Studies on relationship between Na,K-ATPase activity and sperm capacitation in guinea pig

In order to elucidate the biochemical mechanism of sperm capacitation, the relationship between plasmalemma Na,K-ATPase, capacitation and acrosome reaction was investigated. Plasmalemma of guinea pig spermatozoa was isolated by sucrose gradient centrifugation for the determination of Na,K-ATPase activity. As far as the authors are aware, the Na,K-ATPase activity in plasmalemma of the guinea pig has never been detected. By treating sperm plasmalemma with 0.05% DOC (deoxycholate), enzyme activity could be quantitatively measured. After spermatozoa were incubated in capacitation medium for 8 h, Na,K-ATPase activity was found to be decreased to 35.6% as compared with that before incubation. The spermatozoa incubated for 10.5 h in capacitation medium containing 1 and 5 mumol/L ouabain showed 46.5% and 64.4% acrosome reactions respectively, while the acrosome reaction of the control group was only 27.4%. The above results suggest that the decrease in the Na,K-ATPase activity of guinea pig spermatozoa may be a prerequisite for capacitation. Experimental results demonstrated for the first time that Na,K-ATPase activity exists in the sperm plasmalemma of the guinea pig. It was further found that the decrease of Na,K-ATPase activity of plasmalemma enhances sperm capacitation. It is suggested that sperm capacitation in the guinea pig is possibly induced by the decrease in plasmalemma Na,K-ATPase and, as a consequence, the intracellular Na+ is increased, which would benefit the exchange of Na+out/Ca++in and the onset of acrosome reaction.     

The effect of EGTA and Ca++ in regulation of the brain Na/K-ATP-ase by noradrenaline

Background  

The Na/K-ATPase activity of the brain synaptic plasma membranes (SPM) is regulated by noradrenaline (NA) and the synaptosomal factor SF (soluble protein obtained from the synaptosome cytosol). In the absence of SF, NA inhibits Na/K-ATPase, while, on addition of SF to the reaction medium, there is a NA-dependent activation of Na/K-ATPase . On the other hand, EGTA augments the Na/K-ATPase activity and attenuates the ability of NA to inhibit Na/K-ATPase.     

慢性肾衰患者血清中分子物质浓度变化及其对Na,K-ATP酶的抑制作用

慢性肾功能衰竭是指在各种慢性肾脏疾病的基础上,缓慢出现肾功能减退而不可逆转的肾衰竭综合症由于患者肾功能衰竭,使得一些正常人本可以排出体外的代谢产物滞留在体内,导致肌体的一系列病变,涉及到人体的肠胃、免疫、心血管、内分泌、皮肤和骨骼等各个系统．20世纪70年代以来,中分子物质在患者体内的毒性作用开始引起众多研究者的关注．然而,由于这类物质成分复杂,对其分离鉴定工作还面临着很多困难,它们与慢性肾衰患者的临床症状之间的因果关系及致病机理仍不甚明确．在前期研究中,我们采用凝胶色谱法分离尿毒症患者血清,得到两个中分子物质峰A和B,     

Na,K-ATPase reconstituted in liposomes: effects of lipid composition on hydrolytic activity and enzyme orientation

In this paper, the reconstitution of Na,K-ATPase in liposomes (formed by single or mixed phospholipids and cholesterol) was investigated and the enzyme orientation was determined on kinetic basis using only specific inhibitors of ATP hydrolysis.

A condition of foremost importance for enzyme reconstitution is the achievement of complete solubilization of the lipid in the initial stage of the cosolubilization process for the subsequent formation of the liposomes and/or proteoliposomes. PC-liposomes showed that increasing the fatty acid chain length increases the percentage of Na,K-ATPase incorporated. The average diameter of the proteoliposomes also increases in proportion, reaching a maximum with phospholipids with 16 carbon chains, resulting in 75.1% protein reconstitution and 319.4 nm diameter size, respectively. Binary lipid systems with PC and PE were efficient for incorporation of Na,K-ATPase, depending on the lipid:protein ratio used, varying from 15 to 80% recovery of total ATPase activity. The best results for Na,K-ATPase reconstitution using PC and PE mixture were obtained using a lipid:lipid ratio 1:1 (w/w) and lipid:protein 1:3 (w/w). Integrity studies using calcein release mediated by detergent or alamethicin, in association with inhibition of ATPase activity (ouabain and vanadate) showed that the enzyme is oriented inside-out in DPPC:DPPE proteoliposomes. In these vesicular systems, the enzyme is reconstituted with about 78.9% ATPase activity recovery and 89% protein incorporation, with an average diameter of 140 nm. Systems constituted by DPPC:DPPE, DPPC:DLOPE or DLOPC:DLOPE showed approximately 80, 71 and 70% of recovery of total ATPase activity, but no homogeneity in the distribution of Na,K-ATPase orientation. Reconstitution of Na,K-ATPase in DPPC:DPPE:cholesterol or DPPC:DLOPE:cholesterol systems (55% of cholesterol) showed recovery of about 86 and 82%, respectively, of its total ATPase activity.

