Linear Mixed Type Research Articles

Dibucaine inhibition of serum cholinesterase.

The dibucaine number (DN) was determined for serum cholinesterase (EC 3.1.1.8, SChE) in plasma samples. The ones with a DN of 79-82 were used, because they had the "usual" SChE variant. The enzyme was assayed colorimetrically by the reaction of 5,5'-dithiobis-[2-nitrobenzoic acid] (DTNB) with the free sulfhydryl groups of thiocholine that were produced by the enzyme reaction with butrylthiocholine (BuTch) or acetylthiocholine (AcTch) substrates, and measured at 412 nm. Dibucaine, a quaternary ammonium compound, inhibited SChE to a minimum within 2 min in a reversible manner. The inhibition was very potent. It had an IC(50) of 5.3 microM with BuTch or 3.8 microM with AcTch. The inhibition was competitive with respect to BuTch with a K(i) of 1.3 microM and a linear-mixed type (competitive/noncompetitive) with respect to AcTch with inhibition constants, K(i) and K(I) of 0.66 and 2.5 microM, respectively. Dibucaine possesses a butoxy side chain that is similar to the butryl group of BuTch and longer by an ethylene group from AcTch. This may account for the difference in inhibition behavior. It may also suggest the existence of an additional binding site, other than the anionic binding site, and of a hydrophobic nature.

Some Nonclassical Boundary Value Problems for Linear Mixed-Type Equations

Well-posedness of nonlocal boundary value problems is studied for some class of mixed-type equations which includes the Chaplygin equation and parabolic equations with varying time direction.

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid.

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).

Propagation of singularities phenomena for equations of tricome type

A survey of the author's recent results concerning propagation of singularities phenomena for linear mixed type partial differential equations is given. Results described include the interior effects of nontrapping over compact sets and seudo-convexity and the exhibition of the boundary effects of reflection, diffraction, and trapped gliding rays.Implications of the boundary results for selecting domains which can support solutions with isolated parabolic boundary singularities are given. Applications of the interior phenomena to solvability results are discussed as well as interpretations in the context of semi-Riemannian geometry and general relativity.

On the inhibition of camel retina acetylcholinesterase activity by cycloheximide in vitro.

The kinetic parameters of inhibition of camel retinal acetylcholinesterase (AChE) activity by cycloheximide (CH) were investigated. For the control system, the Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.076 mmol/L and the Vmax was 0.547 mumol/min per mg protein. In contrast, these parameters were decreased in the CH-treated systems. Dixon and Lineweaver-Burk plots, and their secondary replots, indicated that the inhibition was of the linear mixed type, which seems to be a combination of partial competitive and pure noncompetitive inhibition. The values of K'i(slope) and KI(intercept) were estimated to be 3.50 and 5.68 mmol/L, respectively. Ki was greater than K'i, indicating that CH has a greater binding affinity for the peripheral site than the active site.

Solvability Theorems for Linear Equations of Tricomi Type

For a large class of linear mixed type partial differential equations, theorems on local and semi-global solvability are stated and rely on a prior result of the author about the non-trapping of null-bicharacteristics over compact sets and the theory of Hörmander. A global solvability result modulo smooth error is demonstrated for a slightly reduced class which includes the equations found most commonly in the literature. The key point is the proof of an additional global convexity property for the bicharacteristic flow which results from the construction of a type of convex hull. All of the results are independent of lower order terms as they depend only on the Hamiltonian system associated to the principal symbol. This independence allows for the interpretation of the results in the context of general relativity and semi-Riemannian geometry.

Effect of sevin on kinetic parameters of camel retina acetylcholinesterase.

The present investigation addresses the effect of sevin (insecticide) on kinetic parameters of camel retina membrane-bound acetylcholinesterase (AChE, EC 3.1.1.7). The Michaelis-Menten constant (Ks) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.072 mM in the control system, a value decreased by 38-55% in the sevin treated systems. The Vmax was 0.967 mumole/min/mg protein for the control system while it was decreased by 50-84% in the sevin treated systems. The Lineweaver-Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the linear mixed type, i.e. uncompetitive and noncompetitive. The values of Ki(slope) and KI(intercept) were estimated as 6.194 and 2.811 microM, respectively. The turnover number (Kcat) and specificity constant (Ksp) were 74.65 min-1 and 10.37 x 10(5) (M.min)-1 in the control system respectively, while the values for both parameters were significantly decreased in the sevin treated systems.

