Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Affinity labeling of spinach ferredoxin-NADP+ oxidoreductase with periodate-oxidized NADP+

Periodate-oxidized NADP⁺ (dialdehyde-NADP⁺) inactivated soluble ferredoxin-NADP⁺ oxidoreductase and combined covalently to the enzyme. This inactivation was first order with respect to dialdehyde-NADP⁺ and followed saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inactivator. NADP⁺ afforded complete protection against inactivation, while spinach ferredoxin was uneffective. In the presence of exogenous ferredoxin and illuminated thylakoids, the nucleotide analog functioned as a coenzyme for the reductase, although with rather lower efficiency than NADP⁺. It also acted as a competitive inhibitor with respect to NADPH in diaphorase activity. Incorporation of radioactivity from periodate-oxidized [³H]NADP⁺ gave a stoichiometry of 0.85 mol of reagent/mol of reductase, indicating that the modification of a single residue in the flavoprotein is responsible for the loss of enzymatic activity.     

Zn2+, Mg2+, and H+ binding to d-fructose 1,6-bisphosphate studied by 31P and 1H NMR

The anomeric composition and mutarotation rates of fructose 1,6-bisphosphate were determined in the presence of 100 mm KCl at pH 7.0 by ³¹P NMR. At 23 and 37 °C the solution contains (15 ± 1)% of the α anomer. The anomeric rate constants at 37 °C are (4.2 ± 0.4) s^?1 for the β → α anomerization and (14.9 ± 0.5) s^?1 for the reverse reaction. A D₂O effect between 2.1 and 2.6 was found. From acid base titration curves it appeared that the pK values of the phosphate groups range from 5.8 to 6.0. Mg²⁺ and Zn²⁺ bind preferentially to the 1-phosphate in the α-anomeric position. Zn²⁺ has a higher affinity for this phosphate group than Mg²⁺ has. At increasing pH the fraction α anomer decreases slightly. At increasing Mg²⁺/fructose 1,6-bisphosphate ratios the fraction α anomer increases till 19% at a ratio of 20. Proton and probably Mg²⁺ binding decreases the anomerization rate. The time-averaged preferred orientation of the 1-phosphate along the C₁O₁ bond of the α conformer is strongly pH dependent, gauche rotamers being predominant at pH 9.4. In the presence of divalent cations the orientation is biased toward trans. A mechanistic model is proposed to explain the Zn²⁺, Mg²⁺, and pH-dependent behavior of the gluconeogenic enzyme fructose 1,6-bisphosphatase.     

Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca2+ homeostasis

The toxicological implications of alterations in intracellular thiol homeostasis during menadione metabolism have been investigated using freshly isolated rat hepatocytes. A strict correlation between depletion of protein sulfhydryl groups and loss of cell viability was observed. Loss of protein thiols preceded cell death, and occurred more rapidly in cells with decreased levels of reduced glutathione. Depletion of protein thiols was also associated with inhibition of Ca²⁺ efflux from the cells and perturbation of intracellular Ca²⁺ homeostasis. It is proposed that the oxidative stress induced by menadione metabolism in isolated hepatocytes results in the depletion of both soluble and protein thiols, and that the latter effect is critically associated with a perturbation of Ca²⁺ homeostasis and loss of cell viability.     

Purification and properties of NADP+:Isocitrate dehydrogenase from lactating bovine mammary gland

NADP⁺:isocitrate dehydrogenase has been purified to homogeneity from lactating bovine mammary gland. Purification was achieved through the use of affinity and DEAE-cellulose chromatography. The isolated enzyme gives one band when stained for protein or enzyme activity on discontinuous alkaline gel electrophoresis. The enzyme has a molecular weight of 55,000 as estimated by sodium dodecyl sulfate-gel electrophoresis and a Stokes radius of 4.1 nm as measured by gel chromatography. The enzyme will not use NAD⁺ in place of NADP⁺ and has an absolute requirement for divalent cations. The apparent K_m values for dl-isocitrate, Mn²⁺, and NADP⁺ were found to be 8, 6, and 11 μm, respectively. The Mn²⁺-d_s-isocitrate complex is the most likely substrate for the mammary enzyme with a K_m of 3 μm. The properties of mammary NADP⁺:isocitrate dehydrogenase are compared with those of the homologous enzymes from pig heart and bovine liver, and its characteristics are discussed with respect to the function of the enzyme in lactating mammary gland.     

