Inci Özer

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.

Resolution of multiple forms of bovine liver arginase by chromatofocusing

1. Bovine liver arginase could be resolved into three distinct peaks by chromatofocusing in the pH range 7-4. 2. In other experimental systems the enzyme appeared to consist of a single active component. 3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a single band which could be assigned to arginase, with no indication of inherent or protease-induced multiplicity. 4. Lineweaver-Burk plots for arginine were linear over a wide concentration range, as were Dixon plots for reversible inhibitors. 5. Covalent inhibition by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide gave semilogarithmic plots of residual activity vs time which were strictly linear. 6. It was concluded that the enzyme was homogeneous with respect to subunit size and kinetic behaviour, but heterogeneous with respect to molecular charge. 7. The charge heterogeneity may have kinetic and regulatory implications, as previously suggested for mouse liver arginase [Z. Spolarics and J. S. Bond (1988) Archs Biochem. Biophys. 260, 469-479].

Inhibition of electric eel acetylcholinesterase by triarylmethane dyes.

The effects of three cationic triarylmethane dyes--pararosaniline (PR), malachite green (MG), methyl green (MetG)--on electric eel AChE (eAChE) activity were tested at 25 degrees C, in 100 mM MOPS buffer (pH 8) containing 0.125 mM 5-5-dithio-bis(2-nitrobenzoic acid), 20-120 microM acetylthiocholine and 0-20 microM dye. All three dyes caused reversible, linear- or hyperbolic-mixed inhibition of esteratic activity. The respective inhibitory parameters for PR, MG and MetG were K(i)=8.4+/-0.67, 1.9+/-0.51 and 0.27+/-0.017 microM; alpha (competitive coefficient)=5.8+/-2.0, 4.8+/-1.8 and 2.7+/-0.32; beta (noncompetitive coefficient)=0, 0 and 0.20+/-0.011. The data were consistent with ligand binding at the peripheral site and a remote effect on substrate binding and turnover.

Resolution and kinetic characterization of glutathione S-transferases from human jejunal mucosa

Cytosolic glutathione S-transferases were purified from human jejunal mucosa by affinity chromatography on S-hexylglutathione-Sepharose 4B. Chromatofocusing in the pH range 7-4 yielded peaks with apparent pIs of 7.2 (peak 1), 5.2 (peak 2), and 4.4 (peak 3). Each enzymatic fraction was shown to have a homodimeric structure, with subunit mass of 24.9 +/- 0.5 (P1), 27.9 +/- 0.9 (P2), and 23.4 +/- 0.8 (P3) kDa, as determined by SDS-PAGE. The substrate specificity of each peak was tested using discriminating substrates for basic, near-neutral, and acidic GSTs. With cumene hydroperoxide, the diagnostic substrate for the alpha (basic) class of GSTs, P1 showed 8- to 36-fold higher activity than P2 and P3. Ethacrynic acid, the selective substrate for the acidic enzyme (pi), gave highest activity with P3. The inhibitory potentials of sulfobromophthalein, cibacron blue, tributyltin acetate, triphenyltin chloride, and bromphenol blue were also tested. A qualitative resemblance between P1 and alpha, and P3 and pi GSTs was noted. The substrate specificity and inhibiton parameters of P2 corresponded most closely to those of mu-GST. The relative abundances of P1, P2, and P3 (based on CDNB-conjugating activity) were 35, 5, and 60%, respectively.

Inhibition of choline oxidase by quinoid dyes

Choline oxidase catalyzes the oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. This study reports on the inhibitory effects of triarylmethanes (cationic malachite green; neutral leukomalachite green), phenoxazines (cationic, meldola blue and nile blue; neutral nile red) and a structurally-related phenothiazine (methylene blue) on choline oxidase, assayed at 25°C in 50 mM MOPS buffer, pH 7, using choline as substrate. Methylene B acted as a competitive inhibitor with Ki = 74 ± 7.2 μM, pointing to the choline–binding site of the enzyme as a target site. Nile B caused noncompetitive inhibition of enzyme activity with Ki = 20 ± 4.5 μM. In contrast to methylene B and nile B, malachite G and meldola B caused complex, nonlinear inhibition of choline oxidase, with estimated Ki values in the micromolar range. The difference in kinetic pattern was ascribed to the differential ability of the dyes to interact (and interfere) with the flavin cofactor, generating different perturbations in the steady-state balance of the catalytic process.

