The isolation and characterization of mouse liver glyoxalase I.

The purification of glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing) EC 4.4.1.5) from DBA/1J mouse liver employing ion exchange and affinity chromatography is described. The enzyme was purified 1140-fold and it exhibits a specific activity of 2200 units/mg of protein. The activity was determined to be homogeneous by sedimentation velocity and sedimentation equilibrium ultracentrifugation and by polyacrylamide electrophoresis. The molecular weight is approimately 43 000 and the sedimentation coefficient is 3.4 S. Kinetic data are consistent with a one-substrate (hemimercaptal) reaction mechanism but do not rule out alternate branches at low substrate and free glutathione concentrations.     

Oxidative phosphorylation of liver mitochondria from mice acclimatized to hypobaric hypoxia

Mice exposed to intermittent hypobaric hypoxia for 20 hours a day, 6 days a week, develop extracellular adaptive responses similar to those found in humans exposed to oxygen tension equivalent to that found at an altitude of 4500 m. Isolated liver mitochondria from these animals show no significant differences in rates of substrate-stimulated respiration, ADP-stimulated respiration and the respiratory control ratio (RCR), when compared with sea level controls. Undetectable or negligible differences in these parameters are also noted when sea level animals are exposed for one hour to severe hypoxia (7% O₂). We therefore conclude that the oxidative phosphorylation capacity of the isolated mouse liver mitochondria remains unaltered in both acute and chronic hypoxia. However thein vivo oxygen consumption by mice at this degree of hypoxia was markedly reduced. Lack of observable changes in oxidative phosphorylation could be accounted for by extracellular adaptations in mitochondria isolated from acclimatized animals. This explanation, however, is not consistent with the lack of changes on oxidative phosphorylation in mitochondria isolated from mice undergoing acute hypoxia at sea level. It is then suggested that isolated mitochondrial preparations are of limited value for investigating biochemical mechanisms underlying the variation of cellular respiration occurringin vivo.     

Nonstereospecific substrate usage by glyoxalase I

Glyoxalase I operates on a mixture of rapidly interconverting diasteriomeric thiohemiacetals, formed in a preequilibrium step between glutathione and alpha-ketoaldehyde. That both diasteriomers are directly used as substrates by the enzyme from yeast and from porcine erythrocytes is an outcome of a series of isotope-trapping experiments in which pulse solutions composed of the two diasteriomeric thiohemiacetals, due to [3H]glutathione and phenylglyoxal, are rapidly mixed with chase solutions containing excess unlabeled glutathione and successively increasing concentrations of glyoxalase I. As the enzyme approaches infinite concentration in the chase solution, the radioactivity incorporated into the S-mandeloylglutathione product approaches 100% of the total radioactivity due to both diasteriomers from the pulse solution. The special properties of the active site that allow the enzyme to accommodate both diasteriomeric substrate forms may also account for the fact that the cis and the trans isomers of various para-substituted S-(phenylethenyl)glutathione derivatives are both strong competitive inhibitors of the enzyme. A catalytic mechanism is proposed for glyoxalase I involving catalyzed interconversion of the bound diasteriomeric thiohemiacetals before transformation to final product.     

Myocardial electrolytes in hypobaric hypoxia

Male, white rats (Holtzmann) were exposed to 7,200 m in a barometric chamber for six hours a day for three weeks. Control rats were kept at a sea level altitude of 200 m in Peoria, Illinois. At the end of the exposure period the rats were guillotined and their hearts removed. Approximately equal size strips were cut out of the right ventricles (RV), septa (S) and left ventricles (LV) and weighed on a Mettler balance. All heart pieces were dried overnight in a 40°C oven and reweighed following cooling. They were digested in concentrated nitric acid, diluted with distilled water; and sodium, potassium, calcium and magnesium were determined by atomic absorption spectrophotometry. RV and S muscle from the high altitude rats (HA) had a significantly higher water content. RV muscle from HA rats contained significantly more Na⁺, K⁺ and Ca⁺⁺ and less Mg⁺⁺ than sea level controls (SL). LV muscle from HA animals contained significantly less Na⁺ than SL controls. Within the HA hearts, the RV contained sïgnificantly more Na⁺, K⁺ and Ca⁺⁺ and significantly less Mg⁺⁺ than the S or LV. Evidence indicates that chronic high altitude exposure results in significant alterations in electrolyte distribution throughout the heart.Presented at the Seventh International Biometeorological Congress, 17–23 August 1975, College Park, Maryland, U.S.A.     

