Unique uncoupler-stimulation pattern of mitochondrial ATPase activity of tumor cells, brain, and fetal liver

Mitochondria were prepared from various kinds of normal tissues and tumor cells of mice, and their ATPase activities were measured in the presence of an uncoupler. The ATPase activities of all mitochondria were stimulated by the uncoupler when it was added to the mitochondrial suspension just before or after addition of ATP. However, when mitochondria were preincubated with the uncoupler for four minutes or more before the addition of ATP, its stimulating effect on mitochondrial ATPase activities was greatly reduced in all tumor cells tested, but not in normal adult liver. Reduction of the stimulating effect of the uncoupler by preincubation with it was also observed with mitochondrial ATPase of brain and fetal liver. Thus this pattern of change in the effect of uncoupler on preincubation may be common to tumor mitochondria, but it is not specific to tumor mitochondria. The pattern of uncoupler stimulation observed in fetal liver changed rapidly to that of adult liver immediately after birth. Thus the difference between the two uncoupler stimulation patterns is probably not due to a difference in molecular species of mitochondrial ATPase.     

Effects of fluorescamine labeling on mitochondrial ATPase activity.

Fluorescamine labeling of rat liver mitochondria enhances the ATPase activity. It reached maximum stimulation when mitochondria were treated with 30–34 nmol fluorescamine per mg of mitochondrial protein. This stimulation is inhibited by N,N′-dicyclohexylcarbodiimide. The maximum stimulation caused by labeling is the same as that obtained from uncoupler with optimum concentration. The chemiosmotic potential (Δμ_H⁺) decreases as the labeling increased. However, Δμ_H⁺ is not abolished completely even when ATPase activity reaches a maximum. The results suggest that primary amino groups may be involved in controlling mitochondrial ATPase activity.     

A proline shuttle in insect flight muscle.

A proline shuttle for oxidation of extramitochondrial NADH was reconstituted from soluble and mitochondrial fractions of blowfly ($\text{Phormia}$$\text{regina}$) flight muscle. The soluble fraction catalyzed reduction of Δ′-pyrroline-5-carboxylate to proline via the action of Δ′-pyrroline-5-carboxylate reductase (EC 1.5.1.2). The reaction required NADH as hydrogen donor, NAD (P) H being ineffective in this regard. Mitochondria catalyzed regeneration of Δ′-pyrroline-5-carboxylate from proline via action of proline oxidase. The capacity of the shuttle to operate under conditions of possible competition for Δ′-pyrroline-5-carboxylate between Δ′-pyrroline-5-carboxylate reductase and Δ′-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was incestigated. Results of these investigations indicate that dehydrogenase activity does not significantly interfere with shuttle activity.     

Reconstitution of succinate-Q reductase.

Reconstitution of succinate-Q reductase is achieved by admixing soluble succinate dehydrogenase (SDH) and ubiquinone-protein-S (QP-S), a new protein isolated from the soluble cytochrome $\text{b-c}{}_1$ complex. The reconstituted reductase catalyzes reduction of Q by succinate. The reaction is fully sensitive to thenoyltrifluoroacetone. The reconstituted reductase (same as succinate-cytochrome $\text{c}$ reductase or submitochondrial particles) does not show “low concentration ferricyanide reductase activity” as soluble dehydrogenase does. In other words, this enzymic site on SDH is occupied by QP-S. When an artificial dye, such as phenazine methosulfate or Wurster's Blue, is used as electron acceptor the rate of oxidation of succinate by SDH is not significantly changed regardless of whether the dehydrogenase is in the free or in the reconstituted succinate-Q reductase forms.     

The effect of Ca2+ on the oxidation of exogenous NADH by rat liver mitochondria.

The respiratory rate of rat liver mitochondria in the presence of NADH as exogenous substrate is enhanced by the addition of CaCl₂ (> 50 μM) when inorganic phosphate is present in the medium. The Ca-induced oxidation of NADH is inhibited by rotenone but is not affected by uncoupling agents. EDTA, which does not reverse the swelling of mitochondria which occurs in the presence of Ca²⁺ and phosphate, is able to inhibit reversibly the Ca-stimulated NADH oxidation. A stimulation of the rate of oxidation of NADH by Ca²⁺ is also observed in mitochondria partially swollen in a hypotonic medium.     

Distribution of gangliosides in parenchymal and non-parenchymal cells of rat liver

Parenchymal and non-parenchymal cells were isolated from rat liver with purities of more than 90%. Total and ganglioside sialic acid contents were higher in non-parenchymal cells than in parenchymal cells. Thin-layer chromatography of gangliosides showed that the main component in rat liver was ganglioside GM3 and that this was abundant in non-parenchymal cells. Parenchymal cells had ganglioside GD1b as the main component and less GM3 than non-parenchymal cells. These results suggested that the main ganglioside of rat liver, GM3, arises mainly from non-parenchymal cells.     