Acetylglyceryl ether phosphorylcholine (AGEPC; platelet-activating factor)-induced stimulation of rabbit platelets: Correlation between phosphatidic acid level, 45Ca2+ uptake,and [3H]serotonin secretion

When ³²P_i-labeled rabbit platelets were incubated with 5 × 10^?10m 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), either in the presence or absence (0.1 mm EGTA) of added Ca²⁺, there was a three- to five-fold increase in the [³²P]phosphatidic acid (PA) pool within 15 to 20 s. This event was followed by a gradual decrease in the [³²P]PA level to near basal level in 5 min. AGEPC effected this change in [³²P]PA in a characteristic dose- and time-dependent manner. Polar head group analogs of AGEPC, such as AGEDME and AGEMME, also effected an increase in PA labeling at levels. comparable to those previously reported for their activity toward rabbit platelets [K. Satouchi, R. N. Pinckard, L. M., McManus, and D. J. Hanahan (1981) J. Biol. Chem.256, 4425–4432]. Other analogs, i.e., lysoGEPC and the enantiomer, sn-1-AGEPC, which are inactive toward rabbit platelets, also showed no effect on the level of [³²P]PA. The finding that the PA level in rabbit platelets could be manipulated by the addition of AGEPC, without any added Ca²⁺, provided an excellent model system for establishinig a correlation between the uptake of Ca²⁺, serotonin release, and PA level. Thus, PA must be regarded as a sensitive indicator of a reaction mechanism important to the platelet response to AGEPC, and could be the focal point in promoting calcium uptake during the stimulation process.     

Heparan sulfate proteoglycans of human neuroblastoma cells: Affinity fractionation on columns of platelet factor-4+

Human neuroblastoma cells (Platt) were detached from tissue culture substrata with a Ca²⁺ chelating agent, and then the suspended cells were extracted with a sodium dodecyl sulfate (SDS)-containing buffer to maximally solubilize their sulfate-radiolabeled proteoglycans. The majority of the high-molecular-weight material in these dissociative extracts was heparan sulfate proteoglycan, which resolves into two heterodisperse size classes upon gel filtration on columns of Sepharose CL4B. After removal of SDS from these extracts by hydrophobic chromatography on Sep-Pak C₁₈ cartridges, extracts were further fractionated on various affinity matrices. All of the sulfate-radiolabeled material eluted as one peak from DEAE-Sephadex ion-exchange columns. In contrast, affinity fractionation on Sepharose columns derivatized with the heparan sulfate-binding protein, platelet factor-4, resolved three major and one minor subsets of these components. The nonbinding fraction contained some heparan sulfate proteoglycan and some chondroitin sulfate. The weak-binding fraction contained principally heparan sulfate proteoglycan, as well as a small amount of chondroitin sulfate proteoglycan; the gel-filtration properties of these proteoglycans before or after alkaline borohydride treatment indicated that they were small in size, containing perhaps 2 to 4 glycosaminoglycan chains. The high-affinity fraction eluted from platelet factor 4-Sepharose was composed entirely of “singlechain” heparan sulfate. A portion of the heparan sulfate proteoglycan of the original extract bound to the hydrophobic affinity matrix, octyl-Sepharose, and this hydrophobic proteoglycan partitioned into the nonbinding and weak-binding fractions of the platelet factor 4-Sepharose affinity columns. These studies reveal that the majority of the proteoglycan made by these neuronal cells in culture is of the heparan sulfate class, is small in size when compared to other characterized proteoglycans, and can be resolved into several overlapping subsets when fractionated on affinity matrices.     

