Quinone interaction with the respiratory chain-linked NADH dehydrogenase of beef heart mitochondria. II. Duroquinone reductase activity

1. Enzymatic reduction of 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone) by NADH can be used in an assay procedure for the NADH dehydrogenase. The reduction of this quinone occurs in the region of the electron transport system between the primary dehydrogenase and the cytochrome system as defined by the almost complete loss of reductase activity following piericidin A treatment.

2. Duroquinone reduction can be distinguished from ubiquinone 2 reduction by the marked inhibition of the former following phospholipase C, poly- -lysine, or chloroquine diphosphate treatment. In addition, duroquinone reduction requires the presence of endogenous ubiquinone 10 specifically whereas ubiquinone 2 reduction does not require the presence of endogenous quinone. These observations are consistent with the nonequivalency of the reduction sites of duroquinone and ubiquinone 2.

3. Duroquinol can be utilized as an electron donor for the energy-linked reduction, of NAD⁺. Duroquinol reduction of NAD⁺ is dependent upon the presence of ATP, is inhibited by oligomycin, carbonyl cyanide p-trifluoro methoxyphenylhydrazone and piericidin A, and is not inhibited by antimycin A at levels which inhibit electron transport.

4. Duroquinone reduction as well as ubiquinone 2 reduction are inhibited almost completely by phospholipase A, p-chloromercuribenzoate, o-phenanthroline, and Triton X100 treatments.     

Interaction of Mg+2 with beef heart mitochondrial ATPase (F1).

The soluble mitochondrial ATPase, F₁, can be slowly inactivated by incubation with Mg⁺² in a manner consistent with the observations of Moyle and Mitchell $\text{(}\text{FEBS}\text{Lett}\text{.}\text{56}$, 55 (1975)). This inhibition results in a low initial rate of ATP hydrolysis upon addition to an ATPase assay medium of F₁ which has been incubated with Mg⁺². This inhibition, however, is completely reversible by Mg·ATP in a time dependent process and results in the rate of ATP hydrolysis increasing during the ATPase assay to reach control levels after 30 sec. The length of the lag is independent of the F₁ concentration in the ATPase assay and the lag is also completely reversed by subsequent incubation with excess EDTA before assay.F₁ is unstable if incubated with EDTA in the absence of free nucleotides or Mg⁺². The rate of inactivation increases with decreasing protein concentration until a limiting rate is reached at high dilution. Mg⁺² in excess of the EDTA or 50 μM ADP stabilize the F₁ against the inactivation but cannot reverse prior denaturation.     

NADH/NAD+ interaction with NADH: ubiquinone oxidoreductase (complex I)

The quantitative data on the binding affinity of NADH, NAD(+), and their analogues for complex I as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. The redox-dependency of the nucleotide binding and the reductant-induced change of FMN affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of complex I.     

Role of Mg2+ in lipid-protein interaction in reconstituted procine heart mitochondrial H+-ATPase

During reconstitution of pig heart mitochondrial H+-ATPase in soybean phospholipid liposomes by the cholate dialysis method, Mg2+ greatly enhances 32Pi-ATP exchange activity, ATPase activity and the sensitivity to oligomycin of the reconstituted enzyme complex. The effect of Mg2+ on the fluidity of the reconstituted proteoliposomes was measured by means of a fluoursecent probe. 1-anilinonaphthalene ?e-8-sulfonate, and spin-label probes, 5-nitroxide stearate, 12-nitroxide stearate and 16-nitroxide stearate. A difference in fluidity seems to be localized near the polar faces of the lipid bilayers of the reconstituted proteolipsomes. Fluidity was less in the presence of Mg2+ than it is absence. The conformations of the Mg2+-containing proteoliposomes was higher. We postulate that Mg2+ may play a role in altering the fluidity of the proteoliposomes, which would favor the formation of a conformation of the reconstituted H+-ATPase with higher activity.     

Mg2+-dependent interaction of DNA with eukaryotic cells

Interaction of DNA with eukaryotic cells under conditions similar to those providing DNA adsorption onto liposomes was studied. It was revealed that mouse fibroblasts (line A9) and myeloma cells bind phage and plasmid DNA in 0.3 M sucrose solution containing Mg2+-ions. Additional pretreatment of the cells by trypsin did not affect DNA adsorption efficiency. The major part of the adsorbed DNA recovered by salt treatment of the cells, but 10-15% of DNA was found to be irreversible. Up to 50% of the irreversibly bound DNA molecules retain their linear size after treatment of cells with DNAse I. Efficiencies of DNA adsorption and irreversibly binding depend on the concentration of Mg2+ in the medium. The process of DNA irreversible binding is not inhibited by drugs affecting cell metabolism. It is assumed that DNA adsorbs onto the phospholipid domains of the cell membrane, and part of the adsorbed DNA is taken up into the interior of the cells.     