间歇性低压低氧与连续性低压低氧对大鼠血液动力学作用的比较(英文)

本研究旨在探讨并比较慢性间歇性低压低氧（intermitten thypobaric hypoxia,IHH）和慢性连续性低压低氧（continuous hypobaric hypoxia,CHH）对大鼠血液动力学作用的影响。40只成年Sprague—Dawley大鼠随机分为5组：对照组（CON）,28天IHH处理组（IHH28）,42天IHH处理组（IHH42）,28天CHH组（CHH28）和42天CHH组（CHH42）。IHH火鼠于低压氧舱分别接受28或42天模拟5000m海拔高度低氧（11．1％O2）处理、每天6h。CHH处理大鼠生活在低压氧舱环境中,除每天半小时常氧供食、供水和清洁外,其余时间均分别接受时程为28或42天的模拟5000m海拔高度低氧（11．1％O2）处理。每周定时测定大鼠体重。通过导管法测定基础常氧和急性低氧状态下的血液动力学,包括半均动脉压（meanartery blood pressure,MAP）、心率（heartrate,HR）、左审收缩峰压（1eft ventricular systolic pressure,LVSP）、正负左率最人压力变化速率（maximum change rate of left ventricular pressure,±LVdP／dtmax）。通过生物化学方法测定大鼠心肌超氧化物岐化酶活性和丙二醛含量。并分别测定全心、左心室和右心室重量。结果显示：（1）CHH42大鼠基础HR和MAP低于CON,IHH和CHH28大鼠（P〈0．05）。（2）IHH大鼠表现出明显的抗心肌缺氧／复氧损伤作用,表现为急性低氧状态下的HR、MAP、LVSP和＋LVdP/dtmax,改变明显低于CON大鼠（P〈0．05）;CHH大鼠表现出更为明显的抗急性低氧心脏保护作用,表现为急性低氧的HR、MAP、LVSP和±LVdP/dtmax;改变明显低于CON和IHH火鼠（P〈0．05）,但出现复氧损伤作用,表现为复氧过程中血液动力学的恢复明显低于CON和IHH大鼠（P〈0．05）。（3）与CON大鼠相比较,IHH和CHH大鼠心肌抗氧化能力明显增强（P〈0．05,P〈0．01）。（4）与IHH和CON大鼠相比较,CHH大鼠表现明显的右心室肥厚（P〈0．01）。结果表明,IHH可诱导有效的心脏保护作用,而无明显的不良反应,因而具有潜在的实际应用价值。     

Lymphopoietic characteristics in acute hypobaric hypoxia

10 healthy men were examined, that underwent a 48 hours hypobaric (acute) hypoxia in the climate chamber "Taba?", with conditions corresponding to those at an altitude of 4,000 m. The "ascent" and "descent" were performed correspondingly within 30 minutes. Increased share of dying lymphocytes and frequency of chromosomal aberrations in these were found in addition to an elevated activity of nucleolar organizers and to changed morphological patterns of argentophilic nucleoli. T- and B-lymphocyte contents remained unchanged. The role of the lymphopoietic alterations in autoimmune complications and of hemopoietic disorders in the mountains is discussed.     

