Rat Liver Mitochondrial Fractions Research Articles

Isolation and characterization of a membrane-bound proteinase from rat liver

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment ( M r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl- l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl- l-tyrosine ethyl ester is 5.0 × 10 −3 m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.

Studies on the conversion of multiple forms of tyrosine aminotransferase in rat liver

Studies from several laboratories have demonstrated the existence of at least three separable forms of the hepatic enzyme, tyrosine aminotransferase. The significance of these separable forms of the enzyme isolated in vitro for the nature and regulation of the enzyme in vivo has been the subject of some controversy. The studies reported in this paper demonstrate the existence of a heat-labile, pH- and temperature-dependent, nondialyzable component associated predominantly with the lysosomal and mitochondrial fraction of rat liver which catalyzes the conversion of form II to forms III and IV of the enzyme. The activity of this conversion factor is not significantly affected by F −, molybdate ions, or two inhibitors of proteases. On the other hand, the cyanate ion completely inhibits the conversion of form II to forms III and IV of tyrosine aminotransferase, as do iodoacetate and oxidized glutathione. p-Chloromercuribenzoate also markedly inhibits the conversion. Kinetic studies suggest that the shift from one form to another follows the pathway: II to III to IV. Titration of the available sulfhydryl groups of the three forms of the enzyme demonstrates that form II possesses between 16 and 17 titratable SH groups per mole, while forms III and IV possess 15 and 13 or 14, respectively. The possible catalytic mechanism by which the conversion of the multiple forms of tyrosine aminotransferase is accomplished is discussed.

Activation of particle associated rat liver guanine deaminase by lecithin and interferences of lecithin in protein precipitation.

Guanine deaminase solubilized from the "light" mitochondrial fraction of rat liver was activated by lecithin. The activation was proportional to the concentration of lecithin taken in the system. A ratio of 1:1 between the two constituents (protein and lecithin) was at least necessary for complete precipitation.

Further characterization of rat liver mitochondrial fractions. Lipid composition and synthesis, and protein profiles

1. Heavy and light mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their lipid composition and synthesis and protein profiles, as seen by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The light mitochondrial fraction was rich in total lipids, phospholipids and cholesterol. The cardiolipin content, however, was low. 3. Rates of [3H]glycerol incorporation into phospholipids of heavy mitochondria and microsomal fractions were almost identical. On the other hand, incorporation into the individual phospholipids in light mitochondria was about 4-6 times higher. Incorporation into cardiolipin of light mitochondria was about 10-fold higher than in the heavy mitochondria. 4. Analysis of protein profiles by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed that the pattern obtained for the light mitochondria was similar to that for heavy mitochondria. However, the light fraction was relatively poor in high-molecular-weight proteins and rich in low-molecular-weight proteins. The microsomal protein profile was altogether different. 5. The significance of these findings is discussed in relation to mitochondrial biogenesis.

Formation, Size, and Solubility in Chloroform/Methanol of Products of Protein Synthesis in Isolated Mitochondria of Rat Liver and Zajdela Hepatoma

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.

Specificity and mechanism of stimulation of adenylate deaminase activity by serotonin

The adenylate deaminase activity of the mitochondrial fraction of rat liver was stimulated in vivo not only by serotonin (5-HT) or other biogenic and synthetic indolylalkylamines, but also by phenyl- and imidazolealkylamines. 5-HT also stimulated adenylate deaminase activity of the liver mitochondria of CBA mice. Actinomycin D or cycloheximide, inhibitors of protein biosynthesis, prevented the stimulation of adenylate deaminase activity by 5-HT. Theophylline, a phosphodiesterase inhibitor, gave a similar effect only in the presence of a high degree of stimulation of adenylate deaminase activity.

The properties of sulfite oxidation in perfused rat liver; interaction of sulfite oxidase with the mitochondrial respiratory chain