Myofibrillar Ca2+-stimulated Mg2+-ATPase from chronically ischemic canine heart

Functional properties of myofibrils from chronically ischemic canine myocardium were evaluated. Ischemia was produced by tight stenosis of left anterior descending artery (LAD), followed by 40 min acute ischemia with prior preconditioning. Animals of the first group were sacrificed after 8 weeks. In the second group, angioplasty of LAD was performed after 8 weeks of ischemia and animals were kept alive for other 4 weeks. Control animals were sham operated. Activity and kinetic parameters of myofibrillar Ca2+-stimulated Mg2+-ATPase were measured in myofibrils isolated from anterior and posterior parts of all hearts. We did not find any differences in maximal velocity (Vmax), half-maximal activation constant for calcium (K(Ca2+)50) and cooperativity coefficient (n(hill)) of myofibrils from different experimental groups as compared to controls, either at pH 7, pH 6.5 (acidosis) or pH 7.5 (alkalosis). K(Ca2+)50 increased in medium simulated acidosis (12.6-33.5 times) and n(hill) decreased significantly in all groups as compared with values obtained at pH 7. These results indicate that activity and Ca2+-sensitivity of myofibrillar Mg2+-ATPase remain unchanged despite deteriorated heart function 8 weeks after LAD obstruction. Experiments have confirmed that Ca2+-stimulated-ATPase from canine heart myofibrils responded to pH decrease by a decreased sensitivity to Ca2+ and a decreased cooperativity. However, sensitivity of the enzyme to the pH changes is unaltered by 8 weeks of chronic ischemia.     

The polypeptide composition of the mitochondrial NADH: ubiquinone reductase complex from several mammalian species.

The polypeptide composition of isolated mitochondrial NADH:ubiquinone reductase (NADH dehydrogenase) is very similar to that of material immunoprecipitated from detergent-solubilized bovine heart submitochondrial particles by antisera to the holoenzyme. The specificity of the antisera for dehydrogenase polypeptides was determined by immunoblotting, which showed that antisera reacting with only a few proteins were able to immunoprecipitate all others in parallel. The polypeptide compositions of rat, rabbit and human NADH dehydrogenase were determined by immunoprecipitation of the enzyme from solubilized submitochondrial particles and proved to be very similar to that of the bovine heart enzyme, particularly in the high-Mr region. Further homologies in these and other species were explored by immunoblotting with antisera to the holoenzyme and monospecific antisera raised against iron-sulphur-protein subunits of the enzyme.     

Strontium ranelate stimulates the activity of bone-specific alkaline phosphatase: interaction with Zn2+ and Mg2+

Strontium ranelate (SR) is an orally administered and bone-targeting anti-osteoporotic agent that increases osteoblast-mediated bone formation while decreasing osteoclastic bone resorption, and thus reduces the risk of vertebral and femoral bone fractures in postmenopausal women with osteoporosis. Osteoblastic alkaline phosphatase (ALP) is a key enzyme involved in the process of bone formation and osteoid mineralization. In this study we investigated the direct effect of strontium (SR and SrCl₂) on the activity of ALP obtained from UMR106 osteosarcoma cells, as well as its possible interactions with the divalent cations Zn²⁺ and Mg²⁺. In the presence of Mg²⁺, both SR and SrCl₂ (0.05–0.5 mM) significantly increased ALP activity (15–66 % above basal), and this was dose-dependent in the case of SR. The stimulatory effect of strontium disappeared in the absence of Mg²⁺. The cofactor Zn²⁺ also increased ALP activity (an effect that reached a plateau at 2 mM), and co-incubation of 2 mM Zn²⁺ with 0.05–0.5 mM SR showed an additive effect on ALP activity stimulation. SR induced a dose-dependent decrease in the Km of ALP (and thus an increase in affinity for its substrate) with a maximal effect at 0.1 mM. Co-incubation with 2 mM Zn²⁺ further decreased Km in all cases. These direct effects of SR on osteoblastic ALP activity could be indicating an alternative mechanism by which this compound may regulate bone matrix mineralization.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at 3000 X g followed by sedimentation of a microsomal fraction at 200 000 X g. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591--1.1083 were characterized by (Na+ + K+)-ATPase activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 micron. These fractions contained (Ca2+ + Mg2+)-ATPase which appreared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5--10 microM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell.     