The results point to an important effect of the lipid acyl chain length and lipid–protein ratio in relation to the composition of the lipid matrix to finely tune the structural asymmetry and the amount of enzyme that can be incorporated a lipid bilayer vesicle while preserving membrane permeability.     

Biostimulation of Na,K-ATPase by low-energy laser irradiation (685 nm, 35 mW): comparative effects in membrane, solubilized and DPPC:DPPE-liposome reconstituted enzyme

OBJECTIVE: The aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of the different forms of the Na,K-ATPase. METHODS: Membrane-bound and solubilized (alphabeta)(2) form of Na,K-ATPase was obtained from the dark red outer medulla of the kidney and proteoliposomes of DPPC:DPPE and Na,K-ATPase was prepared by the co-solubilization method. Irradiations were carried out at 685 nm using an InGaAIP diode laser. RESULTS: The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/cm(2). However, with irradiation doses ranging from 32 to 40 J/cm(2), a 28% increase on the ATPase activity was observed while when using up to 50 J/cm(2) no additional enhancement was observed. When biostimulation was done using the solubilized and purified enzyme or the DPPC:DPPE-liposome reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/cm(2). With irradiation above these values (24 J/cm(2)) no additional increase in the activity was observed. These studies revealed that the biostimulation of ATPase activity from different forms of the Na,K-ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes.     

Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence

A method for the functional immobilization of Na,K-ATPase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent field of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-ATPase was used, which had been labelled covalently within the ATP-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corresponding fluorescence intensity changes found in bulk solution provides information about the side-directed surface binding of the Na,K-ATPase membrane fragments. The affinity constants of K+ ions to the Na,K-ATPase was the same for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functional immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced methods for exploring the molecular mechanisms of membrane receptor targets and drug screening are required.     

Far infrared spectroscopy on hemoproteins: A model compound study from 1800–100 cm

Infrared spectroscopy measurements on different hemoproteins and models of the active side have been completed for the spectral range from 1800 to 100 cm⁻¹ giving an overview on the contributions expected in the low frequency range. Little is known of the low frequency contribution of proteins in infrared. In order to detect the contributions of heme centers and protein moiety, a systematic study of the infrared spectroscopic properties of the porphyrin ring, the ferric porphyrines with different ligands (hemine and hematine), a heme with 11 amino acids (microperoxidase-11), cytochrome c and cytochrome c oxidase are compared at different pD values and an overview on the relative contributions of hemes, their ligands and the protein site can be provided in the low frequency region. Beside the well know amide I and II modes, the low frequency range is found to be dominated by the amide IV and VI mode around 530/580 cm⁻¹ for cytochrome c and cytochrome c oxidase, as well as further proteins like ferrodoxin. Below 300 cm⁻¹ amide VII modes, doming modes of the heme site and hydrogen-bonding signatures overlap to a broad peak with covering 100–250 cm⁻¹. As clear markers for the iron ligands, bands can be depicted at 388/378 cm⁻¹ (FeN, histidine ligand) and 345 cm⁻¹ FeCl. Furthermore the ring vibration of the protonated histidine is determined at 623 cm⁻¹.     

Inhibition of highly purified porcine kidney Na,K-ATPase by steroid glycosides of the spirostan and furostan series and a study of structure-activity relationships

A comparative investigation has been made of the action of 14 steroid glycosides of the spirostan and furostan series on highly purified Na,K-ATPase from the medullary layer of porcine kidneys (∼90% purity in terms of protein). It has been shown that alliospirosides A, B, and D, isolated from the collective fruit ofAllium sepa L., are capable of inhibiting the activity of the Na,K-ATPase The inhibition of the activity of the transport enzyme by alliospirosides A and B is of the uncompetitive type and by alliospiroside D of the competitive type. It is desirable to test alliospirosides on the intact organism. Institute of Physiology, Academy of Sciences of the Republic of Uzbekistan. Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 558–566, July–August, 1993.     

