Adenosine triphosphatase localization in the branchial heart of Sepia officinalis L. (Cephalopoda)

Summary Sodium- and potassium-dependent adenosine triphosphatase (Na⁺–K⁺-ATPase) is demonstrated in the branchial heart of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na⁺–K⁺-ATPase, shown by potassium and magnesium dependency and inhibition by ouabain. Cytochemically and autoradiographically, the enzyme is localized in the sarcolemma of the muscle cells. The positive reaction of the transparent cells (type I cells) is due to activity of alkaline phosphatases. The dark cells (type II cells) react negatively. In addition to the Na⁺–K⁺-ATPase, a magnesium-activated adenosine triphosphatase (Mg²⁺-ATPase) and a bicarbonate-stimulated ATPase (HCO ₃ ^- -ATPase) are localized in the mitochondria.This study was supported by the Deutsche Forschungsgemeinschaft and is part of the doctoral dissertation     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by trypsin digestion

Summary To distinguish ligand-induced structural states of the (Na⁺–K⁺)-ATPase, the purified membrane-bound enzyme isolated from rat kidneys was digested with trypsin in the presence of various combinations of Na⁺, K⁺, Mg⁺⁺ and ATP. It was found that first the large and then the small polypeptide chain of the (Na⁺–K⁺)-ATPase was degraded, indicating that the lysine and arginine residues of the large chain are more exposed than are those of the small one. The (Na⁺–K⁺)-ATPase activity was inactivated in parallel with the degradation of the large polypeptide chain. After the degradation of the large polypeptide chain, about 75% of the (Na⁺–K⁺)-ATPase protein remained bound to the membrane, demonstrating that the split protein segments were only partially released.It was found that the combinations of ATP, Mg⁺⁺, Na⁺ and K⁺ present during trypsin digestion influenced the time course and degree of degradation of the (Na⁺–K⁺)-ATPase protein. The degradations of the large and the small polypeptide chain were affected in parallel. Thus, certain ATP and ligand combinations influenced neither the degradation of the large nor the degradation of the small polypeptide chain, whereas by other combinations of ATP and ligands the degree of susceptibility of both polypeptide chains to trypsin was equally increased or reduced.In the absence of ATP the time course of trypsin digestion of the (Na⁺–K⁺)-ATPase was the same, whether Na⁺ or K⁺ was present. With low ATP concentrations (e.g., 0.1mm), however, binding of Na⁺ or K⁺ led to different degradation patterns of the enzyme. If a high concentration of ATP (e.g., 10mm) was present, Na⁺ and K⁺ also influenced the degradation pattern of the (Na⁺–K⁺)-ATPase, but differentially compared to that at low ATP concentrations, since the effects of Na⁺ and K⁺ were reversed. Furthermore, it was found that the degradation of the small chain was only influenced by certain combinations of ATP, Mg⁺⁺, Na⁺ and K⁺ if the large chain was intact when the ligands were added to the enzyme.The described results demonstrate structural alterations of the (Na⁺–K⁺)-ATPase complex which are supposed to include a synchronous protrusion or retraction of both (Na⁺–K⁺)-ATPase subunits. The data further suggest that ATP and other ligands primarily alter the structure of the large (Na⁺–K⁺)-ATPase subunit. This structural alteration is presumed to lead to a synchronous movement of the small subunit of the enzyme. The structural state of the (Na⁺–K⁺)-ATPase is regulated by binding of Na⁺ or K⁺ to the enzyme-ATP complex. The effects of Na⁺ and K⁺ on the (Na⁺–K⁺)-ATPase structure are modulated by the ATP binding to high affinity and to low affinity ATP binding sites.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Summary As different structural states of the (Na⁺–K⁺)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na⁺, K⁺, Mg⁺⁺, P_i and ATP on the reaction between highly purified (Na⁺–K⁺)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na⁺–K⁺)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na⁺, K⁺, Mg⁺⁺ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg⁺⁺, (ii) Mg⁺⁺ and Na⁺ or (iii) Mg⁺⁺ and K⁺ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na⁺, Mg⁺⁺ and ATP were present together. (c) It was inhibited even more if only (i) Na⁺, (ii) K⁺, (iii) ATP or both (iv) ATP and Na⁺, (v) ATP and K⁺, (vi) ATP and Mg⁺⁺, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg⁺⁺, ATP and K⁺ were present together.In the presence of Mg⁺⁺ plus ADP and in the presence of Mg⁺⁺ plus the ATP analog adenylyl (--methylene) diphosphonate, Na⁺ and K⁺ did not influence the degree of antibody inhibition as they did in the presence of Mg⁺⁺ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg⁺⁺, ATP and K⁺ was affected by the sequence in which K⁺ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na⁺–K⁺)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na⁺ and K⁺ in the presence of Mg⁺⁺ plus ATP are proposed to occur during the Na⁺–K⁺ transport.     