Kinetics of inhibition of thermolysin-catalyzed peptide synthesis by alcohols in aqueous organic one-phase system

The kinetic patterns and parameters of 12 alcoholic organic solvents of different classes inhibiting thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe-Phe-OMe) in aqueous organic one-phase reaction system have been determined. All alcohols showed a linear mixed type inhibition. A kinetic model of inhibition is suggested. It was presumed that alcohols interact with substrate in the active site of thermolysin.

In vitro inhibition of human erythrocyte acetylcholinesterase (EC3.1.1.7) by an antineoplastic drug methotrexate.

This work addresses the kinetic analysis of the interaction of methotrexate (MTX) with human erythrocyte membrane-bound acetylcholinesterase (AChE, EC 3.1.1.7). It was found that the MTX effect was independent of time of incubation with AChE before the addition of substrate which proves its reversible action. The IC50 was determined, by three methods, to be 0.73 mM. The Michaelis-Menten constant (Ks) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.13 mM in the control system, a value decreased by 30-61% in the MTX treated systems. The Vmax was 1.27 mumole/min/mg protein for the control system while it was decreased by 44-77% in the MTX treated systems. The Lineweaver-Burk plot Dixon Plot, and their secondary replots indicated that the nature of the inhibition was of the linear mixed type, i.e. uncompetitive and noncompetitive. The values of Ki(slope) and Ki(intercept) were estimated as 1.67 and 0.34 mM, respectively.

Kinetics for Camel (Camelus dromedarius) Retina Acetylcholinesterase Inhibition by Methotrexate In Vitro

This work addresses the kinetic analysis of the interaction of methotrexate (MTX) with camel retina acetylcholinesterase (AChE, EC 3.1.1.7). It was found that the MTX effect was reversible in nature.The IC50 was determined, by two methods, to be 1.362 mM. The Michaelis-Menten constant (Ks) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.123 mM in the control system and the MTX-treated systems showed a 10–35% decrease in this value. The Vmax was 0.789 μmol/min/mg protein for the control system, while it was decreased by 23–76% in the MTX-treated systems. The Lineweaver-Burk plot, Dixon plot and their secondary replots indicated that the inhibition was a linear mixed type; i.e., uncompetitive and noncompetitive. The values of Ki and Ki were estimated as 0.782 and 0.404 mM, respectively. The use of camel retina as a model for the study of human retina may open new avenues for studying various aspects of AChE.

Inhibition of camel lens zeta-crystallin/NADPH:quinone oxidoreductase activity by Cibacron blue.

Camel lens zeta-crystallin/NADH:quinone oxidoreductase activity was inhibited by Cibacron blue 3GA (CB) with 9.10-phenanthrenequinone (PQ) as an electron acceptor and NADPH as an electron donor in a time-independent and concentration dependent manner. The IC50 value of CB was 50 nM. The Lineweaver-Burk plots and the secondary plots indicated that the inhibition was linear mixed type (partial competitive and pure noncompetitive) with respect to NADPH and noncompetitive with respect to PQ. The estimated inhibition constant (Ki) values were 26.0 nM for NADPH and 55.0 nM for PQ respectively, suggesting that CB has high affinity towards the NADPH binding site. The secondary plots of inhibition with respect to NADPH, also indicate a dissociation constant (Ki) value of 68.0 nM for the zeta-crystallin-NADPH-CB complex. This Ki being greater than the Ki value suggests that noncompetitive inhibition is predominant over competitive inhibition at the NADPH binding site.

Kinetics of the inhibition of acetylcholinesterase from desert cobra (Walterinnesia aegyptia) venom by local anesthetics: procaine and tetracaine.

The kinetic parameters of W. aegyptia venom acetylcholinesterase (AChE) inhibition by procaine and tetracaine hydrochloride were investigated in the present study. Procaine and tetracaine reversibly inhibited the AChE activity in a concentration-dependent manner, the IC50 being about 0.28 and 0.04 mM, respectively. The Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.051 mM with Vmax 10.2 mumole/min/mg protein. Both K(m) and Vmax were affected by procaine while only Vmax decreased with tetracaine. A Lineweaver-Burk plot and its secondary replot indicated that the nature of the inhibition is of the linear mixed type for procaine which is considered to be a mixture of competitive and noncompetitive types while the inhibition was noncompetitive for tetracaine. The values of Ki(slope) and K(intercept were estimated as 0.133 mM and 0.451 mM for procaine and 7.2 x 10(-3) mM for tetracaine, respectively, by the secondary replots of the Lineweaver-Burk plot.

