A new perspective on thermal inactivation kinetics of human serum butyrylcholinesterase.

Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37 degrees, 40 degrees, 45 degrees, and 50 degrees C for 24 hr. It was observed that the enzyme heated at 45 degrees C for 24 hr converted to a stabilized form and followed Michaelis-Menten kinetics, whereas the enzyme samples, heated at the other temperatures for 24 hr, shown negative cooperativity with respect to its substrate, butyrylthiocholine. Even the sample heated at 45 degrees C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40 degrees C for 24 hr, the heated enzyme at 45 degrees C for 24 hr could not be reactivated when it was kept at 4 degrees C for 24 hr. In the kinetic studies, it was found that substrate analogs choline and benzoylcholine inhibited both the native enzyme and the enzyme heated at 45 degrees C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme.

A comparative study on effect of dietary selenium and vitamin E on some antioxidant enzyme activities of liver and brain tissues.

Since selenium and vitamin E have been increasingly recognized as an essential element in biology and medicine, current research activities in the field of human medicine and nutrition are devoted to the possibilities of using these antioxidants for the prevention or treatment of many diseases. The present study was aimed at investigating and comparing the effects of dietary antioxidants on glutathione reductase and glutathione peroxidase activities as well as free and protein-bound sulfhydryl contents of rat liver and brain tissues. For 12-14 wk, both sex of weanling rats were fed a standardized selenium-deficient and vitamin E-deficient diet, a selenium-excess diet, or a control diet. It is observed that glutathione reductase and glutathione peroxidase activities of both tissues of the rats fed with a selenium-deficient or excess diet were significantly lower than the values of the control group. It is also shown that free and bound sulfhydryl concentrations of these tissues of both experimental groups were significantly lower than the control group. The percentage of glutathione reductase and glutathione peroxidase activities of the deficient group with respect to the control were 50% and 47% in liver and 66% and 61% in the brain, respectively; while these values in excess group were 51% and 69% in liver and 55% and 80% in brain, respectively. Free sulfhydryl contents of the tissues in both experimental groups showed a parallel decrease. Furthermore, the decrease in protein-bound sulfhydryl values of brain tissues were more pronounced than the values found for liver. It seems that not only liver but also the brain is an important target organ to the alteration in antioxidant system through either a deficiency of both selenium and vitamin E or an excess of selenium alone in the diet.

Inhibition effects of benactyzine and drofenine on human serum butyrylcholinesterase.

Benactyzine and drofenine are widely used anticholinergic drugs. Benactyzine is used to treat organophosphate poisoning and drofenine acts on smooth muscle to stop muscle spasms. Both of these drugs are esters. After they enter the bloodstream, they will interact with butyrylcholinesterase (BChE; acylcholine acyl hydrolase: EC 3.1.1.8), which has an ability to hydrolyze a wide variety of esters. Therefore, the kinetic analysis of their inhibitory effects on human serum BChE was examined using butyrylthiocholine as substrate. Both drugs were competitive inhibitors of BChE and the Ki values of benactyzine and drofenine were calculated to be 0.010 +/- 0.001 and 0.003 +/- 0.000 mM, respectively, using the Systat (version 5.03, 1991) nonlinear regression analysis software package. According to these parameters, drofenine is a more potent competitive inhibitor of BChE than benactyzine.

A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens.

This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2',5'-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62 +/- 3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2 +/- 3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at pI 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea = 5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows 'Ordered Bi Bi' sequential kinetics. From the graphical and statistical analyses, KmNADP+, KmG-6-P, KiNADPH, Ki6-PGA were estimated to be 0.008 +/- 0.002, 0.035 +/- 0.013, 0.173 +/- 0.007 and 1.771 +/- 0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources.

Inhibition kinetics of human serum butyrylcholinesterase by Cd2+, Zn2+ and Al3+: comparison of the effects of metal ions on cholinesterases.

Butyrylcholinesterase (BChE, EC 3.1.1.8) has been purified about 6600-fold from human serum with a procedure including ammonium sulfate fractionation (55-70%) with acid step at pH 4.5 and procainamide-Sepharose 4B affinity chromatography. The purified enzyme exhibited negative cooperativity with respect to butyrylthiocholine (BTCh) binding at pH 7.5. Ks was found to be 0.128 +/- 0.012 mM. Inhibition kinetics of the enzyme by Cd2+, Zn2+ and Al3+ were studied in detail. The 1/v vs 1/[BTCh] plots in the absence (control plot) and in the presence of different concentrations of cations intersected above 1/[BTCh]-axis. The data were analyzed by means of a nonlinear curve fitting program. The results demonstrated that all of the three cations are the linear mixed-type inhibitors of BChE. Ca2+ and Mg2+ had no effect on the enzyme activity in the experimental conditions. But when the enzyme was inhibited by 0.5 mM Cd2+ or Zn2+, Ca2+ and Mg2+ partially reactivated the inhibited allosteric form of BChE. Results were compared with data obtained from brain BChE purified from sheep.

Amitriptyline: a potent inhibitor of butyrylcholinesterase from human serum.