Amino group modification of (Na++K+)-ATPase

The effects of three amino group reagents on the activity of (Na⁺+K⁺)-ATPase³ and its component K⁺-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na⁺ of Mg²⁺ present, the enzyme inactivation process follows complicated kinetics. In the presence of K⁺, Rb⁺, or Tl⁺, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg²⁺ (P ₅₀ 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ and K⁺ have no effects, and ATP only a minor effect, on the degree of modification. The K⁺-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na⁺+K⁺)-ATPase activity. Half-inhibition of the (Na⁺+K⁺)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ has no effect, and ATP has only a slight protecting effect. The K⁺-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na⁺+K⁺)-ATPase activity. Half-inactivation of the (Na⁺+K⁺)-ATPase activity is obtained after 20% modification of the amino groups.This article is No. 52 in the series Studies on (Na⁺+K⁺)-Activated ATPase.     

Reduction of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to chemically induced hyperhomocysteinemia

Hyperhomocysteinemia occurs in homocystinuria, an inherited metabolic disease clinically characterized by thromboembolic episodes and a variable degree of neurological dysfunction whose pathophysiology is poorly known. In this study, we induced elevated levels of homocysteine (Hcy) in blood (500 M), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 mol/g of body weight) twice a day at 8-hr intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the hippocampus of treated Hcy- and saline-treated rats. Chronic administration of Hcy significantly decreased (40%) Na⁺,K⁺-ATPase activity but did not alter Mg²⁺-ATPase activity. Considering that Na⁺,K⁺-ATPase plays a crucial role in the central nervous system, our results suggest that the brain dysfunction found in homocystinuria may be related to the reduction of brain Na⁺,K⁺-ATPase activity.     

Adenosine triphosphatase localization in the branchial heart appendage of Sepia officinalis L. (Cephalopoda)

Summary Sodium- and potassium-dependent adenosine triphosphatase (Na⁺–K⁺-ATPase) has been demonstrated in the branchial heart appendage (pericardial gland) of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na⁺–K⁺-ATPase, judging from the potassium dependency and, with some restrictions, the inhibition by ouabain. Cytochemically and autoradiographically, the enzyme could be localized on the cytoplasmic surfaces of the lateral plasma membranes and the basal membrane infoldings (basal labyrinth) of the folded epithelium of the branchial heart appendage. The pdocytes of the peripheral zone of the organ reacted negatively. In addition to the Na⁺–K⁺-ATPase, a magnesium-activated adenosine triphosphatase (Mg²⁺-ATPase) was demonstrated in the folded epithelium, localized mainly in the mitochondria but also at the brush border and in the apical intercellular space, whereas a bicarbonate-stimulated ATPase (HCO ₃ ^– -ATPase) was present only in the mitochondria.This study was supported by the Deutsche Forschungsgemeinschaft     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.     

Inhibition of ATPases Enzyme Activities on Brain Disturbing Normal Oestrous Cycle

Wistar rats treated with -methyl- DL-p -tyrosine methylester showed significant level of inhibition in the activity of Na⁺, K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase enzymes in different regions of the brain. The enzyme activity was assayed in cerebral hemispheres, hypothalamus, thalamus, hippocampus, amygdala and septum at proestrous (12 h), estrous (25 h), metestrous (38 h) and diestrous periods (92 h) of the rat. The Na⁺, K⁺-ATPase activity was significantly inhibited in most of the brain regions after treated with -methyl- DL-p -tyrosine methylester (MPT) and this indicated that MPT affected the active transport system and nerve impulse transmission. Mg²⁺-ATPase and Ca²⁺-ATPase was also significantly (P < 0.001) reduced in different regions of the brain. The results revealed that MPT affected active transport system and nerve impulse transmission by inhibiting Na⁺, K⁺-ATPase and Ca²⁺-ATPase. It has induced energy crisis by inhibiting Mg²⁺-ATPase and all these cumulative effects of MPT have adversely affected the female Wistar rats. These effects have been manifested in the form of aberrations in the behavior of MPT treated female rats, which have shown their inability to perform their normal sexual activity.     