Matrix Mg2+ regulates mitochondrial ATP-dependent potassium channel from heart

Mitochondrial ATP-regulated potassium (mitoKATP) channels play an important role in cardioprotection. Single channel activity was measured after reconstitution of inner mitochondrial membranes from bovine myocardium into a planar lipid bilayer. After incorporation, the potassium channel was recorded with a mean conductance of 103+/-9 pS. The channel activity was inhibited by ATP/Mg and activated by GDP. Magnesium ions alone affected, in a dose dependent manner, both the channel conductance and the open probability. Magnesium ions regulated the mitoKATP channel only when added to the trans compartment. We conclude that Mg2+ regulates the cardiac mitoKATP channel from the matrix site by affecting both the channel conductance and gating.     

Electrostatic interaction of internal Mg2+ with membrane PIP2 Seen with KCNQ K+ channels

Activity of KCNQ (Kv7) channels requires binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) from the plasma membrane. We give evidence that Mg(2+) and polyamines weaken the KCNQ channel-phospholipid interaction. Lowering internal Mg(2+) augmented inward and outward KCNQ currents symmetrically, and raising Mg(2+) reduced currents symmetrically. Polyvalent organic cations added to the pipette solution had similar effects. Their potency sequence followed the number of positive charges: putrescine (+2) < spermidine (+3) < spermine (+4) < neomycin (+6) < polylysine (>+6). The inhibitory effects of Mg(2+) were reversible with sequential whole-cell patching. Internal tetraethylammonium ion (TEA) gave classical voltage-dependent block of the pore with changes of the time course of K(+) currents. The effect of polyvalent cations was simpler, symmetric, and without changes of current time course. Overexpression of phosphatidylinositol 4-phosphate 5-kinase Igamma to accelerate synthesis of PIP(2) attenuated the sensitivity to polyvalent cations. We suggest that Mg(2+) and other polycations reduce the currents by electrostatic binding to the negative charges of PIP(2), competitively reducing the amount of free PIP(2) available for interaction with channels. The dose-response curves could be modeled by a competition model that reduces the pool of free PIP(2). This mechanism is likely to modulate many other PIP(2)-dependent ion channels and cellular processes.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Na+-independent Mg2+ efflux from Mg2+-loaded human erythrocytes

Net Mg2+ efflux from Mg2+-loaded human erythrocytes was maximal after reincubation in sucrose. Net Mg2+ efflux was not inhibited by furosemide or bumetanide and, therefore, was not performed by the (Na,K,Cl)- or (K,Cl)-cotransport system. A component of net Mg2+ efflux was inhibited by extracellular NaC1, KCl, LiCl, choline Cl and SITS, in analogy to the inhibition of net Cl- and SITS. Therefore, it was concluded that net Mg2+ efflux is dependent on net Cl- efflux for charge compensation. Cl- -dependent net Mg2+ efflux was inhibited by amiloride. Only 10% of the maximal net Mg2+ efflux may depend on extracellular Na+.     

Cu2+ and Zn2+ inhibit nitric-oxide synthase through an interaction with the reductase domain

Cu(2+) and Zn(2+) inhibit all of the NADPH-dependent reactions catalyzed by neuronal nitric-oxide synthase (nNOS) including ferricytochrome c reduction, NADPH oxidation, and citrulline formation. Cu(2+) and Zn(2+) also inhibit ferricytochrome c reduction by the independent reductase domain. Zn(2+) affects all activities of the full-length nNOS and the reductase domain to the same extent (estimated IC(50) values from 9 to 31 microm), suggesting Zn(2+) occupation of a single site in the reductase domain. Citrulline formation and NADPH oxidation by the full-length nNOS and ferricytochrome c reduction by the reductase domain are affected similarly by Cu(2+), with estimated IC(50) values ranging from 6 to 33 microm. However, Cu(2+) inhibits ferricytochrome c reduction by the full-length nNOS 2 orders of magnitude more potently, with an estimated IC(50) value of 0.12 microm. These data suggest the possibility that Cu(2+) may interact with nNOS at two sites, one composed exclusively of the reductase domain (which is perhaps also involved in Zn(2+)-mediated inhibition), and another that includes components of both domains. Occupation of the second (higher affinity) site could then promote the selective inhibition of ferricytochrome c reduction in full-length nNOS. Neither the inhibition by Cu(2+) nor that by Zn(2+) is dependent on calmodulin.