Rapid purification of lactate dehydrogenase X from mouse testes by two steps of affinity chromatography on oxamate-sepharose

Summary At concentrations of 200 M NADH and 0.5M NaCl LDH-X is separated from the other LDH isozymes of mouse testes on oxamate-sepharose. In a second step LDH-X is bound to the same matrix at lower NADH and NaCl concentrations and the pure enzyme can subsequently be eluted.Acknowledgment. This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29 Embryonale Differenzierung und Entwicklung.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

A logical approach to the isolation of lactate dehydrogenase isozyme X from human tests: A general rationale for the isolation of homotetrameric LDH isozymes

Summary Immunoadsorbent and oxamate-Sepharose chromatography were used to isolate electrophoretically homogeneous LDH-X from human testes with a final specific activity of 125 IU/mg and good yields: other applications of this approach are discussed.This work was supported by the Rockefeller Foundation.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Summary Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8–7.4) and ionic strength (20–50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO ₄ ^2– ) and cations (Mg²⁺, Ca²⁺) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD⁺ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0–37°C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

On the regulatory properties of a halophilic malic enzyme from Halobacterium cutirubrum.

The NADP-linked malic enzyme from Halobacterium cutirubrum is strongly inhibited by acetyl-CoA and NADH, and rather weakly inhibited by oxaloacetate and glyoxylate, in the presence of very high KCl concentrations (3 M), considered physiological for the extremely halophilic bacteria.     

Conformational flexibility of poly (dG-m5dC) under very low salt conditions

Summary The methylated DNA polymer poly (dG-m⁵dC) which exhibits a B helical conformation in solutions containing 20 mM NaCl, undergoes a gradual and reversible transition to the Z conformation as the NaCl concentration is lowered. The midpoint of this transition occurs around 5–6 mM NaCl. The conformational flexibility of this polymer at such low NaCl concentrations opens up the possibility of studying the effects of other perturbants with negligible interference from salt concentration effects.     

Receptor potential of rat taste cell to potassium benzoate.

Rat taste cells responded to relatively low concentrations of K-benzoate with a hyperpolarization and to the high concentrations with a depolarization. During both responses the membrane resistance of a taste cell decreased. Depolarization elicited by application of a combination of 0.25 M NaCl and 0.05 M K-benzoate was smaller than that by the NaCl alone, indicating a depressant action of K-benzoate.     

Receptor potential of rat taste cell to potassium benzoate

Summary Rat taste cells responded to relatively low concentrations of K-benzoate with a hyperpolarization and to the high concentrations with a depolarization. During both responses the membrane resistance of a taste cell decreased. Depolarization elicited by application of a combination of 0.25 M NaCl and 0.05 M K-benzoate was smaller than that by the NaCl alone, indicating a depressant action of K-benzoate.     

Osmotic adaptation of moderately halophilic methanogenic Archaeobacteria,and detection of cytosolicN,N-dimethylglycine

Methanohalophilus mahii SLP andMethanohalophilus halophilus Z-7982, two closely-related, moderately halophilic, methylotrophic methanogens, were tested for their adaptation to saline conditions. They grew in a wider range of salinities than previously reported, in a defined medium with as little as 0.1 M NaCl, and with a high as 4.0 M NaCl forM. halophilus and 4.5 M NaCl forM. mahii. Fastest growth occurred with 1.5 M NaCl forM. mahii and 1.0 M NaCl forM. halophilus. M. mahii also grew in media in which NaCl was replaced by sucrose or KCl as osmolytes up to the osmolal equivalent of 2 and 2.5 M NaCl (these media contained other sodium salts totaling about 0.1 M Na⁺). In media with either sucrose of KCl replacing NaCl,M. mahii grew fastest at osmolalities approximately equiosmolal to 1 M NaCl.M. mahii not only grew well at a wide range of osmosities, it also tolerated rapid shifts in osmolality. Cells subjected to a rapid 10-fold hypertonic shift resumed growth without a prolonged lag. When cells were subjected to a rapid 10-fold hypotonic shift, 90% of cells lysed, but the remaineder continued to swell with little further lysis during the next 45 min. Surviving cells resumed growth.Methanohalophilus strains grown in defined medium had low cytosolic Na⁺ concentrations; K⁺ concentrations were as high as 0.35 M. Organic osmotica in the cytosol include glycine betaine and larger amounts of N,N-dimethylglycine.     

Comparison of glutamate dehydrogenase and glutamine synthetase activities in the roots and aerial organs of an obligate halophyte: Suaeda maritima var. macrocarpa and a glycophyte: Phaseolus vulgaris, grown in presence of different concentration of NaCl]

The glutamate dehydrogenase and glutamine synthetase activities of an obligate halophyte, Suaeda maritima var. macrocarpa and a glycophyte. Phaseolus vulgaris are compared in function of salinity (increasing concentrations of NaCl) of the culturing solution. In culture, addition of NaCl stimulates glutamine synthetase activity and lowers glutamine dehydrogenase activity in the aerial organs and in the roots of Suaeda as opposed to what is observed in the glycophyte. Hence the obligatory halophily of Suaeda is related to an increase of the glutamine synthetase activity in a sal-trich medium corresponding to the stimulation of nitrate reductase and proteogenesis.     

On the regulatory properties of a halophilic malic enzyme fromHalobacterium cutirubrum

Summary The NADP-linked malic enzyme fromHalobacterium cutirubrum is strongly inhibited by acetyl-CoA and NADH, and rather weakly inhibited by oxaloacetate and glyoxylate, in the presence of very high KCl concentrations (3,M), considered physiological for the extremely halophilic bacteria.This work was supported by grants from the consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, and the Consejo de Investigaciones de la Universidad Nacional de Rosario. JJC and MCV are members of the Carrera del Investigador Científico of the former and the latter institutions, respectively.     