Mg2+-dependent (Na+ + K+)- and Na+-ATPases in the kidneys of the gilthead bream (Sparus auratus L.)

• 1.1. The (Na⁺ + K⁺)- and Na⁺-ATPases, both present in kidney microsomes of Sparus auratus L., have different activities and optimal assay conditions as, in the first of the two stocks of fish used (A), the spec. act. of the former is 51.7 μmol P_i mg prot⁻¹ hr⁻¹ at pH 7.5, 100 mM Na⁺, 10 mM K⁺, 17.5 mM Mg²⁺, 7.5 mM ATP and that of the latter is 6.5 μmol P_i mg prot⁻¹ hr⁻¹ at pH 6.5, 40 mM Na⁺, 4.0 mM Mg²⁺, 2.5 mM ATP.
• 2.2. Ouabain and vanadate specifically inhibit the (Na⁺ + K⁺)-ATPase but not the Na⁺-ATPase that is preferentially inhibited by ethacrynic acid.
• 3.3. While the (Na⁺ + K⁺)-ATPase is strictly specific for ATP and Na⁺, Na⁺-ATPase can be activated by various monovalent cations and, apart from ATP, hydrolyses CTP, though less efficiently.
• 4.4. The second stock B, subjected to higher salinity than A, shows an acidic shifted Na⁺-ATPase optimal pH, opposed to the stability of that of the (Na⁺ + K⁺)-ATPase, a decreased (Na⁺ + K⁺)-ATPase and a strikingly depressed Na⁺-ATPase.
• 5.5. The results are compared with literature data and discussed on the basis of the presumptive different roles as well as functional prevalence in various salinities of the two ATPases.

     

Effects of phenylalanine and its deaminated metabolites on Na+, K+-ATPase activity in synaptosomes from rat brain

The effects of phenylalanine (PHE) and its deaminated metabolites phenylpyruvate (PHP), phenyllactate (PHL) and phenylacetate (PHA) on sodium and potassium activated adenosinetriphosphatase (Na⁺, K⁺-ATPase) in synaptosomes from rat brain were investigated. At very low concentrations (5–10 M), PHE, PHL and PHA inhibited the activity, while PHP stimulated the activity. At intermediate concentrations (50–100 M), all compounds had no effect, but at higher (0.5–1.0 mM) concentrations they inhibited the enzyme activity. Thus all the compounds tested showed a biphasic effect on the enzyme activity. Hydroxylamine inhibited the Na⁺, K⁺-ATPase activity when present alone; simultaneous addition of hydroxylamine and PHE, however, eliminated the inhibitory effects of each other. Reversal of mutual inhibition also occurred in the presence of hydroxylamine and very low (5–10 M) concentrations of PHL or PHA. The inhibitory effects of PHE at all concentrations, and of PHL or PHA at low concentrations, were also eliminated in the presence of EGTA. The data indicate that inhibition of brain membrane Na⁺, K⁺-ATPase by PHE and by low concentrations of PHL and PHA may involve metal ions, but that the inhibition by high concentrations of these metabolites must occur by a different mechanism. Since Na⁺, K⁺-ATPase plays a central role in neuronal function, and the presence of excess PHE and its deaminated metabolites occurs in brain tissue under conditions of experimentally induced hyperphenylalaninemia and genetic phenylketonuria, the neurologic impairment in experimental and genetic PKU may in part be related to the deleterious effects of these compounds on brain ATPase.     

The effects of nitric oxide synthesis on the Na+,K+-ATPase activity in guinea pig kidney exposed to lipopolysaccharides