Stereochemistry of Gd3+ and Mn2+ interactions with D-gluconamide derivatives by 13C NMR spectroscopy

Natural abundance ¹³C nuclear magnetic resonance spectroscopy (¹³C NMR) was used to study the mode of binding of Gd³⁺ and Mn²⁺ to the polyol portion of several synthetic D-gluconamides. The results indicate that Gd³⁺ forms a single, unique binding structure requiring three oxygen atoms. The binding of Mn²⁺ to the polyol portion of these compounds appears to be nonspecific. The carbohydrate containing model compounds studied may be used to design new metal-ion chelating agents.     

NMR studies on UO22+ complexes with pyridoxal

The interaction of pyridoxal with dioxouranium(VI) acetate was studied by ¹H and ¹³C NMR measurements in D₂O and CD₃OD.The results indicate that the preferred bonding site is the C-3O^? donor, and the major species obtained under the experimental conditions used is the equimolar complex.     

Differential effects of Mg2+ on various hydrolytic and synthetic activities of multifunctional glucose-6-phosphatase

The adaptive synthesis of fatty acid synthetase in the livers of rats fed a fat-free diet following 48 hr of fasting has been studied using immunochemical methods. The development of fatty acid synthetase activity during adaptive synthesis occurs about 3 hr following feeding, whereas the synthesis of material precipitable by anti-fatty acid synthetase serum, as judged by the incorporation of ³H-labeled amino acids into the immunoprecipitate, commenced within 1 hr. Extracts of liver of rats fed a fat-free diet for 1–3 hr following fasting contain increasing amounts of material which competes with purified fatty acid synthetase for antibody binding sites, even though they have no fatty acid synthetase activity. This suggests the presence of enzymatically inactive precursors of fatty acid synthetase in the liver extracts. The incorporation of [¹⁴C]pantothenate into fatty acid synthetase during adaptive synthesis follows the same pattern as the development of enzyme activity, indicating that these enzymatically inactive precursors of fatty acid synthetase may represent an apoenzyme which is converted to the enzymatically active holoenzyme by the incorporation of the 4′-phosphopantetheine prosthetic group. The subcellular site of synthesis of fatty acid synthetase was shown to be in the pool of polysomes that are not membrane bound, rather than in the rough endoplasmic reticulum.     

Inhibition of the (Na+ + K+)-dependent ATPase by inorganic phosphate

Inhibition of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase by inorganic phosphate, P_i, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na⁺,K⁺-pump that these activities reflect. The K⁺-dependent phosphatase activity of the enzyme was most sensitive to P_i, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if P_i were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K⁺ may not be involved. The Na⁺-dependent exchange activity was unaffected by P_i, in accord with the absence of P_i release in the reaction sequence. For the corresponding Na⁺/Na⁺ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of P_i obviously cannot be required for Na⁺ discharge. With the Na⁺-dependent ATPase activity, measured using micromolar concentrations of ATP, P_i inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na⁺ concentration was raised from 10 to 100 mM. This elevated concentration of Na⁺ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na⁺ efflux, the findings suggest that Na⁺ discharge follows P_i release, in contrast to Na⁺/Na⁺ exchange. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na⁺,K⁺-transport function, was similarly sensitive to P_i, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na⁺ concentration was lowered. For this activity, and the associated transport process, the site of Na⁺ discharge in the overall reaction sequence remains unresolved.     

Determination of the mode of interaction of Mn2+ and Cu2+ with cis-inositol by 13C NMR spectroscopy

Natural abundance ¹³C nuclear magnetic resonance spectroscopy (¹³C NMR) was used to study the mode of binding of Mn²⁺ and Cu²⁺ to the cyclitol, cis-inositol. Resonance linewidths and the electron nuclear relaxation rates [(T₁^e)^?1 values] were used to establish that a unique binding site exists for these metal-ions on this cyclitol involving only the three axial hydroxyl groups. This work may aid in the development of new organometallic complexes used as paramagnetic relaxation agents in magnetic resonance imaging research.     