Sulfate oxidation in the isolated mitochondrial fraction of rat liver proceeds exclusively through the respiratory chain in a sequence of electron flow from sulfite oxidase to cytochrome c and then to cytochrome oxidase. Thus, the SO 3 2− O ratio observed is almost unity and the ATP O ratio with sulfite is half that obtained with succinate as the respiratory substrate. Direct reduction of molecular oxygen to H 2O 2 by sulfite oxidase occurs only when the respiratory chain is inhibited by cyanide. These two reactions differ with respect to the effects of the O 2 concentration and the sulfite concentration on the activity and on the SO 3 2− O ratio. In the perfused liver, infusion of sulfite causes increased uptake of O 2, with concomitant reduction of the mitochondrial components, such as pyridine nucleotides, flavoproteins, cytochrome c and cytochrome oxidase. The SO 3 2− O ratio observed is 1.6−1.4 and no increase in H 2O 2 production is detected during sulfite oxidation, indicating that sulfite oxidase is located between the outer and inner membranes of mitochondrion and in contact with cytochrome c, thus providing reducing equivalents to the respiratory chain. In the presence of excess cyanide, O 2 consumption increases two-fold during sulfite oxidation but the rate of sulfite oxidation does not change significantly. Under these conditions the SO 3 2− O ratio is 0.8−0.6, indicating direct reduction of O 2 to H 2O 2 by sulfite oxidase in the perfused liver. The production of H 2O 2 is accompanied by a remarkable oxidation of pyridine nucleotides which is attributed to the stimulation of the glutathione-peroxidase reaction. The maximal rate of sulfite oxidation is more than 1.2 μmol/min/g wet wt in the control liver, and decreases to 0.6 μmol/min/g wet wt in the liver of the tungsten-pretreated rat which possesses 23% of the sulfite oxidase activity. As the rate of sulfite oxidation reaches its maximum, progressive inhibition of some dehydrogenases appears to occur in the perfused liver.

Determination of the isoelectric point of rat liver mitochondria by cross-partition

The isoelectric points of rat liver mitochondria and mitochondrial membrane fractions determined by two independent methods, cross-partition in sodium salts and isoelectric focusing in Ampholine gradients, are in good agreement. The determined isoelectric points are 5.2–5.4 for whole mitochondria, 5.3 for the inner membrane fraction and 4.8–4.9 for the outer membrane fracton. This work shows that cross-partition which has previously only been applied to proteins can also be used to determine the isoelectric points of cell organelles and membranes. It is suggested that cross-partition in phase systems can be used to study charge and conformational changes in membranes.

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[ 14C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.

Identification of the palmitoyl-CoA synthetase present in the inner membrane-matrix fraction of rat liver mitochondria

1. 1. The palmitoyl-CoA synthetase (palmitate: CoA ligase (AMP)) present in the inner membrane-matrix fraction of rat liver mitochondria was studied after the outer membrane acyl-CoA synthetase (acid:CoA ligase (AMP), EC 6.2.1.3) had been destroyed by the protease Nagarse. 2. 2. When Nagarse-treated liver mitochondria are subfractionated in a membraneous and a soluble fraction, both palmitoyl-CoA synthetase and octanoyl-CoA synthetase (acid: CoA ligase (AMP), EC 6.2.1.2) are distributed between these fractions as malate dehydrogenase. This indicates that both acyl-CoA synthetase activities are localized in the matrix mitochondrialis. 3. 3. When this mitochondrial soluble fraction is submitted to Sephadex G-100 chromatography, both palmitoyl-CoA synthetase and octanoyl-CoA synthetase activities reside in one single peak with a calculated molecular weight of 47000. For acetyl-CoA synthetase (acetate: CoA ligase (AMP), EC 6.2.1.1.), also present in this soluble fraction, a molecular weight of 62000 was calculated. 4. 4. Kinetic experiments performed with fractions purified by Sephadex chromatography clearly showed that palmitate is activated by the same enzyme as octanoate. This indicates that no special palmitoyl-CoA synthetase is present in the inner membrane-matrix fraction of rat liver mitochondria.

Protective effect of polyanions on the constitutents of rat liver lysosomal fraction in acidic pH.

The ability of polyanionic substances such as heparin, chondroitin sulfate, dextran sulfate, polyanethol sulfonic acid and polyvinyl sulfate in preventing the aggregation of constituents of the light mitochondrial fraction of rat liver at pH 3.0, 5.0 and 7.0 was investigated. The polyanions were found to act as potent agents in preventing the aggregation and disruption of the particles as shown by light scattering measurements and electronmicroscopy.

Hepatic subcellular fractions lipids in rats fed millet (Sorghum vulgarie) at different protein levels.

Effect of feeding defatted millet (Sorghum vulgarie) flour at 5, 10 and 14.5% protein levels respectively for six weeks has been studied on rat liver mitochondrial, microsomal and supernatant fractions total lipids, cholesterol, triglycerides, total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine. The results have been compared with rats fed casein at 10% level for the same period. The metabolism of liver subcellular fractions lipids of millet diet and casein diet fed rats has been studied by the incorporation of acetate-1-14C and . A significant increase in mitochondrial triglycerides of rats fed millet diet at 5 and 10% protein level, in microsomes of rats fed millet diet at 5, 10 and 15% protein levels and in supernatant fractions of rats fed millet diet at 5 and 15% protein levels was observed. A significant increase in total cholesterol in mitochondria and microsomes and a significant decrease in supernatant fraction of rats fed millet diet at 10% protein level was observed. A signi...

