Inhibition of rat testis microsomal 3beta-hydroxysteroid dehydrogenase activity by tributyltin

In this study, we have examined the effects of a range of organotin compounds (mono-, di-, tributyltin, mono-, di-, trioctyltin) on the activities of rat testis microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17-hydroxylase (17-OHase) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). 17-OHase activity was inhibited by more than 50% compared with the control rate by 59 microM tributyltin (TBT) but other organotin compounds showed no inhibition. 17beta-HSD activity was unaffected by all organotins tested. 3beta-HSD was inhibited by monooctyltin (81 microM) and by TBT at all concentrations tested in a dose-dependent manner, with almost complete loss of activity at TBT concentrations of 12 microM. The mechanism of inhibition of 3beta-HSD was investigated in kinetic analysis with 0-12 microM TBT. Three rat testis microsomal preparations were incubated with dehydroepiandrosterone as the steroid substrate ranging from 1 to 10,000 nM. Tributyltin was primarily a competitive inhibitor of 3beta-HSD activity, causing an increase in the value of the K(m(app)). However, the mechanism was not entirely competitive as while there was an increase in K(m(app)), a decrease in the V(max(app)) was also observed with increasing concentrations of TBT. Slope and intercept replots demonstrated that the K(i)((app)) from slope replots was around 2.7 microM whereas the K(i)((app)) value from intercept replots was around 30 microM. When compared with the K(m(app)) for 3beta-HSD of around 0.42 microM, TBT could be an effective inhibitor of this enzyme.     

Interaction of lactate dehydrogenase with skeletal muscle troponin

Rabbit muscle troponin complex covalently bound to CNBr-activated Sepharose 4B was shown to interact with soluble lactate dehydrogenase with a stoichiometry of 2 mol lactate dehydrogenase/mol of troponin. The presence of Ca2+ influenced the strength of association (the KD values of 0.73 and 2.3 microM were determined in the presence of 200 microM EGTA or 100 microM Ca2+, respectively). In the absence of Ca2+, the affinity of lactate dehydrogenase to troponin was strongly pH-dependent, reaching a maximum in the region of pH 6.0-7.0. No change of catalytic activity was observed as a result of interaction between lactate dehydrogenase and troponin, the enzyme appeared capable of functioning in the bound form.     

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.     

Histochemical localization of lactate dehydrogenase isoenzymes in human skeletal muscle fibers.

Lactate dehydrogenase (LDH) activity was histochemically localized in fibers of the vastus lateralis muscle of men and for comparative purpose in the soleus and plantaris muscleo of rats. Human muscle fibers were identified as fast twitch (FT) or slow twitch (ST) from the histochemical stain for myofibrillar adenosine triphosphatase activity. Rat skeletal muscle fibers were classified as fast-twitch-oxidative-glycolytic (FOG), fast-twitch-glycolytic (FG), or slow-twitch-oxidative (SO) on the basis of NADH-diaphorase and myofibrillar adenosine triphosphatase activities. Heart-type (H) LDH was identified by inhibition of the muscle-type (M) isozyme with 4 M urea. Total LDH as estimated histochemically was highest in the human FT and rat FG fibers. This was predominantly the M-LDH isozyme. ST fibers of human and SO fibers of rat skeletal muscle had the least total LDH but the most H-LDH activity. The FOG fibers of rat muscle contained a total LDH activity intermediate to that of the FG and SO fibers and a combination of H- and M-LDH. There were no fibers in the human muscle samples studied that had LDH activities similar to the FOG fibers of rat muscle.     

藏羚羊骨骼肌肌红蛋白含量及乳酸脱氢酶、苹果酸脱氢酶活力的研究

目的:探讨藏羚羊骨骼肌对低氧环境的适应机制。方法:以生活在同海拔高度(4 300 m)的藏绵羊和低海拔绵羊(1 800 m)为对照,用分光光度法测定三种动物骨骼肌中肌红蛋白(Mb)含量、乳酸(LA)含量,酶活力法测定三种动物骨骼肌中乳酸脱氢酶(LDH)和苹果酸脱氢酶(MDH)活力。结果:藏羚羊骨骼肌中Mb含量明显高于藏绵羊和低海拔绵羊(P<0.05),而藏绵羊和低海拔绵羊间无明显差异。LA含量和LDH活力明显低于藏绵羊和低海拔绵羊(P<0.05),而MDH活力及MDH/LDH比值显著高于藏绵羊和低海拔绵羊(P<0.05),藏绵羊和低海拔绵羊间无明显差异。结论:藏羚羊可能通过增加骨骼肌中Mb的含量,提高其在低氧环境获取氧的能力,且藏羚羊骨骼肌组织中有氧代谢比例高,这可能与肌肉中Mb含量较高有关,推测藏羚羊较高的Mb含量可能是其适应高原缺氧条件的分子基础之一。     

