal plots of initial-rate data for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH by human placental GSH S-transferase pi were linear for both substrates. Computer modelling of the initial-rate data using nonlinear least-squares regression analysis favoured a rapid equilibrium random sequential bi-bi mechanism, over a steady-state random sequential mechanism or a steady-state or rapid equilibrium ordered mechanism. KGSH was calculated as 0.125 +/- 0.006 mM, KCDNB was 0.87 +/- 0.07 mM and alpha was 2.1 +/- 0.3 for the rapid equilibrium random model. The product, S-(2,4-dinitrophenyl)glutathione, was a competitive inhibitor with respect to GSH, and a mixed-type inhibitor toward CDNB (KP = 18 +/- 3 microM). The observed pattern of inhibition is consistent with a rapid equilibrium random mechanism, with a dead-end enzyme.CDNB.product complex, but inconsistent with the inhibition patterns of other bireactant mechanisms. Since rat liver GSH S-transferase 3-3 acts via a steady-state random sequential mechanism [1], while human placental GSH S-transferase and perhaps also rat liver GSH S-transferase 1-1 [2] exhibit rapid equilibrium random mechanisms, we conclude that the kinetic mechanism of the GSH S-transferases is isoenzyme-dependent.     

Kinetics of rat liver GM2 ganglioside-beta-D-N-acetylhexosaminidase

The kinetics of beta-D-N-acetylhexosaminidase against GM2 ganglioside were examined. We used a crude preparation of rat liver as the enzyme source because purification of beta-D-N-acetylhexosaminidase results in a decrease in specific activity against GM2 ganglioside. Kinetic plots were not linear but showed a break. At substrate concentrations less than 50 microM the Vmax was 6 pmol GM2 hydrolyzed per hour per micromole 4-MU-GlcNAc hydrolyzed per hour (pmol GM2/mumol 4-MU-GlcNAc) and the Km was 5 microM.At substrate concentrations greater than 50 microM, the Vmax was 7 pmol GM2/mumol 4-MU-GlcNAc and the Km was 14 microM. The critical micelle concentration of GM2 ganglioside was 20-25 microM as determined by spectral shifts of the dye pinacyanol chloride in association with GM2, and 10-15 microM from electrical conductivity measurements which also showed the end of the monomer-micelle transition to occur at 40-50 microM GM2. The increasing excess of micellar substrate at greater than 50 microM GM2 explains the discontinuity in the kinetic plots. Sodium taurocholate had a critical micelle concentration of 9-11 mM using pinacyanol chloride and 2.5-3 mM using electrical conductivity. When included in the assay mixture at a concentration of 10 mM, sodium taurocholate produced a linear kinetic plot. This is probably due to the formation of mixed micelles of detergent and GM2 ganglioside. The Vmax was 200 pmol GM2/MUmol 4-MU-GlcNAc and the Km was 93 microM. The data suggest that ganglioside hydrolysis occurs more readily when the substrate is incorporated into a membrane-like environment.     

A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes.

1. Approx. 10% of the rat liver cellular cyclic AMP phosphodiesterase activity was associated with a plasma-membrane fraction. 2. Lineweaver-Burk plots of this activity were clearly non-linear, yielding extrapolated Km values of 0.7 and 60.6 microns. 3. Treatment of these membranes with high-ionic-strength NaCl solutions apparently released 80% of this activity assayed at 0.4 micron-cyclic AMP, and 15% of the activity assayed at 1 mM-cyclic AMP. 4. The high-salt-solubilized enzyme gave a non-linear Lineweaver-Burk plot. 5. The cyclic AMP phosphodiesterase activity of the washed high-salt-treated membranes exhibited a linear Lineweaver-Burk plot, yielding a Km of 60 microns. 6. The high-salt-solubilized enzyme exhibited a single peak of activity upon polyacrylamide-gel electrophoresis, a single peak upon sucrose-density-gradient centrifugation (3.9 S) and decayed as a single exponential upon heat-treatment (half-life 1 min at 55 degrees C). 7. The activity of washed high-salt-treated membranes decayed as a single exponential upon heat-treatment (half-life 42 min at 55 degrees C), and was solubilized in the detergent Triton X-100. 8. Cytosol-derived cyclic AMP phosphodiesterase activity could bind to washed high-salt-treated plasma membranes, but was totally eluted by washing with 1 mM-KHCO3, unlike the high-salt-solubilized enzyme, which required high salt concentrations to elute it. 9. We suggest that the cyclic AMP phosphodiesterase activity of rat liver plasma membranes can be resolved into two components: a single peripheral protein exhibiting apparent negative co-operativity, that is distinct from cytosol forms, and an intrinsic protein exhibiting normal Michaelis kinetics.     

Rabbit erythrocyte purine nucleoside phosphorylase. Initial-velocity studies.

1. Concave-downward double-reciprocal plots were obtained for rabbit erythrocyte purine nucleoside phosphorylase when the concentration of Pi was varied over a wide range at a fixed saturating concentration of either inosine or deoxyinosine. Similar behaviour was also displayed by the calf spleen enzyme. 2. The degree of curvature of double-reciprocal plots was greatly modified by the presence of SO42-, introduced into the assay mixture with the linking enzyme xanthine oxidase; competitive inhibition by SO42- was observed over a narrow range of high Pi concentrations. 3. Partial inactivation with 5,5'-dithiobis-(2-nitrobenzoic acid) resulted in a marked alteration in the kinetic properties of the enzyme when Pi was the variable substrate. 4. Initial-velocity data are expressed in the form of Hill plots, and the significance of such plots is discussed.     

The regulation of N-acetylglutamate synthetase in rat liver by protein intake

Urea cycle enzymes are subjected to regulation by dietary proteins. We have shown that this is also the case for N-acetylglutamate synthetase (EC 2.3.1.1.) (NAGS). Four different groups (n = 7) of male Wistar rats received either a low protein (8.7%) or a high (32% and 51%) protein diet and a control diet of 17% protein. The NAGS-activity in the liver, assayed after 15 days of feeding the different diets, increased from 25 +/- 7 (controls, 17% protein) to 31 +/- 5 (32% protein) and to 52 +/- 17 (51% protein) nmoles.min-1.g-1 wet weight. It decreased in the group with low protein diet (8.7%) to 5 +/- 3. The ratio of the arginine stimulated to the unstimulated enzyme activity remained constant over the range of protein intake. Similar changes were observed for carbamylphosphate synthetase, ornithine carbamyltransferase and arginase. As it is known for these enzymes adaptive mechanisms in relation to variations in dietary protein consumption also could be demonstrated for the enzyme NAGS.     

Enzyme electrode-based kinetic assays of enzyme activities.

Potentiometric enzyme electrodes for urea, AMP, and adenosine have been used for the kinetic assays of arginase (EC 3.5.3.1.), 3′:5′-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17), and 5′-nucleotidase (EC 3.1.3.5), respectively. The initial rate of potential change, after addition of the enzyme to the substrate solution, is direetly proportional to the enzyme activity present. Arginase assays were found to be reproducible to a relative precision of 13% or better and applicable to the direct measurement of arginase activity in beef liver homogenates.     

Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines.

1. Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.