Rapid isolation of pancreatic islets from collagenase digested pancreas by sedimentation through Percol at unit gravity.

A Percoll solution with a density of 1.045 g/ml was used to separate pancreatic islets and exocrine tissue from collagenase-digested human and $\text{ob/ob}$ mouse pancreases by sedimentation at unit gravity. Most exocrine tissue from the mouse was found to range in density from 1.015 to 1.045 g/ml whereas the denser islets lay in a narrower range of 1.065–1.070 g/ml. Up to 400 islets were obtained from each mouse pancreas and 140 islets from 4 g of human pancreas; the isolated islets being essentially free from contamination with exocrine tissue. Glucose-stimulated insulin release was the same whether the mouse islets were isolated with or without Percoll. The simplicity of the method makes it suitable for large-scale islet isolation, a feature of potential importance for the treatment of diabetes by islet transplantation.     

Guanosine 3',5'-monophosphate and the action of alkylating agents.

The intracellular level of guanosine 3',5'-monophosphate (cGMP) has been measured in Walker carcinoma cells in tissue culture after treatment with various alkylating agents. At concentrations which caused a rise in the level of adenosine 3',5'-monophosphate (cAMP) chlorambucil and 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) produced only a small (35%) elevation of cGMP, while merophan had no such effect. This suggests that any effect of cAMP will not be outweighed by an equivalent rise in cGMP. Sepcific cytosolic binding of cGMP decreased with increasing resistance of Walker cells to alkylating agents, while the dissociation constant, KD, for binding increased. This was also observed with cAMP binding which suggests that the same protein in responsible for binding both nucleotides.     

Regulation of ketone body production from (14C)palmitate in rat liver mitochondria: effects of cyclic nucleotides and unlabeled fatty acids.

N⁶′, O²′-dibutyryl adenosine 3′, 5′-cyclic monophosphoric acid, but not other cyclic nucleotides stimulates [¹⁴C]ketone body production from [¹⁴C]palmitate in isolated rat liver mitochondria. Butyrate alone, as well as unlabeled acetate, octanoate and palmitate had similar effects. This redistribution of the oxidative products of [¹⁴C]palmitate can best be explained by exceeding the capacity of the Krebs cycle and/or changes in the acetyl coenzyme A/coenzyme A ratio. In contrast to [¹⁴C]palmitate, [¹⁴C]octanoate oxidation to [¹⁴C]O₂ and [¹⁴C]ketone bodies was inhibited by the addition of unlabeled fatty acids. This suggests that an additional mechanism by which unlabeled fatty acids may stimulate [¹⁴C]ketone body production is by enhancing the carnitine-dependent transport of [¹⁴C]palmitate into mitochondria.     

Inhibition of ruthenium red-induced Ca2+ efflux from liver mitochondria by the antibiotic X-537A

It has been reported (Becker, G.L., Fiskum, G. and Lehninger, A.L. (1980) J. Biol. Chem. 255, 9009-9012) that respiring rat liver mitochondria suspended in KC1 medium containing ATP, Mg2+ and phosphate, maintain a steady state extramitochondrial free Ca2+ concentration of about 0.5 microM. The results reported here show that the addition of the antibiotic X-537A, at concentrations far below those required for ionophorous activity, caused a perturbation in this steady state, lowering the extramitochondrial free Ca2+ concentration by about 0.20 microM. This shift in steady state was clarified by a study of X-537A inhibition of the Ca2+ efflux induced by ruthenium red; a half-maximum effect was observed at approximately 25 nM X-537A. No effect on Ca2+ transport through the influx uniporter was observed. The possibility of a generalized stabilizing action of the antibiotic on the mitochondrial membrane seems to be ruled out by its effectiveness at very low concentrations.     

Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli.

The three major subunits (α, β and γ) of the coupling factor, F₁ ATPase, of $\text{Escherichia coli}$ were separated and purified by hydrophobic column chromatography after the enzyme was dissociated by cold inactivation. The ability to hydrolyze ATP was reconstituted by dialyzing the mixture of subunits against 0.05 M Tris-succinate, pH 6.0, containing 2 mM ATP and 2 mM MgCl₂. A mixture containing α, β and γ regained ATP hydrolyzing activity. Individual subunits alone or mixtures of any two subunits did not develop ATPase activity, except for a low but significant activity with α plus β. The reconstituted ATPase had a K_m of 0.23 mM for ATP and a molecular weight by sucrose gradient density centrifugation of about 280,000.     