Inhibition of skeletal muscle activity by lung expansion in the dog

The ability of lung expansion to reflexly decrease skeletal muscle activity was tested in anesthetized dogs. In animals whose left lung was vascularly isolated but neurally intact, the left lung was inflated statically to 40 cmH2O pressure or cyclically with tidal volumes of 10, 20, or 30 ml/kg. Responses to these stimuli were compared with those of injecting 120 or 240 micrograms capsaicin into the left pulmonary artery. Skeletal muscle activity was assessed from the electromyogram (EMG) response of the left hindlimb muscles and from the monosynaptic reflex response to a periodic patellar tendon tap of the right leg (knee jerk). Static inflation and cyclic inflations above 10 ml/kg resulted in significant decreases in both EMG and knee jerk responses. The results indicate that lung expansion is capable of initiating a reflex decrease in skeletal muscle activity. Capsaicin injections caused responses that were similar to those caused by lung inflation, suggesting that at least part of this skeletal muscle reflex response to lung inflation can be attributed to the stimulation of pulmonary C-fibers that could be caused by stretch of the lung.     

Inhibition of Rhizopus lactate dehydrogenase by fructose 1,6-bisphosphate

The closely related fungi Rhizopus oryzae and Rhizopus delemar are often used for the production of lactic and fumaric acid, respectively. These organisms differ primarily by their ability to regenerate NAD through alternative fermentative routes. R. oryzae contains an NAD-dependent l-lactate dehydrogenase enzyme, RO-LdhA, that is primarily responsible for production of lactic acid, while both organisms contain another enzyme, LdhB that is thought to be involved in lactic acid production only under certain growth conditions. We have characterized LdhB from both R. oryzae and R. delemar, respectively referred to as RO-LdhB and RD-LdhB in this study, and have determined that RO-LdhB is significantly more effective than RD-LdhB with regard to k_cat/K_m with reductive LDH activity. Only negligible oxidative LDH activity could be measured with both enzymes; however, the presence of an amino terminal fusion with a small ubiquitin-related modifier, SUMO, increased the oxidative activity per μmol protein by more than 100-fold, while having little effect on the reductive LDH activity. We also determined that RO-LdhA, RO-LdhB, and RD-LdhB were all significantly inhibited in a non-competitive manner by fructose 1,6-bisphosphate (FBP) with K_i values of 1.2, 3.2, and 28.8 mM. Intracellular concentrations of FBP were tested with fermentative conditions to demonstrate that this metabolic intermediate does accumulate to levels that would likely cause inhibition of the R. oryzae LDH. Possible reasons for the significant K_i differences between the nearly identical LdhB proteins are discussed.     

Characterization of a lactate dehydrogenase inhibitor protein from rabbit skeletal muscle mitochondrial fraction

A lactate dehydrogenase inhibitor protein is isolated from rabbit skeletal muscle crude mitochondrial fraction. The molecular weight of the inhibitor is approximately 20,000 as determined by size exclusion HPLC. The inhibitor isoelectricpH is 5.3 as determined by agarose or polyacrylamide gel isoelectric focusing. The amino acid composition of the inhibitor is given. The presence of the inhibitor gives an acidic characteristic to the alkaline M₄ lactate dehydrogenase isozyme and the lactate dehydrogenase-inhibitor complex is more stable than the enzyme alone.     