Assay of Sodium and Potassium Activated Adenosine Triphosphatase in Submicrogram Fragments of Renal Tubules

Abstract

An improved method for measuring Na, K-ATPase in submicrogram fragments of single renal tubules approximately one millimeter long is described. The activity is determined by coupling ATP hydrolysis stoichiometrically to pyruvate kinase and the oxidation of NADH by lactic dehydrogenase. NADH oxidation is followed fluorimetrically using an instrument specially modified for increased sensitivity and stability.     

Inhibition of highly purified porcine kidney Na,K-ATPase by steroid glycosides of the spirostan and furostan series and a study of structure-activity relationships

A comparative investigation has been made of the action of 14 steroid glycosides of the spirostan and furostan series on highly purified Na,K-ATPase from the medullary layer of porcine kidneys (90% purity in terms of protein). It has been shown that alliospirosides A, B, and D, isolated from the collective fruit ofAllium sepa L., are capable of inhibiting the activity of the Na,K-ATPase The inhibition of the activity of the transport enzyme by alliospirosides A and B is of the uncompetitive type and by alliospiroside D of the competitive type. It is desirable to test alliospirosides on the intact organism.Institute of Physiology, Academy of Sciences of the Republic of Uzbekistan. Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 558–566, July–August, 1993.     

Membrane proteins in thin films

Molecular functions and structural changes of membrane proteins in an aqueous environment can be elucidated by reaction-induced FTIR difference spectroscopy upon photolysis of caged compounds. The achieved detection of IR band changes even due to single amino acid residues is, however, only possible in the presence of very high protein concentrations, implying that a low water content must be present. In general, the films are formed by controlled dehydration of membrane protein suspensions at reduced pressure and low temperature. For the retention of enzymatic activity of Na,K-ATPase, for example, a cosolvent such as glycerol is required. In order to interprete the results obtained by FTIR spectroscopy, it is important to know whether essential properties of the proteins such as hydration are changed upon film formation. Therefore, a differential scanning calorimetry (DSC) study has been carried out with purified Na,K-ATPase and Ca-ATPase in suspension, in form of pellets obtained by high-speed ultracentrifugation and in thin films. As relevant thermoanalytical properties, the endothermic denaturation transitions of the proteins have been studied. For Na,K-ATPase in the presence of 20% glycerol as cosolvent, a single, comparatively narrow endothermic and irreversible denaturation transition with a denaturation enthalpy of about 1.7 MJ mol⁻¹ and transition temperatures of about 65 and 70°C is found in concentrated suspension and in the state of the pellet, respectively. In the case of thin films suitable for IR spectroscopy, a characteristic change is observed in a reproducible manner. The enthalpy change of the remaining transition around 70°C is reduced but an additional transition at about 77°C is observed. Based on control experiments, the new high temperature transition is attributed to a partially dehydrated state of the protein. Furthermore, a comparatively broad endothermic transition around 20°C is found under conditions of high protein concentrations (film), which is tentatively assigned to a transition of the lipid environment of this integral membrane protein. Similar results are found for Ca-ATPase films. In the absence of glycerol, the deoxycholate treated enzyme in suspension exhibits a narrow endothermic main transition at 52°C with a denaturation enthalpy around 0.9 MJ mol⁻¹. For the film of this protein, two almost equally large endothermic transitions are found at 59 and 77°C. Also here, the data are characteristic of partial protein dehydration. These results show clearly that DSC can easily be applied in a sensitive manner to control and characterize the integrity and hydration properties of concentrated protein samples in thin films.     