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. LPS triggers the synthesis and release of cytokines and the vasodilatör nitric oxide (NO). A major contributor to the increase in NO production is LPS-stimulated expression of inducible nitric oxide synthase (iNOS). This occurs in vasculature and most organs including the kidney. During endotoxemia, NO and superoxide react spontaneously to form the potent and versatile oxidant peroxynitrite (ONOO^–) and the formation of 3-nitrotyrosine (nTyr)-protein adducts is a reliable biomarker of ONOO^– generation. Therefore, the present study was aimed at investigating the role of endogenous nitric oxide in regulating Na⁺,K⁺-ATPase activity in the kidney, and at investigating the possible contribution of reactive nitrogen species (RNS) by measuring of iNOS activity. In addition, the present study was aimed at investigating the relationship between nTyr formation with iNOS and Na⁺,K⁺-ATPase activities. Previously in our study, nTyr was not detectable in kidney of normal control animals but was detected markedly in LPS exposed animals. In this study, kidney Na⁺,K⁺-ATPase activity were maximally inhibited 6 h after LPS injection (P:0.000) and LPS treatment significantly increased iNOS activity of kidney (P:0.000). The regression analysis revealed a very close correlation between Na⁺,K⁺-ATPase activity and nTyr levels of LPS treated animals (r = –0.868, P = 0.001). Na⁺,K⁺-ATPase activity were also negatively correlated with iNOS activity (r = –0.877, P = 0.001) in inflamed kidney. These data suggest that NO and ONOO^– contribute to the development of oxidant injury. Furthermore, the source of NO may be iNOS. iNOS are expressed by the kidney, and their activity may increase following LPS administration. In addition, NO and ONOO^– formation inhibited Na⁺,K⁺-ATPase activity. This results also have strongly suggested that bacterial LPS disturbs activity of membrane Na⁺,K⁺-ATPase that may be an important component leading to the pathological consequences such as renal dysfunction in which the production of RNS are increased as in the case of LPS challenge. (Mol Cell Biochem 271: 107–112, 2005)     

The role of monovalent cations for photosynthesis of isolated intact chloroplasts

The role of monovalent cations in the photosynthesis of isolated intact spinach chloroplasts was investigated. When intact chloroplasts were assayed in a medium containing only low concentrations of mono- and divalent cations (about 3 mval l^-1), CO₂-fixation was strongly inhibited although the intactness of chloroplasts remained unchanged. Addition of K⁺, Rb⁺, or Na⁺ (50–100 mM) fully restored photosynthesis. Both the degree of inhibition and restoration varied with the plant material and the storage time of the chloroplasts in low-salt medium. In most experiments the various monovalent cations showed a different effectiveness in restoring photosynthesis of low-salt chloroplasts (K⁺>Rb⁺>Na⁺). Of the divalent cations tested, Mg²⁺ also restored photosynthesis, but to a lesser extent than the monovalent cations.In contrast to CO₂-fixation, reduction of 3-phosphoglycerate was not ihibited under low-salt conditions. In the dark, CO₂-fixation of lysed chloroplasts supplied with ATP, NADPH, and 3-phosphoglycerate strictly required the presence of Mg²⁺ but was independent of monovalent cations. This finding excludes a direct inactivation of Calvin cycle enzymes as a possible basis for the inhibition of photosynthesis under low-salt conditions.Light-induced alkalization of the stroma and an increase in the concentration of freely exchangeable Mg²⁺ in the stroma, which can be observed in normal chloroplasts, did not occur under low-salt conditions but were strongly enhanced after addition of monovalent cations (50–100 mM) or Mg²⁺ (20–50 mM).The relevance of a light-triggered K⁺/H⁺ exchange at the chloroplast envelope is discussed with regard to the light-induced increase in the pH and the Mg²⁺ concentration in the stroma, which are thought to be obligatory for light activation of Calvincycle enzymes.     

Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Salinity-dependence of the properties of gill (Na++K+-ATPase in rainbow trout Oncorhynchus mykiss

• 1.1. The expected higher gill (Na⁺+K⁺)-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected
• 2.2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg²⁺, 50 mM Na⁺, 2.5 mM K⁺, pH 7.0 in FW, and 3 mM ATP, 10 mM Mg²⁺, 100 mM Na⁺, 10 mM K⁺, pH 7.5 in BW.
• 3.3. The change of the enzyme activation kinetics by Mg²⁺, ATP, Na⁺ and K⁺ from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity.
• 4.4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.

