Effect of calcium phosphate and alumina C γ gels on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system

When CaCl₂ was added in increasing concentrations to a rat liver metabolic activation system (S9) buffered with sodium phosphate, the mutagenic activity and cytotoxicity of dimethylnitrosamine (DMN) in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system were greatly increased. This effect was not observed with an S9 mix buffered with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). The calcium phosphate gel precipitate of the S9 mix possessed approximately $\text{built1}\text{3}$ of the total activity of the mix, while the supernatant had only slight activity. However, when the calcium phosphate gel precipitate of a solution of S9 salts (without S9 protein) was added to the supernatant, the remaining $\text{2}\text{3}$ of the activity was recovered. Commercially obtained calcium phosphate, tricalcium phosphate, and alumina C γ gels could substitute for CaCl₂ in the S9 mix, but diethylaminoethyl cellulose (DEAE cellulose) could not. Alumina C γ gel can exert its effect in the absence of both CaCl₂ and phosphate in the S9 mix. Increasing the time of contact between the S9 protein and the S9 salts increased the efficacy with which the S9 mix activated DMN; this is indicative of an adsorptive process by calcium phosphate gel.     

The recognition of two specific binding sites of the adenine nucleotide translocase by palmitoyl CoA in bovine heart mitochondria and submitochondrial particles.

Palmitoyl CoA which is an effective inhibitor of adenine nucleotide transport is able to remove bound [¹⁴C]ADP and [³H]atractylate from the translocator on the outer side of the inner mitochondrial membrane. Bongkrekic acid, when added to the incubation medium prior to palmitoyl CoA, can prevent the removal of bound [¹⁴C]ADP from the membrane by palmitoyl CoA, however, bongkrekic acid is ineffective if palmitoyl CoA is added first. Upon incubation with inverted submitochondrial particles, both palmitoyl CoA and bongkrekic acid prevent the uptake and transport of [¹⁴C]ADP by the particles. Moreover, when the submitochondrial particles are preincubated with [¹⁴C]ADP, palmitoyl CoA, like bongkrekic acid, is unable to remove the bound nucleotide from the inner face of the carrier. Thus, palmitoyl CoA which has a high affinity for the translocator on both sides of the inner mitochondrial membrane, nevertheless, interacts differently with the carrier on each side of the membrane. This suggests that the translocase contains binding sites in two specific states both of which accommodate palmitoyl CoA.     

Interaction of membrane surface charges with the reconstituted ADP/ATP-carrier from mitochondria

Various modulating influences of negative and positive membrane charges on binding and transport properties of the reconstituted ADP/ATP carrier from mitochondria were investigated. The results are interpreted in terms of functional and structural asymmetries of the adenine nucleotide carrier embedded in the liposomal membrane. The surface potential of liposomes was measured directly either by potential-dependent adsorption of the fluorescent dye $\text{2-p-}\text{toluidinylnaphthalene}$ 6-sulfonate (TNS) or by the $\text{p}\text{K}$ shift of the lipophilic pH indicator pentadecylumbelliferone. These results were correlated with the following observations. (1) Negative surface potentials increase the apparent dissociation constant, $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$, for binding of the negatively charged inhbitor carboxyatractylate to the reconstituted carrier protein. (2) Surface potentials modulate the apparent transport affinity, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, of the reconstituted adenine nucleotide carrier for ADP and ATP. The interaction of surface charges with the transport function was investigated with carrier proteins oriented both right-side-out and inside-out. Thus the influence of the surface potential on the function of the ADP/ATP carrier could be determined for the internal and external active sites of the translocator on the outer side of the membrane. Large discrepancies were observed not only between the potentials measured directly (fluorescent dyes) and those measured indirectly (binding and transport affinities), but also between the different surface potentials determined from the influence on the alternatively oriented carrier proteins. The effect of surface charges was rather weak on the cytosolic side of the translocator, whereas there was a strong influence of surface charges on the active site at the matrix side. The most obvious explanation, i.e., screening of negative membrane charges by positively charged amino acid residues at the protein surface, could be ruled out. Besides the modulation of binding affinities for substrates and inhibitors, an additional side-specific effect of surface charges on the transport velocity was observed. Again, the influence on the internal active site of the ADP/ATP carrier was found to be much higher than that on the cytosolic site. The observed effects can be explained by a definite structural asymmetry of the carrier embedded in the liposomal membrane. That site which is physiologically exposed to the cytosol is located at a considerable distance from the plane of the membrane, whereas the opposite site seems to be in close proximity to the membrane surface. Moreover, a spatial equivalence of carboxyatractylate binding site and nucleotide binding site at the external side of the carrier protein was concluded.     

