E. Ferhan Tezcan

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.

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.

Total Calcium Content of Sacs Associated with Inguinal Hernia, Hydrocele or Undescended Testis Reflects Differences Dictated by Programmed Cell Death

Introduction: Inguinal hernia and hydrocele are suggested to result from the persistence of smooth muscle (SM) which should undergo programmed cell death (PCD) after presenting transiently to propel the testis. Since Ca2+ is involved in PCD, the Ca2+ contents of the peritoneum and sacs associated with undescended testis, inguinal hernia and hydrocele were determined and compared. Materials and Methods: Sacs were obtained from boys with undescended testis (n = 11), inguinal hernia (n = 22) and hydrocele (n = 10), and girls with inguinal hernia (n = 7). The calcium content of the sacs and peritoneal samples (n = 6) was determined through atomic absorption spectrophotometry. Calcium contents were compared according to their sources using the Mann-Whitney U test and p values of <0.05 were considered significant. Results: While revealing similar Ca2+ contents as the peritoneum, sacs associated with undescended testis and hydrocele contained more Ca2+ contents than the sacs of boys and girls with inguinal hernia (p < 0.05). Conclusions: Sacs associated with inguinal hernia, which are known to contain SM all around the mesothelial layer, contain the least Ca2+. Despite the decrease in SM, sacs associated with hydrocele contain more Ca2+. Since PCD is associated with Ca2+ overload and inhibition of Ca2+ load inhibits PCD, differences in Ca2+ content may reflect the inhibition of PCD at different stages and for different reasons in inguinal hernia or hydrocele of childhood.

Less Calcium in Cremaster Muscles of Boys with Undescended Testis Supports a Deficiency in Sympathetic Innervation

In addition to an increase in contractility, contracted fibers and small and electron-dense mitochondria have suggested an increase of cytosolic Ca2+ within the cremaster muscles (CM) associated with undescended testis. Therefore, the Ca2+ content of CM associated with an undescended testis was determined and compared with the Ca2+ content associated with inguinal hernia and hydrocele and in internal oblique muscles. CM samples from boys with undescended testis (n = 9), inguinal hernia (n = 15), and hydrocele (n = 7) and from girls with inguinal hernia (n = 8) were obtained. The calcium contents of these samples and of samples from internal oblique muscle (n = 6) were determined by atomic absorption spectrophotometry. While the Ca2+ contents of CM from boys with undescended testis and from girls with inguinal hernia were similar (p > 0.05), the internal oblique muscle and the CM from boys with inguinal hernia and hydrocele contained more Ca2+ than CM from boys with undescended testis (p < 0.05). Despite evidence of an increase in cytosolic calcium, the decrease in total calcium content suggests a decrease in the adenylyl cyclase activity, thus inhibition of influx of Ca2+, but an increase in phospholipase C activity, and generation of more inositol 1,4,5-trisphosphate and mobilization of calcium from internal stores. Since the sympathetic system acts through adenylyl cyclase in striated muscles and is sexually dimorphic, similar Ca2+ contents as encountered in girls suggest an alteration in the balance of autonomic innervation against the sympathetic system in boys with undescended testis.

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.

Inhibition of glutathione reductase by cadmium ion in some rabbit tissues and the protective role of dietary selenium

This study is aimed at investigating the inhibitory effect of cadmium ion on glutathione reductase activity of rabbit brain and liver and the relationship of this effect with dietary selenium. For this purpose, one group of New Zealand rabbits were fed a selenium-deficient diet, another group was fed a selenium-rich diet, and the control group was fed a normal diet. The brain and liver tissues of these groups were investigated for the in vitro inhibitory effects of Cd2+ on glutathione reductase activity. For liver, the percentage inhibition of glutathione reductase by 40 nmol/mg protein of Cd2+ was similar for selenium-deficient and control groups, but significantly lower in the selenium-rich group. For brain tissues, there was no difference with respect to cadmium inhibition of glutathione reductase in all three groups.

The effects of Ni2+, Co2+, and Mn2+ on human serum butyrylcholinesterase

The effects of Ni2+, Co2+, and Mn2+ on human serum butyrylcholinesterase (BChE, acylcholine acylhydrolase E.C. 3.1.1.8) were investigated in this study. Inhibition kinetics of BChE were studied using butyrylthiocholine (BTCh) as substrate. The “1/v” versus “1/[BTCh]” plots in the absence (control plot) and in the presence of the metal ions intersected above 1/[BTCh]-axis for all trace elements. In addition, when the concentrations of the cations were increased at 4 mM BTCh, velocities decreased and drove to zero at high concentrations of the trace elements. These results demonstrate that Ni2+, Co2+, and Mn2+ are linear mixed-type inhibitors of BChE. αKi values have been determined as 53.20 mM, 152.25 mM, and 190.24 mM for Ni2+, Mn2+, and Co2+, respectively, by using nonlinear regression analysis. From the comparison of αKi values of the trace elements, it can be said that BChE has more affinty to binding Ni2+ than Co2+ and Mn2+.

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.

Purification and subunit structure of glutathione reductase from human leukocytes

Abstract Glutathione reductase from human leukocytes has been purified 570-fold by using the methods of ammonium sulfate fractionation (35–70%), CM-Sephadex column chromatography, and affinity chromatography. The molecular weight of the enzyme was determined by gel filtration on Sephadex G-200 and found to be 120,000 ⊣ 5000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme had a molecular weight of 19,000 ± 2000 when dissolved in 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol. Velocity versus pH curve indicated that the enzyme may be a diprotic system.

Effects of Ni2+, Co2+, and Mn2+ on desensitized butyrylcholinesterase prepared from human serum

Human serum butyrylcholinesterase (BChE) has been converted into a stable but less active desensitized form when heated at 45°C for 24 h. The desensitized BChE follows Michaelis-Menten kinetics, whereas native enzyme exhibits slightly negative cooperativity with respect to butyrylthiocholine binding. In this study, we investigated the effects of Ni2+, Co2+, and Mn2+ on the desensitized BChE. It is found that all three ions were noncompetitive inhibitors of the desensitized BChE, and Ki values have been determined as 7.816±1.060 mM, 48.722±4.635 mM, and 84.795±5.249 mM for Ni2+, Co2+, and Mn2+, respectively. In our previous study, these ions were linear mixed-type inhibitors of the native BChE. This finding confirms that desensitized BChE changes to a different conformation than native BChE. From the comparison of Ki values of the trace elements, it can be said that Ni2+ is a more effective inhibitor of the desensitized BChE than Co2+ and Mn2+.