The nature of the inhibition of camel retina acetylcholinesterase (EC 3.1.1.7) activity by tetrahydroaminoacridine.

The nature of the inhibition of camel retina acetylcholinesterase (AChE) activity by tetrahydro-aminoacridine (THA, tacrine) has been investigated in the present study. The non-significant change of the percent inhibition of AChE by THA with respect to various lengths of the preincubation period showed the type of the reversible inhibition. THA reversibly inhibited AChE activity in a concentration dependent manner; IC50 was 0.23 microM while the IC100 was 14.22 microM. The K(m) for the hydrolysis of acetylthiocholine iodide was found to be 62.6 microM in the control system; a value increased in the THA treated systems. The Vmax was 0.472 mumole/min/mg protein for the control system, while it decreased in the THA treated systems. Dixon, as well as Lineweaver-Burk, plots and their secondary replots indicated that the nature of the inhibition is of the linear mixed type, which is considered to be a partial competitive and pure non-competitive mixture. The values of Ki(slope) and K'i(intercept) were estimated as 0.068 microM and 0.181 microM, respectively. The K'i was greater than Ki indicating that THA has a greater affinity of binding for the peripheral site than the active site of the camel retina AChE. The use of camel retina as a good experimental animal model may open new avenues for studying acetylcholine and AChE metabolism.

Inhibition of human acetylcholinesterase by cyclophosphamide

The inhibitory effect of cyclophosphamide (CP) on human erythrocyte membrane bound acetylcholinesterase (AChE) was investigated in the present study. It was found that CP inhibits the AChE reversibly with an IC 50 of 511 μM. The Michaelis-Menten constant ( K m) was 132 μM for AChE in the control system; a value increased by 78% in the CP treated system. The V max was 73.8 μmol/h/mg protein for the control system. The Lineweaver-Burk plot and Dixon plot indicated that the nature of this inhibition is of the linear mixed type, i.e., partially competitive and purely noncompetitive. The values of K i and K I were estimated as 378 and 582 μM, respectively. The K Iwas greater than K i indicating that noncompetitive inhibition was predominant over competitive.

Kinetics of the inhibition of acetylcholinesterase from pigeon brain by procaine hydrochloride

The kinetic parameters of the inhibition of pigeon brain acetylchlolinesterase (AChE) by procaine hydrochloride were investigated. Procaine (0·083–1·67 mM) reversibly inhibited AChE activity (15–83 percent) in a concentration dependent manner, the IC50 being about 0·38 mM. The Michaelis-Menten constant (Km) for the hydrolysis of acetylthiocholine iodide was found to be 1·53 × 10−4 M and the Vmax was 1·06 μmol min−1 mg−1 protein. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition is of the linear mixed type which is considered to be a mixture of partial competitive and pure non-competitive. The values of Ki(slope) and Ki (intercepts) were estimated as 0·14 mM and 0·22 mM respectively by the primary Dixon and by the secondary replots of the Lineweaver-Burk plot. The Ki′/Ki ratio shows that procaine has a greater affinity of binding for the peripheral than for the active site.

Investigation of the reversible inhibition of camel ( Camelus dromedarius) acetylcholinesterase by tetracaine

1. The camel erythrocyte membrane bound acetylcholinesterase (AChE) was extracted with the non-ionic detergent Triton X-100 and some of its kinetics parameters were studied. In addition the effect of tetracaine hydrochloride on AChE was also investigated. 2. The Michaelis-Menten constant ( K m ) for the hydrolysis of acetylthiocholine iodide was found to be 7 × 10 −5M and the V max was 21.2 μmol/hr/mg protein. 3. Tetracaine (0.025–0.80 mM) reversibly inhibited the AChE activity (25–82%) in a concentration-dependent manner, the ic 50 being about 0.12 mM. 4. The Lineweaver-Burk plot and its secondary plots indicated that the nature of this inhibition is of the linear mixed type. This mixed type inhibition system is considered to be composed of partial competitive and pure non-competitive in nature. 5. The values of K i(slope) and K ii(intercept) were estimated as 0.127 mM and 0.263 mM, respectively, by a secondary replot of primary double reciprocal plot of Lineweaver-Burk plot and Dixon plot. 6. K ii K i ratio shows that tetracaine has a greater affinity of binding to the active site than to a peripheral site.

