Effect of hemopexin on the efflux of metalloporphyrin from isolated rat liver mitochondria

When rat liver mitochondria were incubated with ⁵⁷Co²⁺ and deuteroporphyrin (or protoporphyrin) the efflux of metalloporphyrin was markedly increased when hemopexin was included. The effect of hemopexin could be abolished by adding hemin, or in part by high concentrations of K⁺. Globin behaved essentially as hemopexin. The results could not be ascribed to the removal of metalloporphyrin from de-energized, leaky mitochondria. The results are strong evidence for a protein-facilitated transport of metalloporphyrin from the mitochondria to the cytosol.     

Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H⁺). Although its acid-base properties were indistinguishable from those of δ-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the δ-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0.It is apparent that the assay of δ-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236–250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.     

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.     

Developmental changes in the form of the Arrhenius plots of rat liver plasma membrane adenylate cyclase and 5'-nucleotidase activities

Human α-1-proteinase inhibitor is inactivated by human myeloperoxidase in the presence of hydrogen peroxide and chloride ion. Several antiarthritic drugs and related compounds, including many containing gold, were tested as inhibitors of the myeloperoxidase system. Of the twenty-six compounds used, twenty-two inhibited. The most important feature of these was the presence of a sulfhydryl group. The most effective compounds also were the most hydrophobic. The presence of gold, on the other hand, made little difference to the amount of inhibition. These drugs appear to have many effects, and their inhibition of the myeloperoxidase system suggests that this could be one of them.     

Increase in polymerized liver tubulin during stimulation of hepatic plasma protein secretion in the rat

Involvement of hepatic microtubules in plasma protein secretion by the liver was investigated by stimulating protein secretion in rat liver and then measuring the different forms of tubulin. Total and free tubulin were estimated in liver supernatants by the [³H] colchicine-binding assay. Polymerized tubulin, assumed to reflect the presence of microtubules, was calculated from the difference between total and free tubulin. To enhance liver plasma protein secretion, an acute inflammatory reaction was induced in one group of rats and a nephrotic syndrome in another. In both cases, total liver tubulin increased significantly compared to normal animals, but free tubulin was unchanged. Accordingly, polymerized tubulin rose by 50% during the inflammatory reaction and by 90% during the nephrotic syndrome. These results support the hypothesis that hepatic microtubules are involved in plasma protein secretion by the liver and also suggest that enhanced secretion requires additional microtubules.     

Characterization of cytochalasin B binding to adult rat liver parenchymal cells in primary culture

The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-³H]cytochalasin B and transport of $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-[}{}^{14}\text{C]glucose}$ into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a K_D of 2.86 · 10^?7 M, while the low-affinity site had a K_D of 1.13 · 10^?5M. Sugar transport was monitored by $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-}\text{glucose}$ uptake and it was found that cytochalasin B (10^?5M) drastically inhibited transport. However, D-glucose (10^?5M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier.     

Ca2+ transporting activity of membrane fractions isolated from the post-mitochondrial supernatant of rat liver

The post-mitochondrial supernatant of rat liver contains two vesicular fractions which transport Ca2+ actively. The heavier fraction, sedimenting at 17.500 xg, 20 min, is enriched in plasma membrane markers and apparently contains both a Ca2+ pumping ATPase and a Na+/Ca2+ exchanger. These activities have been attributed to the plasma membrane vesicles. The lighter fraction, sedimenting at 100.000 xg, 60 min, is enriched in endoplasmic reticulum markers, and contains only a Ca2+ pumping ATPase, which can be differentiated from that of the heavier fraction on the basis of the sensitivity to vanadate. The Ca2+ pumping activity of endoplasmic reticulum appears to be regulated by both a cAMP-dependent, and a calmodulin-dependent system. The former system involves a heat-stable protein fraction from the cytosol. The regulation by the cAMP and the calmodulin-dependent systems involves the phosphorylation of several proteins in the endoplasmic reticulum membrane.     

Cytosolic pH regulation in perfused rat liver: role of intracellular bicarbonate production