Membrane receptor calorimetry: cardiac glycoside interaction with Na,K-ATPase

The receptor–ligand interaction between the cardiac glycoside Ouabain and purified, membrane-bound as well as micellar Na,K-ATPase is investigated. Calorimetric titrations are carried out with micromolar concentrations of the phosphorylated protein in the presence of Mg²⁺. The measured heat changes provide evidence for an exothermic, high affinity and specific receptor binding process as well as for a low affinity, nonspecific binding to the lipid part of the nanoparticulate membrane fragments. The degree of lipid binding markedly depends on the lipid composition of the tissue. The measured time course of the heat change resulting from specific binding to the receptor site is unusually slow and is limited by the binding kinetics of the ligand. A course estimation of the Ouabain binding kinetics leads to a rate constant around 10⁴ mol⁻¹ l s⁻¹. Receptor binding is characterized by affinities ranging between 10⁷ and 10⁸ mol⁻¹ l, ΔH values around −95 kJ mol⁻¹ and ΔS values of about −130 J K⁻¹ mol⁻¹ at 25°C. The enthalpic contribution is assumed to be mainly due to hydrogen bond formations between the ligand and the receptor site whereas the large, negative entropy change may be attributed to an increased interaction between water and the protein as a consequence of a conformational transition. The evaluation of the titrations provides stoichiometric coefficients around 0.55, which implies that only about 50–60% of the Na,K-ATPase protomers are capable to bind the cardiotonic steroid. This result is consistent with radioactive phosphorylation studies and appears to be a typical feature of kidney-type Na,K-ATPase preparations. Possible implications of this finding are discussed. As a general result, this study demonstrates how simple and suitable calorimetric titrations with micromolar protein concentrations can be for the purpose of a quantitative characterization of a receptor in nanoparticulate membrane systems.     

Mechanistic principles of ion transport in the Na,K-ATPase

The Na,K-ATPase is a member of the P-type ATPase family and a primary active ion transporter for Na⁺ and K⁺ ions in the cytoplasmic membrane of virtually all animal cells. Considerable progress in understanding the ion-pump mechanism of the Na,K-ATPase was gained by combining biophysical and biochemical studies of more than 30 years with structural information at atomic resolution available since recent years. Biophysical studies have revealed detailed properties of the ion movements that led to a gated-channel model which is strongly supported by structural findings obtained for the sodium pump. The basic question of how the free Gibbs energy released by ATP hydrolysis is transferred to the protein and transformed into uphill transport of the ions is still without reply.     

EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE CELLULAR ENERGY METABOLISM IN THE ABSENCE AND PRESENCE OF LIGHT

Effects of Photofrin II on energy metabolism and metabolic viability were studied in a mammalian transformed cell line (BHK-21) in dark and after photo-irradiation with visible light. Cells were allowed to accumulate Photofrin by incubating for 4 h in buffer containing Photofrin (5-60 micrograms/ml). The results show that Photofrin significantly affects the cellular energy metabolism even in the absence of light; activity of cytochrome c oxidase is decreased and glucose utilization and lactate production (glycolysis) are increased. Irradiation with light resulted in a significant decrease in the activity of cytochrome c oxidase, glycolysis, ATP content, energy charge, ratios of adenine nucleotides like ATP/ADP, ATP/AMP and cell viability (dye exclusion test). Presence of inhibitors of energy metabolism, potassium cyanide (respiration) and 2-deoxyglucose (glycolysis), further enhanced the cytotoxic effects induced by hematoporphyrin derivative and light.     

The indirect coulometric titration of cytochrome c oxidase with cytochrome c

The indirect coulometric titration of cytochrome c oxidase and dioxygen using cytochrome c as a mediator is described. Results of both the indirect coulometric titrations and the cyclic voltammetric experiments reported herein verify that the reaction mechanism involves the catalytic regeneration of the electroactive species, the cytochrome c mediator, with the selective reduction of cytochrome c oxidase alone. During the indirect coulometric titrations dioxygen is reduced to water only by cytochrome c oxidase and not by either direct reduction at the electrode surface or reaction with cytochrome c. This system utilizes the electron transfer selectivity of cytochrome c for cytochrome c oxidase over dioxygen and offers a means by which the reaction of cytochrome c oxidase and dioxygen can be examined.     

In Vivo Low-level Light Therapy Increases Cytochrome Oxidase in Skeletal Muscle

Low-level light therapy (LLLT) increases survival of cultured cells, improves behavioral recovery from neurodegeneration and speeds wound healing. These beneficial effects are thought to be mediated by upregulation of mitochondrial proteins, especially the respiratory enzyme cytochrome oxidase. However, the effects of in vivo LLLT on cytochrome oxidase in intact skeletal muscle have not been previously investigated. We used a sensitive method for enzyme histochemistry of cytochrome oxidase to examine the rat temporalis muscle 24 h after in vivo LLLT. The findings showed for the first time that in vivo LLLT induced a dose- and fiber type-dependent increase in cytochrome oxidase in muscle fibers. LLLT was particularly effective at enhancing the aerobic capacity of intermediate and red fibers. The findings suggest that LLLT may enhance the oxidative energy metabolic capacity of different types of muscle fibers, and that LLLT may be used to enhance the aerobic potential of skeletal muscle.     