Fatty acid utilization and purine nucleotide binding in brown adipose tissue of genetically obese (ob/ob) mice

The activities of the main enzymes involved in fatty acid utilization i.e. palmitoyl CoA synthetase as well as peroxisomal and mitochondrial β-oxidation were measured in brown adipose tissue homogenates of lean and $\text{ob}$/$\text{ob}$ mice kept at 23°C or acclimated at 4°C. The proton conductance pathway, i.e. the number of purine nucleotide (GDP) binding sites and the percentage of 32,000 polypeptide in brown adipose tissue mitochondria were also measured. In the if$\text{ob}$/$\text{ob}$ mice at 23° C, the specific activities of the palmitoyl CoA synthetase and of the β-oxidation as well as the number of GDB binding sites were lower than in the lean mice by 26%, 43% and 37%, respectively. The percentage of 32, 000 polypeptide, however, was the same in both groups. In the $\text{ob}$/$\text{ob}$ mice at 23° C, the lower homogenate β-oxidation specific activity was due to the fact that the peroxisomal and mitochondrial specific activities were 44% and 37% lower, respectively. Cold acclimation at 4° C was found to cause an increase of the palmitoyl CoA synthetase specific activity, of the palmitoyl CoA synthetase and peroxisomal β-oxidation total activities and of the number of GDP binding sites, in both lean and $\text{ob}$/$\text{ob}$ mice. Cold acclimation increased the percentage of 32,000 polypeptide in the $\text{ob}$/$\text{ob}$ mice only.     

Adenine nucleotide translocase activity and sensitivity to inhibitors in hepatomas. Comparison of the ADP/ATP carrier in mitochondria and in a purified reconstituted liposome system

Adenine nucleotide uptake was found to be lower in mitochondria from hepatoma 7777, 7800, and 9618A than in the host livers. Moreover, in the fast-growing hepatoma 7777 the sensitivity of the adenine nucleotide translocase to inhibition by carboxyatractylate and bongkrekic acid was considerably decreased. Purification of the ADP/ATP carrier from hepatoma 7777 mitochondria and its reconstitution into an artificial liposome system reversed the abnormal kinetics in that the adenine nucleotide uptake and response to inhibitors were identical in proteoliposome preparations from host liver and tumor mitochondria. Analysis of the lipids of the hepatoma inner mitochondrial membrane indicated considerable differences from normal in the levels of phospholipids and cholesterol. Most striking was the increase in cholesterol and sphingomyelin of the hepatoma 7777 inner membrane. An artificial liposome system containing cholesterol in addition to the standard phospholipids could produce alterations in kinetics of the purified ADP/ATP carrier from heart mitochondria similar to those seen in the hepatoma 7777. In general, these results support the suggestion that alterations in the lipid environment of the inner mitochondrial membrane rather than intrinsic changes in the carrier protein itself produce the aberrant observations of adenine nucleotide translocase activity in hepatoma mitochondria.     

The effect of long chain fatty acyl CoA esters on the adenine nucleotide translocase and myocardial metabolism.

The adenine nucleotide translocase, the transport protein for ADP and ATP, located in the inner mitochondrial membrane is an important site for the regulation of cell metabolism. Inhibition of the adenine nucleotide translocase by long chain fatty acyl CoA esters demonstrated $\text{in}$$\text{vitro}$ may also occur $\text{in}$$\text{vivo}$ when the complete oxidation of fatty acids by the myocardium has been compromised during ischemia. Reversal of this biochemical lesion may be of benefit in the preservation of the ischemic myocardium.     