Enzymatic Characteristics of CO‐Sensitive 26‐Hydroxylase System for 5β‐Cholestane‐3α, 7α, 12α‐triol in Rat‐Liver Mitochondria and Its Intramitochondrial Localization

The enxymatic characteristics of mitochondrial 26-hydroxylase system for 5β-cholestane-3α,7α,12α-triol were studied with rat liver mitochondria and the inner membrane-matrix fractions with special reference to its intramitochondrial localization. 1 Conditions for the assay of enzymatic activity of the hydroxylase system were established with rat liver mitochondrial preparations and inner membrane-matrix fractions derived by digitonin treatment. 2 It was established that rat liver mitochondria, as well as microsomes, possessed the hydroxylase system for 5β-cholestane-3α,7α,12α-triol as an intrinsic constituent. Product analyses revealed that only the mitochondrial hydroxylase system was specifically active for C-26 position. In contrast, the microsomal system was found to be apparently unspecific on C-26 position but rather more active for other adjacent positions. 3 Among the tested citric acid cycle intermediates and nicotinamide coenzymes, isocitrate and NADPH exerted the most remarkable enhancement of the 26-hydroxylase activity. NADP and NADH were totally inert under the present assay conditions. 4 Partial rupture of the mitochondrial structure by hypotonic or digitonin treatment was essential for the reactivity of 26-hydroxylase system under the present experimental conditions. Accessibility to the intramitochondrial reacting site of the exogenous reactants such as NADPH, isocitrate and 5β-cholestane-3α,7α,12α-triol in particular was apparently improved by the partial rupture. 5 The NADPH-dependent or isocitrate-dependent 26-hydroxylase system was totally insensitive to inhibitors of the respiratory electron transfer such as potassium cyanide, antimycin A, rotenone and amytal. However, it was specifically inhibited by phenyl isocyanide. 6 Remarkable sensitivity to carbon monoxide of the 26-hydroxylase system was demonstrated. Apparent Michaelis constant for oxygen and partition coefficient (between carbon monoxide and oxygen) of the 26-hydroxylase system were estimated to be approximately 10–20 μM and 0.1, respectively. Thus, the possible functioning of a “cytochrome P-450”-like entity in intramitochondrial 26-hydroxlase system was proposed. 7 The localization of 26-hydroxylase system was assigned to be in the inner membranematrix region, on the basis of the distribution of intramitochondrial marker enzymes during the course of serial solubilization by digitonin.

Localization of enzymes by means of proteases

A method is described which makes it possible to determine whether a given enzyme is located on the outer surface of a mitochondrial membrane, or wether it is localized within a mithondrial compartment. The method combines the use of proteases with digitonin. Depending on the concentration of digitonin, enzymes behind the microsomal membranes and the outer mitochondrial membrane may be successively exposed to the action of proteases. Thus, enzymes located within microsomes contaminating the mitochondrial fraction may be easily distinguished from true intramitochondrial enzymes. Applying this technique to the mitochondrial fraction of rat liver, it is shown that lactate dehydrogenase ( l-lactate: NAD + oxidoreductase, EC 1.1.1.27) is located on the outer surface of the mitochondria and within microsomal contaminants.

An NAD- and NADP-dependent malic enzyme with regulatory properties in rat liver and adrenal cortex mitochondrial fractions

The NAD- and NADP-dependent malic enzymes from rat liver and adrenal mitochondrial fractions were separated and partially purified by gel filtration on Sepharose 6B. Two activity peaks were observed. The first contained a malic enzyme capable of reducing either NAD or NADP. This enzyme showed sigmoid kinetics in plots of activity versus the malate concentration. Succinate was an allosteric activator and ATP was a competitive inhibitor of malate. The second peak showed hyperbolic kinetics in plots of activity versus the malate concentration and was unaffected by either succinate or ATP. The relative activities of the two malic enzymes were quite constant in the adrenal mitochondrial fractions. In the liver mitochondrial fractions, the activity of the first peak varied and was sometimes absent while the activity of the second peak was quite constant. The kinetic properties of the first malic enzyme implicate it as an important regulator of malate oxidation.