Fe,Cu-codoped metal-nitrogen-carbon catalysts with high selectivity and stability for the oxygen reduction reaction

Metal-nitrogen-carbon materials(M-N-C) are non-noble-metal-based alternatives to platinum-based catalysts and have attracted tremendous attention due to their low-cost,high abundance,and efficient catalytic performance towards the oxygen reduction reaction(ORR).Among them,Fe-based materials show remarkable ORR activity,but they are limited by low selectivity and low stability.To address these issues,herein,we have synthesized FeCu-based M-N-C catalysts,inspired by the bimetal center of cytochrome c oxidase(CcO).In acidic media,the selectivity was notably improved compared with Febased materials,with peroxide yields less than 1.2%(<1/3 of the hydrogen peroxide yields of Fe-N-C catalysts).In addition to Cu-N-C catalysts which can catalytically reduce hydrogen peroxide,the reduction current of hydrogen peroxide using FeCu-N-C-20 exceeded that of Fe-N-C by about 6% when the potential was greater than 0.4 V.Furthermore,FeCu-based M-N-C catalysts suffered from only a15 mV attenuation in their half-wave potentials after 10,000 cycles of accelerated degradation tests(ADT),while there was a 30 mV negative shift for Fe-N-C.Therefore,we propose that the H₂O₂ released from Fe-Nx sites or N-doped carbon sites would be reduced by adjacent Cu-Nx sites,re sulting in low H₂O₂ yields and high stability.     

Electron transfer between cytochrome c and copper enzymes

The electron transfer between cytochrome c and ascorbate oxidase or laccase from Coriolus hirsutus was investigated using both an electrochemical and a spectrophotometric method. A quasi-reversible cyclic volammogram of cytochrome c was observed on a gold electrode modified with 4,4′-dithiodipyridine. The addition of laccase resulted in the appearance of a catalytic current due to the regeneration of ferricytochrome c by laccase in the presence of oxygen. The second-order rate constant of the reaction between cytochrome c and laccase is calculated to be 9.2 × 10³ M⁻¹ s⁻¹ in 50 mM phosphate buffer of pH 5.8. The reaction rate with ascorbate oxidase is almost three orders of magnitude slower. The difference in the redox potential is considered to be the driving force of the reaction between cytochrome c and the copper proteins investigated.     

Inhibition of mitochondrial respiratory chain by arylthiolated 2,3-ethylenedioxy-1,4-benzoquinones

A series of arylthiolated 2,3-ethylenedioxy-1,4-benzoquinones as a coenzyme Q (CoQ) antagonist was tested for inhibition of succinate oxidase and reduced nicotinamide adenine dinucleotide (NADH) oxidase systems in the mitochondrial respiratory chain. The following characteristics were revealed: (1) 2,3-ethylenedioxy, 5-arylthio and 5,6-diarylthio groups were confirmed to be favorable for inhibition of both systems; (2) these analogs were more effective in the succinate oxidase system than in the NADH oxidase system; (3) 4' substituents on the benzene side ring had little effect on inhibitory activity; (4) the acting sites of these analogs had no strict stereospecificity. The reduced minus oxidized difference spectra revealed that these analogs inhibited the succinate oxidase system at the site between succinate and CoQ, and the NADH oxidase system at the site after cytochrome a + a3, suggesting these analogs might act as antagonists of CoQ in the succinate oxidase system. However, 5-(4'-nitrophenylthio)-2,3-ethylenedioxy-1,4-benzoquinone (If) strongly inhibited only the succinate oxidase system at the site after cytochrome a + a3.     

Resonance Raman imaging of the NADPH oxidase subunit cytochrome b558 in single neutrophilic granulocytes

We have employed confocal resonance Raman (RR) imaging to visualize the subcellular distribution of the NADPH oxidase subunit cytochrome b558 in both resting and phagocytosing neutrophils. Our Raman microscopic technique is a label-free, chemical (vibrational) imaging method that can be applied to individual, intact cells, thus probing cytochrome b558 in its native environment. The Raman signal from cytochrome b558 is resonantly and selectively enhanced in neutrophils by using 413 nm excitation. Experiments on resting neutrophils show a cytoplasmic distribution of cytochrome b558, with several areas of high content. Upon phagocytosis of polystyrene particles, we found that part of the cytochrome b558 is translocated toward the ingested beads. This is in accordance with immunocytochemistry studies combined with electron and fluorescence microscopy. As compared to these methods, RR microscopy requires minimal sample preparation and disturbance. Moreover, it allows the determination of the redox state of cytochrome b558 inside the cell, which reflects its NADPH oxidase activity.