ADP and ATP transport in mitochondria evidence for a metal-ion involved in transport catalysis by use of metallochromic indicators

This study introduces a new class of active-site directed probes with respect to ADP and ATP transport catalysis in rat liver mitochondria. The anionic monoazo dyes, e.g., $\text{p-(2-}\text{hydroxy}\text{-1-}\text{naphthylazo}\text{)}\text{naphtholsulfonic acid}$, are competitive inhibitors of carrier-mediated ADP uptake ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ 20–30 μM). The azo dyes also can displace the same amount of carrier-specific bound ADP as does carboxyatractyloside. Two essential substructures could be derived from a structure-activity study. Firstly, a sulfonic acid group in the para position relative to the azo bridge which becomes neutralized upon binding by a specifically located positive charge of the carrier protein. This electrostatic binding component, which presumably is represented by a strategic arginyl residue, seems to be essential for substrate binding as well as inhibitor binding. The second structural requirement for effective inhibition was found to be the $\text{o-}\text{hydroxy}$ or $\text{o,o′-}\text{dihydroxyazo}$ system, which is known to form stable complexes with metal ions by chelation. Experiments on prevention and reversal of dye-mediated inhibition revealed that the metal-chelating properties are responsible for the effects observed. In addition, using bovine serum albumin or the synthetic polymer Kollidone, inhibition could be prevented as well as abolished. It is postulated that a metal ion, possibly $\text{Mg}{}^{\mathrm{2+}}$, which is bound to the carrier protein plays an essential role for transport catalysis. The metal ion is assumed to form a functional ternary complex, i.e., a metal bridge complex between the carrier protein and its substrate.     

Adriamycin inactivates cytochrome c oxidase by exclusion of the enzyme from its cardiolipin essential environment

The regulation of ligand binding to the muscarinic acetylcholine receptor in developing chick heart has been studied using the radiolabeled antagonist [³H]quinuclidinyl benzilate (QNB). In assays containing only buffer and a source of receptor protein, the antagonist radioligand bound to a single, high affinity state of the receptor. If Mg²⁺ and EDTA were added, [³H]QNB bound to a single, low affinity state. The guanine nucleotide analog, guanylylimidodiphosphate [Gpp(NH)p], reversed the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ so that [³H]QNB again bound only to a single, high affinity state. Sodium could also reverse the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ on antagonist binding but the effects of sodium and Gpp(NH)p on [³H]QNB binding were not additive.     

Adaptive synthesis of rat liver fatty acid synthetase: evidence for in vitro formation of active enzyme from inactive protein precursors and 4'-phosphopantetheine

Evidence is presented in support of the hypothesis that an important step in the adaptive synthesis of fatty acid synthetase is the conversion of inactive enzyme precursors to active enzyme via the incorporation of the 4′-phosphopantetheine prosthetic group. Fatty acid synthetase activity was generated $\text{in vitro}$ when CoA or $\text{E. coli}$ acyl carrier protein was incubated with enzymatically inactive extracts from livers of rats fed a fat-free diet for 0–5 hr following starvation, and a factor present in liver extracts from rats refed for more than 6 hr. When (¹⁴C)-CoA, labelled in the pantetheine moiety, was used in the above system, radioactivity was incorporated into a protein bound form, from which it could be released by mild alkaline hydrolysis.     

Regulation of fatty acid oxidation by adenosine at the level of its extramitochondrial activation

Conditions for the conversion of palmitate into CO₂ and acetoacetate by liver homogenates and isolated liver mitochondria are described. In this system, using liver homogenates, adenosine inhibited the conversion of palmitate into CO₂ and acetoacetate. The inhibition was not observed if the homogenate was substituted by mitochondria or if palmitate was substituted by palmitoyl CoA or palmitoyl carnitine. Intraperitoneal injection of adenosine produced a marked decrease in the level of acetoacetate and β-hydroxybutyrate in plasma, without changing the concentration of serum free fatty acids. Thus, the nucleoside depressed $\text{in vivo}$ the oxidation of long chain fatty acids in liver by inhibiting the extramitochondrial acyl CoA synthase(s). The paramount importance of the extramitochondrial activation of fatty acids as a key control in their oxidation and in the production of ketone bodies is discussed.     