Protein turnover of the lysosomal and mitochondrial fractions of rat liver during aging

In vivo studies on protein turnover and amino acid incorporation in the mitochondrial, lysosomal and crude microsomal fractions of rat liver have been conducted by a double isotope procedure, using 14C-lysine and 3H-lysine, in 1, 4–6, 12 and 20–26 month-old male animals. The apparent turnover rate of mitochondrial proteins, purified from lysosomal contamination by gentle digitonin treatment, and estimated by their 3H/ 14C ratios, is slower than that of the other protein fractions examined and does not vary appreciably with age. The apparent turnover of lysosomal proteins, purified from mitochondrial and microsomal contamination by centrifugation in sucrose density gradients, appears higher than that of the mitochondrial fraction and similar to that of the crude microsomal proteins. Both these fractions show apparent diminutions of protein turnover with age that are statistically significant when differences are compared between young and senescent animals. The degree of amino acid incorporation into protein is also affected by age. It is significantly lower in young rats, reaches the maximum in adult 12 month-old animals and decreases slightly in the older group.

Effect of tri-iodothyronine treatment on electrophoretic mobilities of rat liver mitochondrial fractions

Heterogeneity of mitochondrial fractions, observed earlier in respect of membrane-bound enzyme profiles and protein turnover, is not reflected in their electrophoretic mobilities. Administration of tri-iodothyronine to thyroidectomised rats results in decreased electrophoretic mobilities of all the mitochondrial fractions.

Halidohydrolytic conversion of N, N′-BIS-(dichloroacetyl)- N, N′-diethyl-1,4-xylylenediamine in rat liver

N, N′-bis-(dichloroacetyl)- N, N′-diethyl-1,4-xylylenediamine (WIN 13099) was synthesized with tritium on the xylylene carbon which did not exchange with body water. This compound was metabolized by 140,000 g supernatant liquid and the mitochondrial fraction of rat liver. Deproteinized 140,000 g supernatant liquid was required for the mitochondrial activity. Although WIN 13099 effectively inhibits drug metabolism by rat liver microsomes in vitro and in vivo, it is not metabolized by rat liver microsomes in vitro. The major metabolite in vitro of WIN 13099, N-dichloroacetyl- N′-hydroxyacetyl- N, N′-diethyl-1,4-xylylenediamine, was isolated and characterized and found not as effective as the parent compound in inhibiting electron transport by sub-mitochondrial particles or drug metabolism by rat liver microsomes.

Demonstration and analysis of ribosomes and RNA associated with mitochondria of mammalian cells

Mitochondrial fraction prepared from mammalian cells (rat liver) by high and slow speed centrifugation and purified by sedimentation in an isopycnic sucrose gradient are shown to contain ribosomes.These ribosomes have a sedimentation coefficient of 83 S (measured in the absence of KCl) and a buoyant density of 1.54 g/cm3 as compared to 80S and 1.55 g/cm3 for cytoplasmic ribosomes.The sedimentation behaviour of mitochondrial ribosomes varies as a function of KCl concentration. The dissociation of cytoplasmic and mitochondrial ribosomes into sub‐units begins towards 0.1 M KCl and is complete in 1 M KCl. The sedimentation coefficient of the intact ribosomes and their dissociated units fall as the concentration of KCl is increased between 0 and 1 M.RNA prepared from rat liver mitochondrial fraction contains a low molecular weight component and two high molecular weight species whose sedimentation coefficients 26 S and 17 S correspond to molecular masses of 1.51 × 106 and 0.61 × 106 daltons. These mitochondrial RNA can be separated by sucrose gradient sedimentation from cytoplasmic ribosomal RNA species sedimenting at 28 S and 18 S.Mitochondrial RNA were also characterized by acrylamide gel electrophoresis. In 2.50% gels, the electrophoretic mobility of high molecular weight mitochondrial RNA was found to be slightly higher than that of their cytoplasmic counterparts. Moreover, the RNA corresponding to the smaller units was separated into two distinct peaks.Electrophoresis in 8% acrylamide gels revealved 5 S and 4 S RNA with a migration pattern similar to that of cytoplasmic 5 S and transfer RNA but present in different amounts and additional RNA with an electrophoretic mobility of 0.31 and 0.85 if that of 4 S RNA is equalled to 1.

Deterioration of rat-liver mitochondria during centrifugation in a sucrose gradient.

The distribution of mitochondrial enzymes after isopyenic centrifugation of a rat‐liver mitochondrial fraction in a sucrose gradient depends on the speed of centrifugation. From the distribution changes, it may be deduced that when centrifugation reaches a certain speed there is an increase in the equilibrium density of some mitochondria; after this the mitochondria undergo disruption. The hydrostatic pressure to which the granules are subjected in the gradient seems to be the main cause of these phenomena. The results of morphological examinations are in good agreement with those suggested by biochemical determinations.