The contribution of metabolic bicarbonate to cytosolic pH (pH_cyto) regulation was studied on isolated perfused rat liver using phosphorus-31 NMR spectroscopy. Removal of external HCO^?₃ decreased proton efflux from 18.6±5.0 to 1.64±0.29 μmol/min per g liver wet weight (w.w.) and pH_cyto from 7.17±0.06 to 6.87±0.06. In the nominal absence of bicarbonate, inhibition of carbonic anhydrase by acetazolamide induced a further decrease of proton efflux of 0.69±0.26 μmol/min per g liver w.w. reflecting a reduction in metabolic CO₂ hydration, and hence a decrease of H⁺ and HCO^?₃ supplies. Even though 27% of the proton efflux was amiloride-sensitive under bicarbonate-free conditions, amiloride did not change pH_cyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and amiloride since pH_cyto decreased by 0.16±0.05 and proton efflux by 0.60±0.14 μmol/min per g liver w.w. The data suggest that amiloride-sensitive or SITS-sensitive transport activities could achieve, by themselves, pH_cyto regulation. The involvement of two mechanisms, most likely Na⁺/H⁺ antiport and Na⁺:HCO^?₃ symport, was confirmed in the whole organ under intracellular and extracellular acidosis. The evidence of Na-dependent transport of HCO^?₃ in the absence of exogenous bicarbonate implies that the amount of metabolic bicarbonate is sufficient to effectively participate to pH_cyto regulation.     

In vivo incorporation of cytidine-monophosphate-ciliatine into rat liver lipids.

1. In vivo this investigation was carried out in order to compare the incorporation into rat lipids of free [1,2-minus 14C]-ciliatine and CMP-[1,2-minus 14C]-ciliatine which is the precursor in phosphonolipid biosynthesis. 2. The incorporation of the radioactivity from CMP-[1,2-minus 14C]-ciliatine took place more rapidly than that from free [1,2-minus 14C]-ciliatine in both liver and kidney. The amount of radioactivity from the CMP-[1,2-minus 14C]-ciliatine incorporated into total liver lipids was about 5 times higher than that incorporated into total liver lipids of rat two hrs after injecting free-[1,2-minus 14C]-ciliatine. 3. The amount of [1,2-minus 14C]-ciliatine incorporated into total liver lipids was 15 and 21 times higher than that incorporated into total kidney lipids of rat two and four hrs after injecting free [1,2-minus 14C]-ciliatine. 4. If the main pathway for the phosphonolipid biosynthesis is via CMP-ciliatine, the rate of phosphonolipid formation from CMP-ciliatine must therefore be higher than that from free-ciliatine. The results obtained here indicate therefore that the main pathway for phosphonolipid biosynthesis is a pathway involving CMP-ciliatine. 5. An unknow compound was detected in the water soluble fraction of the acid hydrolyzate of liver phosphonolipids. This material migrated with the N-trimethyl-derivative of ciliatine on the thin-layer chromatogram. The result shows that there is therefore a possibility of methylation of exogenous ciliatine to the phosphonate analogue of choline in the mammalian body.     

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.     

Oxygen radical formation induced by gossypol in rat liver microsomes and human sperm

Gossypol, a polyphenolic compound found in cotton plants, has many potential uses, including use as a male antifertility drug and spermicide. Gossypol affects a variety of cell processes and many of these effects may be explained by a common underlying mechanism. Here we report that gossypol promotes the formation of oxygen radicals when incubated with rat liver microsomes and human sperm suggesting that oxygen radical production may be the underlying basis of its biological activity.     

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.     

Effect of clofibrate treatment on carnitine acyltransferases in different subcellular fractions of rat liver

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyltransferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fraction.In normal liver 48% of total carnitine acetyltransferase activity was peroxisomal, 36% of the activity located in mitochondria and 16% in a membranous fraction containing microsomes. Carnitine octanoyltransferase and carnitine palmitoyltransferase were confined almost totally (77–81%) to mitochondria in normal liver.Clofibrate treatment increased the total activity of carnitine acetyltransferase over 30 times, whereas the total activities of the other two transferases were increased only 5-fold.From the three different subcellular carnitine acetyltransferases the mitochondrial one was not responsive to clofibrate treatment, i.e. the rise in mitochondrial activity was over 70-fold as contrasted to the 6- and 14-fold rises in peroxisomal and microsomal activities, respectively. After treatment mitochondria contained 79% of total activity.It is concluded that the clofibrate-induced increase of carnitine acetyltransferase activity is not due to the peroxisomal proliferation that occurs during clofibrate treatment. The rise in peroxisomal activity contributed only 8% to the total increase.After clofibrate treatment the greatest part of carnitine octanoyl- and palmitoyltrnasferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.     

Inhibition by polyamines of lipid peroxide formation in rat liver microsomes.

Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

Cytoplasmic proteases of rat liver parenchymal cells

Soluble extracts of isolated rat liver parenchymal cells contained three proteases with alkaline pH optima. One protease was a high molecular weight (Mr = 500,000) enzyme which was stimulated by ATP. The other two proteases were totally dependent on calcium for activity and displayed different calcium concentration requirements. One was half-maximally activated by 150 μM Ca²⁺ while the other required only 10 μM Ca²⁺ for half-maximal activation.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.