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.

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.

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 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.

Lymphocyte gamma-glutamyltranspeptidase activity in ataxia-telangiectasia.

gamma-Glutamyltranspeptidase (GGTP) is a membrane-bound enzyme, that catalyzes gamma-glutamyl transfer from gamma-glutamyl compounds to amino acid and peptide acceptors. One of the most important clinical findings about ataxia-telangiectasia (A-T), a multisystemic and autosomal-recessive disease, is dysfunction of the immune system. In this study, the activity of GGTP was determined in the lymphocytes from patients with A-T. Lymphocyte GGTP activity in A-T patients was found to be significantly lower than that of control lymphocytes (P less than 0.001). This change may be due to the abnormality in the membrane of lymphocytes of A-T patients.