Separation of pigeon liver apo- and holo- fatty acid synthetases by affinity chromatography.

Apo- and holo-fatty acid synthetases of pigeon liver were separated by affinity gel chromatography under conditions similar to, but not identical to, those used in separating subunits I and II of [¹⁴C]pantetheine-labeled fatty acid synthetase complex [Lornitzo $\text{et al.}$, J. Biol. Chem. $\text{249}$, 1654 (1974)]. When [¹⁴C]pantetheine-labeled fatty acid synthetases were separated, the enzymatically active holo form contained all of the [¹⁴C] label. Incubation of the apo-pigeon liver fatty acid synthetase complex with CoA, ATP and a partially purified pigeon liver soluble enzyme system, from which fatty acid synthetase had been removed, resulted in the formation of holo-enzyme. Activation of apo-fatty acid synthetase could also be achieved by replacing the apo-(4′-phosphopantetheine-less) acyl carrier protein with holo-acyl carrier protein. It is evident, therefore, that the inactive apo-fatty acid synthetase lacks a 4′-phosphopantetheine group.     

The losses of adenine nucleotide accompanying efflux of Ca2+ from heart,liver and kidney mitochondria

Mitochondria prepared from either rat liver, kidney or heart were loaded with Ca²⁺ and then the efflux was observed after adding Ruthenium Red. The efflux of Ca²⁺ was stimulated by adding either NaCl, lysolecithin, or palmitoyl CoA or oleate. Measurements were made of the quantities of previously [¹⁴C]-labelled adenine nucleotide lost from the mitochondria as function of the loss of Ca²⁺. The two losses are correlated with coefficients between 0.85 and 0.92 in absence of bongkrekic acid. With bongkrekic acid present the adenine nucleotide loss tends to be diminished with respect to the Ca²⁺ loss; the most significant effect is seen with heart mitochondria. The bongkrekic acid potentiates the stimulation of Ca²⁺ efflux by Na⁺     

Acetylaspartate as an extramitochondrial physiological carrier of acetyl coA for fatty acid synthesis

Aminooxyacetate, a transaminase inhibitor, suppresses the enrichment in radioactivity found in the fatty acids of animals receiving 2, 4-¹⁴C citrate in comparison with 1, 5-¹⁴C citrate. On the other hand 3H from N-acetyl-³H aspartate is significantly incorporated into fatty acids $\text{in vivo}$ or in presence of liver supernatant fractions. Our results indicate that citrate seems to be an effective carrier of acetyl CoA for fatty acid synthesis mainly in the rat liver and that acetylaspartate may be an other physiological carrier of acetyl CoA outside the mitochondria.     

Regulation of the mitochondrial adenine nucleotide pool size

A mechanism by which normal adult rat liver mitochondria may regulate the matrix adenine nucleotide content was studied in vitro. If mitochondria were incubated with 1 mm ATP at 30 ° C in 225 mm sucrose, 2 mm K₂HPO₄, 5 mm MgCl₂, and 10 mm Tris-Cl (pH 7.4), the adenine nucleotide pool size increased at a rate of 0.44 ± 0.02 nmol/mg mitochondrial protein/min. The rate of adenine nucleotide accumulation under these conditions was concentration dependent and specific for ATP or ADP; AMP was not taken up. The rate of net ADP uptake was 50–75% slower than that for ATP. The K_m values for net uptake of ATP and ADP were 2.08 and 0.36 mm, respectively. Adenine nucleotide uptake was stoichiometrically dependent on Mg²⁺ and stimulated by inorganic phosphate. Net uptake was inhibited by n-ethylmaleimide, or mersalyl, but not by n-butylmalonate. Nigericin inhibited net uptake, but valinomycin did not. In the presence of uncouplers, net uptake was not only inhibited, but adenine nucleotide efflux was observed instead. Like uptake, uncoupler-induced efflux of adenine nucleotides was inhibited by mersalyl, indicating that a protein was required for net flux in either direction. Carboxyatractyloside, bongkrekic acid, or respiratory substrates reduced the rate of adenine nucleotide accumulation, however, this did not appear to be a direct inhibition of the transport process, but rather was probably related indirectly to an increase in the matrix $\text{ATP}\text{ADP}$ ratio. The collective properties of the transport mechanism(s) for adenine uptake and efflux were different from those which characterize any of the known transport systems. It is proposed that uptake and efflux operate to regulate the total matrix adenine nucleotide pool size: a constant pool size is maintained if the rates of uptake and efflux are equal. Transient alterations in the relative rates of uptake and efflux may occur in response to hormones or other metabolic signals, to bring about net changes in the pool size.     