3H-cimetidine and the H2-receptor

The H₂-antagonist cimetidine is widely employed in biochemical and pharmacological studies of the H₂-receptor. These studies include the use of ³H-cimetidine in radioligand binding experiments. Confirming our previous finding as to the unsuitability of this ligand in these types of investigations, we now report data showing the lack of correlation between the displacement of specific ³H-cimetidine binding and histamine stimulated adenylate cyclase activity, and the displacement of specific binding by imidazoles devoid of H₂-receptor activity. Results are also presented which question the use of copper ions in ³H-cimetidine binding studies. Our conclusions are discussed in relation to the work carried out by a number of laboratories where ³H-cimetidine is reported to label the H₂-receptor.     

Spreading of biomembranes at the air/water interface1

This paper presents the compression isotherms obtained by spreading membranes of intestinal brush border, human erythrocyte and Escherichia coli (cytoplasmic) at the air/water interface. Unilamellar membrane films were formed, with a good yield, at zero surface pressure, whereas multilamellar structures were formed at high surface pressure. Once formed, the films were particularly stable and could be manipulated without any detectable loss. With doubly-labelled E. coli cytoplasmic membrane, we could show that phospholipids and proteins spread, with the same yield, as a single unit. Moreover, we studied the influence of hydrolytic enzymes, chemical agents and cations on the compression isotherm of biomembranes. The resultant change sin architecture of membrane films can provide a very simple method of studying the influence of membrane packing on catalytic activity and protein conformation of membrane-bound proteins.     

A general characteristic of tumor mitochondria: Leakage of endogenous Mg2+ on incubation with uncoupler and resultant reduction of uncoupler-stimulated ATPase activity

Previous studies showed that stimulation of mouse mitochondrial ATPase activity of tumor cells, fetal liver, and adult brain by the uncoupler 2,4-dinitrophenol was markedly suppressed during incubation of the mitochondria with the uncoupler (J.-I. Hayashi et al., 1980, Biochem. Biophys. Res. Commun.92, 261–267). The present work showed the reason for this suppression. More than half the endogenous Mg²⁺ leaked from mitochondria of all tumor cells tested, and of fetal liver and adult brain during incubation with the uncoupler, while only about 30% of the endogenous Mg²⁺ leaked from mitochondria of other normal tissues. The effect of the uncoupler on Mg²⁺ leakage from liver mitochondria changed from the fetal to the adult type within about 30 min after birth. In hypotonic medium, normal liver mitochondria also lost more than half their total Mg²⁺ and concomitantly stimulation of their ATPase activity by uncoupler was considerably reduced. Exogenously added Mg²⁺ could reverse this reduced effect of the uncoupler on ATPase activity of mitochondria from normal tissues and tumor cells. These results show that the endogenous Mg²⁺ content of mitochondria directly affects the stimulation by uncoupler of ATPase activity of mitochondria from both normal tissues and tumor cells. Thus, mitochondria of all tumor cells tested, and of fetal liver and adult brain are leaky to Mg²⁺ during incubation with uncoupler and as a result of the leakage, the stimulatory effect of the uncoupler on their ATPase activity is greatly reduced.     

Continuous fluorometric assay of phospholipase A2 with pyrene-labeled lecithin as a substrate

1,2-Bis[4-(1-pyreno)butanoyl]-sn-glycero-3-phosphorylcholine was synthesized as a fluorogenic substrate for phospholipase A₂. It has a critical micellar concentration of 7.3 μm and gives only excimer fluorescent emission at 480 nm in aqueous micellar dispersion. When hydrolyzed by phospholipase A₂, the products give only monomer emission which is monitored best at 382 and 400 nm. Conditions were developed for an assay for phospholipase A₂ using this substrate. The assay was sensitive to as little as 8 ng of pure porcine pancreatic phospholipase A₂.