     

ATPases of Synaptic Plasma Membranes from Hippocampus After Ischemia and Recovery During Ageing

Plasticity and relationships between individual ATPases linked to energy-utilizing systems of hippocampus, a very sensitive functional area to both age and ischemia, were studied during ageing on synaptic plasma membranes of 1-year-old adult and 2-year-old aged rats after 15 min of complete cerebral ischemia and different reperfusion times (01, 24, 48, 72, and 96 h). Activities of Na⁺, K⁺, Mg²⁺-ATPase, Mg²⁺-ATPase ouabain insensitive, Na⁺,K⁺-ATPase, direct or basal Mg²⁺-ATPase, and acetylcholinesterase (AChE) were evaluated in synaptic plasma membranes, where they play the major role in the regulation of presynaptic nerve ending homeostasis. This in vivo study of recovery time-course from 15 mins of cerebral ischemia indicated specific biochemical assessments of functional meaning: (a) Na⁺K⁺-ATPase of synaptic plasma membranes in adult and aged animals is stimulated by ischemia; (b) this hyperactivity is more markedly related to adult than to aged animals; (c) these abnormalities still persist after 72 and 96 h during the recirculation times, indicating the delayed postischemic suffering of the brain; (d) specific Mg²⁺-ATPase enzyme system possess a lower catalytic power in aged animals than in adult ones, but remained unaltered in adult animals by ischemia and reperfusion; (e) Mg²⁺-ATPase is stimulated in aged animals by ischemia, further increasing during reperfusion up to 72–96 h, indicating the delayed hyperactivity of hippocampus; (f) the increased metabolic activity of hippocampus is indicated by the increased activity of cholinergic system; (g) integrity of synaptic plasma membranes seems not to be altered by 15 min ischemia to a critical extent to compromise their catalytic functionality during reperfusion; (h) AChE activity increases in both adult and aged at some survival times. There are logical reasons for the hypothesis that the modifications in ATPase's catalytic activities in synaptic plasma membranes, which have been modified by ischemia in presynaptic terminals, may play important functional role during recovery time in cerebral tissue in vivo, especially as regards its responsiveness to noxious stimuli, particularly during the recirculation period from acute (or chronic) brain injury.     

Erythrocyte Membrane Digoxin-Sensitive (Na+–K+)-ATPase of Non-Insulin Dependent Diabetic Humans

Erythrocyte plasma membranes of non-insulin dependent diabetic humans (NIDDM) and healthy humans were prepared by hypotonic lysis. The specific activity of (Na⁺–K⁺)-ATPase of NIDDM membranes, both in the absence and presence of digoxin were lower than the specific activity of normal enzymes (83.6 percent and 74.0 percent of the normal enzyme respectively). Addition of digoxin decreased the activity of this enzyme (38.0 percent in NIDDM and 30.0 percent in normal enzyme).Although the affinity of the pump for ATP was similar in both membranes of NIDDM and normal humans (K_m for ATP=19.9±0.24M ATP and 20.0±0.21 M ATP respectively), the V_max of NIDDM membranes was more than 20 percent lower than that of the normal enzyme. The specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺–Mg²⁺)-ATPase) of NIDDM membrane was lower than 80 percent of the specific activity of the normal enzymes. While the affinity of the pump for ATP was lower in the membranes of NIDDM (K_m for ATP=50.0±4.3 M ATP) in comparison to normal membranes (K_m for ATP=63.1±38M ATP), the V_max of NIDDM membranes was similar to the normal enzyme. Altogether, these findings suggest that both the (Na⁺–K⁺)-ATPase and Ca²⁺-pumping ATPase of NIDDM membranes are less functional than the enzymes in normal erythrocytes.     

Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP,K+, Na+, and strophanthidin in vitro

The effect of lipid peroxidation on the affinity of specific active sites of Na⁺, K⁺-ATPase for ATP (substrate), K⁺ and Na⁺ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100M ascorbate and 25M FeCl₂ at 37°C for time intervals from 0–20 min. The level of thiobarbituric acid reactive substances and the activity of Na⁺, K⁺-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0±3.8 to 8.8±0.9 mol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2±0.1 to 16.5 ±1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K⁺ and Na⁺ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na⁺, K⁺-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K⁺ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na⁺, K⁺-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.     

Inhibition of red cell Ca-ATPase by vanadate

1. 1. The Mg²⁺- plus Ca²⁺-dependent ATPase (Ca²⁺-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.

2. 2. Several natural activators of Ca²⁺-ATPase (Mg²⁺, K⁺, Na⁺ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.

3. 3. Among the ligands tested, K⁺, in combination with Mg²⁺, had the most pronounced effect on inhibition by vanadate.

4. 4. Under conditions optimal for inhibition of Ca²⁺-ATPase, the K for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.

5. 5. There are similarities between the kinetics of inhibition of red cell Ca²⁺-ATPase and (Na⁺ + K⁺)-ATPase prepared from a variety of sources; however, (Na⁺ + K⁺)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.