On the activity of ribulosediphosphate carboxylase with CO2 and O2 from leaf extracts of Zea mays

Ribulosediphosphate carboxylase, partially purified from corn leaves, demonstrates a low K_m(CO₂) of 19 μM if stabilized with ribose-5-phosphate during extraction. It also exhibits a ribulosediphosphate dependent uptake of oxygen, similar to that observed with spinach carboxylase. The low K_m(CO₂) is similar to the apparent K_m(CO₂) for photosynthesis by intact corn tissue and requires reconsideration of the hypothesis that CO₂ is concentrated in the bundle sheath cell by the C₄ pathway during photosynthesis.     

Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

Altered metabolism of ppGpp in quinone-treated E. coli: Possible role of aminoacyl-tRNA synthetases

The bacteriostatic quinone 6-amino-7-chloro-5,8-dioxoquinoline inhibits leucyl-tRNA synthetase in vivo and in vitro (Ogilvie et al. Biochim. Biophys. Acta $\text{407}$, 357–364; 1975). In this report it is shown that the quinone also interferes with the metabolism of ppGpp. Quinone treatment of E. coli MRE 600 causes the same phenotypic pattern as found in spoT^? mutants: overproduction of ppGpp and a drastic increase of its half-life; the formation of pppGpp, the possible degradation product of ppGpp, is blocked. A model is discussed to explain how the inhibition of leucyl-tRNA synthetase could account for the altered metabolism of ppGpp.     

Inhibition of ruthenium red-insensitive mitochondrial Ca2+ release and its pyridine nucleotide specificity

Isolated, intact rat liver mitochondria, without extraneous substrates but loaded with Ca²⁺ (20 nmol/mg), can be observed to release Ca²⁺ when treated with ruthenium red. Such release can be inhibited by 0.33 mM dlisocitrate but not by 10 mM dl-β-hydroxybutyrate. Assays of NADP⁺, NADPH, NAD⁺, and NADH revealed that only the reduction of NADP⁺ can be linked with such inhibition of Ca²⁺ release, not that of NAD⁺. Since ruthenium redinsensitive Ca²⁺ release is a physiological (but normally masked) process, this experimental approach avoids some potential problems ascribed to strong pyridine nucleotide oxidation. It is suggested that specific NADP⁺:NADPH dependent reactions are part of a physiological mechanism regulating Ca²⁺ release/retention.     

Inhibition of ADP-ribosylation of histone by diadenosine 5', 5"' -p (1), p(4)-tetraphosphate

Diadenosine 5′, 5?-p¹, p⁴-tetraphosphate (Ap4A) strongly inhibited ADP-ribosylation reaction of histone by purified bovine thymus poly(ADP-ribose) polymerase. This compound showed a relatively weak inhibitory effect on Mg²⁺-dependent, enzyme-bound poly(ADP-ribose) synthesis. Among various adenine nucleotides tested, including several diadenosine nucleotides with varying phosphate chain length, Ap4A was the most effective inhibitor of the histone-modification reaction. Ap5A and Ap6A showed slightly lower inhibitory effect than Ap4A. Kinetic analysis of the inhibitor (Ap4A) with varying concentration of substrate (NAD⁺) revealed that this compound is a “mixed type inhibitor”, with a Ki value of 5.1 μM.     

The mechanisms of the cyclic AMP-dependent regulation of the enzymes of the 2',5'-oligoadenylate system

The cAMP-dependent induction of 2,5-oligoadenylate (2-5A) synthetase and cAMP-dependent inhibition of 2-5A phosphodiesterase are shown. Variations in activities of cAMP-dependent protein kinase and the enzymes of 2-5A metabolism in the cells deepening into the resting state were found to be compatible with the above finding. A scheme of coordinated action of cAMP and 2-5A is proposed.     

Bull sperm plasma and acrosomal membranes: Fluorescence studies of lipid phase fluidity

Bull sperm plasma and outer acrosomal membranes have been isolated by discontinuous sucrose density gradient centrifugation and Ca²⁺-ATPase activity has been determined for both the membranes. Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization studies show that the lipid phase of the sperm plasma membranes is more fluid than the lipids of the outer acrosomal membranes. Approximately, a three fold increase in the cholesterol content has been found in the outer acrosomal membranes as compared to that in the plasma membranes.     

Effect of hemin and isonicotinic acid hydrazide on the uptake of iron from transferrin by isolated rat liver mitochonodria

Isolated rat liver mitochondria accumulate iron from the suspending medium when [⁵⁹Fe] transferrin is used as a model compound. The accumulation proceeds by two different mechanisms, i.e. by an energy-dependent and an energy-independent mechanism. The energy-dependent uptake of iron from transferrin is inhibited by hemin and stimulated by isonicotinic acid hydrazide. The energy-independent uptake of [⁵⁹Fe] transferrin is influenced neither by hemin nor by isonicotinic acid hydrazide.