Sepsis alters pyruvate dehydrogenase kinase activity in skeletal muscle

Chronic sepsis promotes a stable increase in pyruvate dehydrogenase kinase (PDHK) activity in skeletal muscle. PDHK is found tightly bound to the pyruvate dehydrogenase (PDH) complex and as free kinase. We investigated the ability of sepsis to modify the activity of the PDHK intrinsic to the PDH and free PDHK. Sepsis was induced by the intraabdominal introduction of a fecal-agar pellet infected with E. coli and B. fragilis. Five days later, mitochondria were isolated from skeletal muscle and PDHK measured in mitochondrial extracts. Sepsis caused an approximate 2-fold stimulation of PDHK. The mitochondrial extracts from control and septic rats were fractionated by gel chromatography on Sephacryl S-300 to separate PDHK intrinsic to PDH complex and free PDHK. PDH complex eluted at void volume and was assayed for PDHK intrinsic to the complex. The activity of PDHK intrinsic to PDH complex was a significantly increased 3 fold during sepsis. Free PDHK activity eluted after the PDH complex and its activity was enhanced by 70% during sepsis. Incubation of PDHK intrinsic to PDH with dichloroactate, an uncompetitive inhibitor of PDHK, showed the PDHK from septic rats relatively less sensitive to inhibition than controls. These results indicate that sepsis induces stable changes in PDHK in skeletal muscle.     

Modulation of lactate dehydrogenase activity by enzyme-protein interaction

Some lactate dehydrogenase modulator proteins have been isolated from the lactate dehydrogenase-free crude mitochondrial fraction of rabbit muscle, beef liver and chicken liver. It was shown that beef and chicken liver mitochondrial extracts exhibited activatory capacity in contrast to the inhibitory capacity of rabbit muscle mitochondrial extracts. All modulators can be precipitated by 80% ammonium sulphate saturation and show high anodic electrophoretic mobility and heat stability. Modulators have higher affinity for alkaline pI lactate dehydrogenase isoenzymes, independent of whether the M and H subunits are predominant. The inhibitor and the activator molecules compete for lactate dehydrogenase since their modulatory capacity was nullified when similar relative amounts were used. This study shows the existence of analogous proteins with an acidic pI in the different mitochondrial fractions which modify lactate dehydrogenase activity.     

Inhibition kinetics of lactate dehydrogenase isoenzymes by gossypol acetic acid

The effect of gossypol acetic acid, a potent male sterilent was studied on LDH from goat liver (LDH-A4), heart (LDH-B4) and testis (LDH-C4) in vitro. All the preparations of LDH were inhibited by gossypol when the reaction was carried out in pyruvate-lactate (direct) or lactate to pyruvate (reverse) directions. The IC50 of gossypol for the pyruvate oxidation by LDH isozymes varied between 16 and 42 microM in presence of 0.27 mM pyruvate and 0.15 mM NADH at 25 degrees C and pH 7.4 whereas for the lactate oxidation, IC50 was 125 microM in a system containing 3.3 mM lactic acid and 1.8 mM NAD at 25 degrees C and pH 9.0. Reciprocal plots due to Lineweaver-Burk showed that these isozymes are inhibited in a non-competitive manner with respect to pyruvate and lactate, and in a competitive fashion when NAD and NADH were varied as substrates. Ki values of LDH-A4, -B4 and -C4 isozymes in presence of gossypol were 20, 34 and 29 microM against pyruvate; 33, 43 and 45 microM against NADH; 85, 85 and 125 microM against lactate and 94, 108 and 83 microM against NAD respectively.     

Thermodynamics of complex formation between nicotinamide adenine dinucleotide and pig skeletal muscle lactate dehydrogenase.

Formation of the binary complex between the reduced coenzyme nicotinamide adenine dinucleotide (NADH) and pig skeletal muscle lactate dehydrogenase (LDH, EC 1.1.1.27) has been investigated by calorimetric and equilibrium dialysis techniques in 0.2 M potassium phosphate buffer (pH 7.0) at various temperatures. Analysis of thermal titration curves at two temperatures (25 and 31.5 degrees) shows that the experimental enthalpy data can be rationalized assuming four independent and equivalent binding sites for the tetrameric enzyme. Binary complex formation is characterized by a negative temperature coefficient, delta cp, of the binding enthalpy, which amounts to -1300 plus or minus 53 cal/(deg mol of LDH) in the temperature range of 5-31.5 degrees. Despite the slightly smaller standard deviation resulting when polynomial regression analysis of the second degree is applied to the temperature dependence of the enthalpy values, binding enthalpies seem to be adequately represented in the temperature range studied by the equation delta H = -1.3T + 2.3, kcal/mol of LDH, T referring to the temperature in degrees C. By combination of the results obtained from equilibrium dialysis and calorimetric studies a set of apparent thermodynamic parameters for binding of NADH to LDH in 0.2 M potassium phosphate buffer at pH 7 has been established.     

Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke

Summary SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2R1, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy.LDH activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of LDH activity.Supported by a research grant from the ASFC (Association Suisse des Fabricants de Cigarettes), Switzerland