Influence of acid-secretion blockers on gastric and hepatic alcohol dehydrogenase in rat.

The effects of Cimetidine, Ranitidine, and Omeprazole on gastric and hepatic alcohol-dehydrogenase (ADH) activity was studied in rat. Two apparent values for Km were found for gastric ADH (220 mmol l-1 and 1043 mmol l-1 respectively) and one for hepatic ADH (0.54 mmol l-1). Cimetidine was shown to exert an uncompetitive inhibition of low Km gastric ADH with a Ki of 0.167 mmol l-1 and a competitive inhibition of high Km gastric ADH with a Ki 2.3 mmol l-1. Ranitidine was found to present non-competitive inhibition only on low Km gastric ADH with a Ki of 12 mmol l-1. Omeprazole affects only low Km gastric ADH with a Ki of 5.6 mmol l-1 and presents a linear-mixed type of inhibition. Hepatic ADH was shown to be competitively inhibited only by Cimetidine with a Ki of 6.0 mmol l-1 whereas no inhibition for either Ranitidine and Omeprazole was observed. These results confirm the inhibitory action of Cimetidine on both gastric and hepatic ADH; Ranitidine and Omeprazole show minor effects on ADHS activity and probably on first-pass metabolism.

Kinetic characterization of branched chain ketoacid dehydrogenase

Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD + and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a ping-pong mechanism and are similar to pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD + and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD +) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD + at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition.

Cholesterol side-chain cleavage and 11β-hydroxylation are inhibited by gossypol in adrenal cortex mitochondria

Cholesterol side-chain cleavage and 11β-hydroxylation were assessed in isolated adrenal cortex mitochondria by formation of pregnenolone and corticosterone, respectively, in the presence and absence of gossypol. Pregnenolone biosynthesis was inhibited when increasing concentrations of gossypol were added. The control value of 4 nmol min −1 mg −1 dropped to 2 nmol min −1 mg −1 with 30 μM of the drug in the incubation medium. A more pronounced inhibitory effect was observed upon 11β-hydroxylation of steroids; I 50 was 11 μM. Seventy-five percent of corticosterone production was impaired when 30 μM of gossypol were present. Bovine serum albumin prevented and reversed the inhibitory action of the drug. Kinetic studies showed a linear mixed type inhibition, suggesting a direct action of the drug upon the enzymatic complex. This study demonstrates a direct inhibitory effecr of gossypol upon the steroidogenic enzymes located in the inner mitochondrial membrane of the adrenal cortex.

Aminopeptidase M from human liver. II. Kinetic analysis of inhibition of the enzyme by bile acids.

The mechanism of inhibition of aminopeptidase M by bile acids was analyzed by application of the specific velocity plot that was introduced by Baici [Eur. J. Biochem. 119, 9-14 (1981)]. Kinetic studies with three bile acids (cholic acid, deoxycholic acid, and chenodeoxycholic acid) and three substrates (Leu-Met, Leu-Gly, and Leu-pNA) showed that the inhibition constants Ki for the bile acids were appreciably different from each other, but that the Ki for each was not affected by the substrates used, being 0.89-1.03 mM for cholic acid, 0.42-0.66 mM for deoxycholic acid, and 0.24-0.31 mM for chenodeoxycholic acid. The values of the kinetic coefficient alpha [(apparent Ks in the presence of inhibitor)/Ks] for cholic acid with Leu-Met and Leu-Gly were 9.0 and 2.5, respectively. These values were very similar to those for chenodeoxycholic acid (7.0 and 2.7) but smaller than those for deoxycholic acid (21 and 11). The values of the other kinetic coefficient beta [(apparent kp in the presence of inhibitor)/kp] were 0 except in the case of the combinations of Leu-Gly with cholic acid (0.33) and Leu-Gly with chenodeoxycholic acid (0.13). On the basis of these kinetic parameters, the inhibitions by bile acids were classified into 4 types: competitive-noncompetitive linear mixed type (1 less than alpha less than infinity, beta = 0), noncompetitive-uncompetitive linear mixed type (0 less than alpha less than 1, beta = 0), pure noncompetitive type (alpha = 1, beta = 0), and hyperbolic mixed type (1 less than alpha less than infinity, 0 less than beta less than 1).(ABSTRACT TRUNCATED AT 250 WORDS)