1. The effect of amitriptyline on human serum butyrylcholinesterase (acylcholine acylhydrolase E.C.3.1.1.8) has been investigated. From the Lineweaver-Burk plot and the plot of v versus amitriptyline concentration, it was concluded that amitriptyline inhibition is partially competitive, and the kinetic parameters have been calculated as Ks = 0.11 mM, alpha = 1425 and Ki = 0.01 mM. 2. Because amitriptyline is a partial competitive inhibitor of butyrylcholinesterase, acquired deficiency may be seen in patients treated with amitriptyline and may cause complications in operations.

Histidine modification of human serum butyrylcholinesterase.

The effects of histidine-modifying reagents on human serum butyrylcholinesterase (BChE) were investigated. The commercially available enzyme was further purified by chromatography on a Sepharose CI-6B column prior to use. In the modification studies, we found that the histidine-specific reagents tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCE) did not modify the enzyme; however, they inhibited the enzyme reversibly. The kinetic parameters of enzyme inhibition calculated were alpha = 10.8, beta = 0.26, and Ki = 0.016 mM for TPCK. TLCK inhibition gave similar kinetic behavior, with alpha = 41.6, beta = 0.065, and Ki = 0.039 mM. Tosyllysine, an analog of TLCK, did not inhibit the enzyme. Removal of TPCK and TLCK by dialysis resulted in significant reactivation of the enzyme. From kinetic studies, it was found that the inhibitions were hyperbolic mixed-type inhibitions. We concluded that the reagents competed with substrate for hydrophobic binding sites and inhibited the enzyme reversibly. On the other hand, in the modification studies with diethyl pyrocarbonate (DPC), it was observed that inactivation of the enzyme was irreversible and time-dependent. In the protection studies, the activity of the enzyme was partially protected from inactivation by DPC even at a 50 mM concentration of butyrylthiocholine. The results indicate that DPC modifies some essential histidine side chains in BChE, including the functional histidyl residue found at the active site.

Inhibition kinetics of sheep brain glutathione reductase by cadmium ion.

Glutathione reductase participates in preventing lipid peroxidation by oxygen radicals which results in cellular damage. The brain is among the organs most susceptible to cadmium-induced lipid peroxidation. The mechanism of free radical generation by Cd2+ is not well understood, but it is known that Cd2+ is an inhibitor of glutathione reductase. In this study, inhibition kinetics of the brain glutathione reductase by Cd2+ was investigated. Sheep brain enzyme (11,000-fold purified) was used for this purpose. The data were analyzed by a nonlinear curve fitting program. It was found that the inhibition was competitive with respect to oxidized glutathione and uncompetitive with respect to NADPH. Inhibition constants were found as 12.3 and 9.4 muM, respectively. These findings might contribute to the understanding of the mechanism of lipid peroxidation by Cd2+ in brain.

Inhibition kinetics of brain butyrylcholinesterase by Cd2+ and Zn2+, Ca2+ or Mg2+ reactivates the inhibited enzyme.

1. The inhibition kinetics of sheep brain butyrylcholinesterase (BChE) (acylcholine acylhydrolase, EC 3.1.1.8) by Cd2+ and Zn2+ has been studied. 2. KS has been determined as 0.14 mM. Cd2+ and Zn2+ were the hyperbolic mixed-type inhibitors of BChE. Ca2+ and Mg2+ had no effect on the enzyme activity in the experimental conditions. 3. But when the enzyme was inhibited by 0.1 mM Cd2+ or Zn2+, Ca2+ and Mg2+ reactivated the inhibited form of BChE.

Sheep brain pseudocholinesterase: inhibition kinetics of the partially purified enzyme by some substrate analogues.

Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been partially purified (about 270-fold) from sheep brain. The procedure included ammonium sulfate fractionation (20-80%), DEAE-Trisacryl M chromatography and procainamide-Sepharose 4B affinity chromatography. The molecular weight of purified ChE was found to be 290,000 by gel filtration. Kinetic properties of the enzyme have been studied using the substrate analogues choline, succinylcholine and benzoylcholine. It was shown that the inhibition was partially competitive.

Kinetic properties of sheep brain glutathione reductase.

The kinetic properties of sheep brain glutathione reductase (GSSGR) were investigated. The enzyme showed Ping-Pong kinetics with double substrate inhibition in the forward direction. Km values for NADPH and GSSG were found to be 60.9 and 116.9 mumol/l, and Ki values were found to be 42.1 and 347.3 mumol/l, respectively. NADP+ inhibition at low fixed concentration of NADPH was mixed-type with a Ki of 281.5 mumol/l and alpha of 0.048. It is concluded that the enzyme shows a hybrid Ping-Pong-ordered branched mechanism.

Sheep brain glutathione reductase: purification and general properties.

Sheep brain glutathione reductase was purified about 11,000-fold with an overall yield of 40%. The method included ammonium sulphate fractionation, heat denaturation, 2',5'-ADP Sepharose 4B and Sephadex G-200 chromatography steps. Specific activity at the final step was 193 IU/mg. The Mr of the enzyme was found to be 116,000 by gel filtration chromatography. On SDS-PAGE, two identical subunits of Mr 64,000 were obtained. From the spectral data, about 2 mol FAD per mol of enzyme were calculated.