Cellular and secreted tumor plasminogen activator: The effects of NaCl

Summary Plasminogen activator, secreted by metastatic tumor cells, was strongly inhibited in buffer or tissue culture medium containing physiological concentrations of NaCl. Intact cells, however, expressed strong activity under similar conditions. Thus, if plasminogen activator is involved in invasion and metastasis, the cellular activity, acting as an ectoenzyme, may be more important than secreted enzyme under physiological conditions.     

Cellular and secreted tumor plasminogen activator: the effects of NaCl

Plasminogen activator, secreted by metastatic tumor cells, was strongly inhibited in buffer or tissue culture medium containing physiological concentrations of NaCl. Intact cells, however, expressed strong activity under similar conditions. Thus, if plasminogen activator is involved in invasion and metastasis, the cellular activity, acting as an ectoenzyme, may be more important than secreted enzyme under physiological conditions.     

Lithium and rubidium: effects on the rhythmic swimming movement of jellyfish (Aurelia aurita).

The effects of adding LiCl, RbCl, KCl or NaCl to sea water at concentrations up to 30 mmoles/1 on the frequency of contraction of jellyfish (Aurelia aurita) suggest that studies on phylogenetically low animals with relatively simple nervous systems may be of use to determine mechanisms of action of lithium and rudidium on movements.     

Some properties of external NADH oxidation by human placental mitochondria.

Isolated human term placenta mitochondria catalyse oxidation of external NADH in the presence of cytochrome c. This reaction is insensitive to the respiratory chain inhibitors such as rotenone and antimycin A, and is not coupled to phosphorylation. Comparison of the effect of Mg++ ion on NADH plus cytochrome c oxidation by human term placental, human skeletal muscle and rat skeletal mitochondria showed that Mg++ ion exerts an inhibitory effect in the case of human mitochondria and a stimulatory effect in the case of rat skeletal muscle mitochondria.     

Inactivation of N-assimilating enzymes and proteolytic activities in wheat leaf extracts: Effect of pyridine nucleotides and of adenylates

Summary Nitrate reductase was protected from inactivation in wheat leaf extracts by NADH, while NADPH was less effective. NAD, NADP or adenylates did not affect nitrate reductase inactivation in vitro. Glutamine synthetase was more stable than nitrate reductase and was protected from inactivation by ATP. ADP, AMP or pyridine nucleotides had no or only a minor effect on the decrease of glutamine synthetase activity in extracts. The caseolytic activity extracted from senescing leaves was slightly decreased by NADH and NADPH but this effect was not sufficient to explain the stabilization of nitrate reductase by NADH. Oxidized pyridine nucleotides and adenylates had no major effect on the caseolytic activity under the conditions used.This work was supported by grant 3.067-0.81 from the Swiss National Science Foundation. Author for correspondence: U.F.     

Plasma adenosine concentrations are elevated in Dahl salt-sensitive rats

We measured plasma levels of adenosine in Dahl salt-sensitive rats (DS) and Dahl salt-resistant rats (DR) to examine the potential role of adenosine in cardiovascular regulation in this type of hypertension. Plasma adenosine concentrations were significantly higher in DS than in DR. The NaCl content in the diet did not affect plasma adenosine concentration in either DS or DR. Significant positive correlation was found between adenosine concentrations and systolic blood pressure when the data for DS and DR were analyzed together. These results suggest that adenosine may play an important role in the pathophysiology of hypertension in DS.     

Some properties of external NADH oxidation by human placental mitochondria

Summary Isolated human term placenta mitochondria catalyse oxidation of external NADH in the presence of cytochrome c. This reaction is insensitive to the respiratory chain inhibitors such as rotenone and antimycin A, and is not coupled to phosphorylation. Comparison of the effect of Mg⁺⁺ ion on NADH plus cytochrome c oxidation by human term placental, human skeletal muscle and rat skeletal mitochondria showed that Mg⁺⁺ ion exerts an inhibitory effect in the case of human mitochondria and a stimulatory effect in the case of rat skeletal muscle mitochondria.This work has been supported by a grant from Ministry of Higher Education Science and Technology within the project No. 01.02.     

固废制备的填充剂中NaCl对橡胶品质的影响

将碱厂白泥、造纸白泥、含油污泥等固体废弃物加工为橡胶填充剂是一种较好的资源化利用技术,但这些填充剂中往往残留有一定量的NaCl,可能对橡胶制品的性能造成一定影响.本文通过向天然橡胶中添加含有NaCl的填充剂研究了NaCl对橡胶制品性能的影响.实验表明,NaCl在橡胶加工过程中作为晶核起异相结晶作用,可提高橡胶的交联性和热稳定性;在实验的含量范围内,NaCl对填充剂粉体的分散性、硫化时间稍有影响,但橡胶交联性明显提高;硬度、拉伸强度、拉断伸长率等主要力学性能变化不大;老化后的橡胶力学性能基本不变,且部分力学性能高于对照样品.研究揭示,只要NaCl的含量控制在4%以内,基本不会对橡胶工程和橡胶制品性能产生影响;该研究成果有利于含NaCl固体废弃物资源化利用新技术的开发与应用.     

Effect of salt concentration on binding of proteins to a non-ionic adsorbent

Summary The effect of NaCl concentration on the adsorption of several proteins to palmityl-substituted Sepharose 4B has been investigated. It has been observed that the degree of adsorption first decreases and then increases with increasing salt concentrations, followed by total immobilization. The results are qualitatively explained by the simple theory of Debye-Hückel-Kirkwood, as applied to poly-electrolytes in the presence of salt.Acknowledgments. The authors wish to thank Ms H. Keshavarzi, Ms J. Nobakht and Mr F. Mohanazadeh for their technical assistance.