The Trypsin-Inhibitory Efficiency of Human α2-Macroglobulin in the Presence of α1-Proteinase Inhibitor: Evidence for the Formation of an α2-Macroglobulin-α1-Proteinase Inhibitor Complex

AbstractThe inhibition of bovine pancreatic trypsin was studied at pH 7, 25°C, using mixtures of purified human α2-macroglobulin (α2M) and α1-proteinase inhibitor (α1PI). The partitioning of the enzyme between the two inhibitors was determined by comparing control esterase activity, assayed with N-benzoyl-L-arginine ethyl ester as substrate, with that remaining after incubation with inhibitory mixtures. (At [I]0 > [E]0, remaining esteratic activity reflects the concentration of α2M-associated enzyme (α2M-E*) and the concentration of α1PI-associated, inactive enzyme (α1PI-E*) is given by the difference, [E]0— [α2M-E*].) The pattern of product distribution was found to be incompatible with an inhibitory model involving parallel, second-order reactions of E with α2M and α1PI. The data pointed to complex formation between the two inhibitors, limiting the level of α2M readily available for reaction with E. Analysis based on the binding equilibrium, α2M (dimeric unit) + α1PI⇆α2M —α1PI, yielded Kd= 2.1 ± 0.3 μM....

Differential reactivity of active sites in human plasma cholinesterase toward 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Abstract Human plasma cholinesterase was found to be inhibited by 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide in a biphasic manner. The faster phase of the inhibition led to loss of approximately 50% of the activity (measured at pH 7.0, 30 °C, using 2.5 m m butyrylthiocholine) and was irreversible. Inhibition in the slower phase was reversible by 0.25 m hydroxylamine. The protective effect of 1 m m propranolol indicated that the target residue in both phases was localized at the active site. Lineweaver-Burk plots for butyrylthiocholine were obtained at different times during the course of inactivation. It was found that for both native and partially inactivated enzymes the plots could be analyzed in terms of two activities showing hyperbolic saturation with the substrate, with K m values of 0.055 ± 0.015 and 2.0 ± 0.2 m m . The carbodiimide affected the maximal velocities of the component activities, leaving the K m s unchanged. The low- K m component was lost in the first phase of the inactivation. The loss of the high- K m component paralleled the second phase. It was concluded that the active sites in the tetrameric enzyme form two classes, differing in their affinity for butyrylthiocholine and their susceptibility to inhibition by the active site-directed carbodiimide.

Mixed-type inhibition of bovine lung angiotensin converting enzyme by lisinopril and its dansyl derivative

The steady-state inhibition of bovine lung angiotensin converting enzyme (ACE; EC 3.4.15.1) by the slow-binding inhibitor lisinopril and its dansyl derivative conformed to a linear mixed inhibition model with inhibitor binding to ES as well as to E. Studied at pH8, 35 degrees, and using N-(3-[2-furyl]-acryloyl)phe-gly-gly as substrate, the approach to steady-state activity at different substrate concentrations pointed to slow isomerizations in both EI and EIS. While an inhibitory scheme involving a single I-binding site adequately accounts for the data presented, information relating to the primary structure of ACE brings up a two-site alternative which remains to be tested.

A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions

Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human alpha(1)-proteinase inhibitor (alpha(1)-PI). The enzyme was maleimidylated using N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and then allowed to react with thiolated BSA or reduced alpha(1)-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. The two methods of evaluating conjugative processes were compared with respect to information provided in relation to the behaviour of the products in solution. The results showed that neither SDS-PAGE nor SEC alone provides sufficient information about conjugate structure. The basic conjugate units observed in electrophoresis tend to form dimeric or higher-order aggregates under gel chromatographic conditions.

Fluorescence Monitoring of the Conformational Change in α2-Macroglobulin Induced by Trypsin Under Second-Order Conditions: The Macroglobulin Acts Both as a Substrate and a Competitive Inhibitor of the Protease

The reaction of bovine pancreatic trypsin with human plasma α2-macroglobulin (α2M) was studied at 25°C, using equimolar mixtures of E and I in 50 mM potassium phosphate buffer, pH 7. The conformational change in α2M was monitored through the increase in protein fluorescence at 320 nm (exc λ, 280 nm). At [α2M]0 = [E]0 = 11.5-200 nM, the fluorescence change data fit the integrated second-order rate equation, (F∞ - F0)/(F∞ - F1) = 1 + ki,obsd [α2M]0t, indicating that cleavage of the bait region in α2M was the rate-determining step. The apparent rate constant (ki,obsd) was found to be inversely related to reactant concentration. The kinetic behavior of the system was compatible with a model involving reversible, non-bait region binding of E to α2M, competitively limiting the concentration of E available for bait region cleavage. The intrinsic value of ki was (1.7±0.24) × 107 M-l s -1. Kp, the inhibitory constant associated with peripheral binding, was estimated to be in the submicromolar range. The results of the present study point to a potential problem in interpreting kinetic data relating to protease-induced structural changes in macromolecular substrates. If there is nonproductive binding, as in the case of trypsin and α2M, and the reactions are monitored under pseudo first-order conditions ([S]0 ≫ [E]0), an intrinsically second-order process (such as the rate-limiting bait region cleavage in α2M) may become kinetically indistinguishable from an intrinsically first-order process (e.g. rate-limiting conformational change). Hence an excess of one component over the other should be avoided in kinetic studies addressing such systems.