Induction of abscission at hypobaric pressures

The use of hypobaric pressures has increased the precision of abscission research by enabling us to differentiate between abscission action of ethylene and abscisic acid. When cycloheximide is sprayed on fruit attached to trees, enhanced levels of ethylene occur in the fruit and, subsequently, the fruit abscises. When ethylene in the fruit is eliminated by hypobaric pressures, the fruit does not abscise. Thus, ethylene is the effector of fruit abscission that results from cycloheximide treatment. When abscisic acid is applied to the fruit through stem uptake and ethylene is removed by hypobaric pressures, rapid fruit abscission occurs, which is presumably caused by abscisic acid itself. Thus, either ethylene or abscisic acid will induce abscission of citrus. Likewise, the abscission of debladed petioles of Coleus plants appears to be effected either by ethylene or abscisic acid.     

Preservation of TSPO by chronic intermittent hypobaric hypoxia confers antiarrhythmic activity

Abnormal activation of mitochondrial translocator protein (TSPO) contributes to arrhythmogenesis during cardiac metabolic compromise; however, its role in the antiarrhythmic activities of chronic hypoxia adaptation remains unclear. Our results demonstrated that 80% of normoxic rats developed ischaemic VF, whereas this condition was seldom observed in rats with 14 days of chronic intermittent hypobaric hypoxia (CIHH). TSPO stimulation or inhibition affected the arrhythmias incidence in normoxic rats, but did not change the CIHH‐mediated antiarrhythmic effects. Abrupt and excessive elevation of TSPO activity was positively linked to ischaemic VF, and CIHH preserved TSPO activity during ischaemia. The preservation of TSPO activity by CIHH also contributed to the maintenance of intracellular Ca homeostasis. These results suggest that the blunt sensitivity of TSPO to ischaemic stress may be responsible for the antiarrhythmic effects by CIHH.     

Two affinity chromatography methods for the purification of glyoxalase I from rabbit liver

The purification of Glyoxalase I from rabbit liver using Blue Dextran-Sepharose-4B and S-hexyl Glutathione Sepharose-6B is described. Elution of Glyoxalase I from both the columns was accomplished with S-hexyl glutathione, a competitive inhibitor of the enzyme. The purified enzyme gave two bands on disc electrophoresis. After treatment with glutathione, only one band was found. Except for these interconvertible forms, the purified enzyme was homogeneous as shown by disc electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Effect of antidiabetic compounds on glyoxalase I activity in experimental diabetic rat liver

The activity of glyoxalase I from the soluble fraction of diabetic rat liver was found to decrease as compared to the control. Sodium orthovanadate in drinking water and Trigonella foenum graecum seed powder when administered to these diabetic animals were found to reverse the activity of glyoxalase I to control values. A combination of the above two antidiabetic compounds showed a better reversal. Vanadate and Trigonella seed powder treatment separately to diabetic rats also normalized hyperglycemia together with glyoxalase I activity. A combination of vanadate and Trigonella seed powder also restored the other general parameters of the diabetic animals.     

Investigation on glyoxalase I inhibitors

Several classes of compounds - flavones, coumarins, S- and N-substituted glutathione analogs, transition state analogs, porphyrins, nucleotides and nucleosides - have been reported to inhibit the enzyme gloxalase I. In the current study, examination of some of the aforementioned compounds has revealed that squaric acid does not function as an inhibitor of glyoxalase I and several other compounds are much less effective in this regard than previously reported. Several new potent inhibitors of yeast glyoxalase I have been identified. Compounds containing the tropolone structure were especially inhibitory. Glutathione adducts of benzoquinone and naphthoquinone were also inhibitory and may be of particular interest with regard to the toxicology of normal aromatic metabolites in vivo.     

Assay of glyoxalase I in blood

Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) was assayed using alcoholic, acidic 2,4-dinitrophenylhydrazine to follow the disappearance of methylglyoxal over time, with the absorbance of formed methylglyoxal bis-hydrazone measured at 432 nm. Erythrocyte glyoxalase I activities were found to be 64, 41, and 18 mumole of S-lactoyl glutathione formed min-1 X ml-1 of red blood cells in rat, human, and rabbit blood and 174 mumole X min-1 X mg-1 of protein for yeast. The Km values found in millimolar hemimercaptal were about 0.5. Glyoxalase I activity can be determined in crude tissue preparations without interference from biological materials.