Formation of the 11,12-diol as a metabolite of benzo(a)pyrene by rat skin in vivo

The dihydrodiols present as metabolites in rat skin after topical application of ³H-labelled benzo(a)pyrene included a significant amount of radioactivity that cochromatographed with synthetic $\text{trans}$-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene. Treatment of the radioactive metabolite with hot mineral acid gave a product that had chromatographic properties identical to those of the phenol similarly formed from the synthetic dihydrodiol and acetylation of the metabolite yielded a product that cochromatographed with the diacetate of the synthetic dihydrodiol. These observations show that the 11,12-dihydrodiol is formed as a metabolite of BP in rat skin $\text{in vivo}$. The metabolite was not detected in mouse skin.     

Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue

Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing ³H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl-$\text{trans}$-zeatin, $\text{trans}$-zeatin, N⁶-(Δ²-isopentenyl) adenosine and N⁶-(Δ²-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N⁶-(Δ²-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of $\text{cis}$-zeatin and ribosyl-$\text{cis}$-zeatin.     

Bound nucleotides and phosphorylation in Rhodospirillum rubrum.

Chromatophores from $\text{Rhodospirillum rubrum}$ contain 12 × 10^?3 mol ATP and 8.3 × 10^?3 mol ADP per mol chlorophyll, tightly bound to the coupling ATPase. Under energised conditions, these exchange slowly with added nucleotide. Using single turnover light flashes, it is demonstrated that the release of bound ATP is too slow to be on the direct pathway of photophosphorylation.     

Citrulline synthesis: Regulation by alterations in the total mitochondrial adenine nucleotide content

The total adenine nucleotide content of rat liver mitochondria was varied in vitro over a wide range in order to investigate a possible relationship between net changes in the total matrix ATP + ADP + AMP content and the overall rate of citrulline synthesis. Isolated mitochondria were specifically depleted of matrix adenine nucleotides by incubating with inorganic pyrophosphate (G. K. Asimakis and J. R. Aprille, 1980, Arch. Biochem. Biophys.203, 307–316); alternatively, matrix adenine nucleotides were increased by incubating mitochondria with 1 mm ATP at 30 °C. No exogenous ATP or ADP was included in the subsequent incubations for the determination of citrulline synthesis. Rates varied from 0.1 to 1.6 μmol citrulline/mg protein/h as a linear function of total adenine nucleotide content in the range 2–15 nmol (ATP + ADP + AMP)/mg protein. Further increases in the matrix ATP + ADP + AMP content caused no further increase in citrulline synthesis rates. Changes in the total adenine nucleotide content were reflected in proportional changes in both the ATP and ADP content of the matrix. The $\text{ATP}\text{ADP}$ ratio did not change significantly. Therefore, the variations in citrulline synthesis were most simply explained as the effect of different concentrations of ATP on the activity of carbamoyl-phosphate synthetase. It was concluded that net changes in the total adenine nucleotide content can contribute to the control of citrulline synthesis. These findings are significant in the context of recent evidence which shows that the matrix adenine nucleotide pool size is under hormonal control.