Light Mitochondrial Fraction Research Articles

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)

Properties of porcine white adipose tissue and liver mitochondrial subpopulations

Properties of porcine white adipose tissue heavy and light mitochondrial subpopulations were investigated so as to identify any functional heterogeneity. Liver mitochondrial subpopulations were concurrently evaluated since their properties have been studied in some detail. Mitochondrial subpopulations were isolated by means of differential centrifugation and the relative purity estimated using marker enzymes. Due to the greater contamination of the light mitochondrial fractions, mtDNA content, determined by PCR analysis, was used as a basis to demonstrate any mitochondrial heterogeneity. Enzymatic activity, electron microscopy, lipid analysis and Western blotting were used to characterise the different populations. With the exception of liver cytochrome c oxidase, the enzymatic capacity of adipose and liver heavy mitochondria ranged between approximately two- and threefold higher than the corresponding light fraction. The cardiolipin content and mean mitochondrial diameters paralleled these differences, suggesting an increased mitochondrial mass rather than a functional difference. However, the cytochrome c oxidase activity of the liver heavy mitochondria was 4.75-fold higher relative to the light fraction. A strong correlation between cytochrome c oxidase activity and the subunit I content was evident. Adipose tissue mitochondrial subpopulations would seem to possess a comparable oxidative capacity per gram mitochondrial protein, while liver heavy mitochondria possess an increased oxidative capacity and mass.

Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation

This unit describes the isolation of Golgi membranes on a preparative basis. Methods are provided for rapid isolation of dextran-treated Golgi stacks from rat liver using a sucrose density barrier, and Golgi isolation by floatation from a light mitochondrial fraction, along with an alternate procedure using a self-generated iodixinol gradient. If the Golgi tends to vesiculate during homogenization (commonly the case with cultured cells), a primary requirement is to separate these vesicles from other microsomal compartments, so a procedure is described for a discontinuous gradient of sucrose for cultured cells, and an alternate protocol describes a continuous iodixinol gradient that may provide greater resolution. A self-generated iodixinol gradient is described to prepare Golgi membranes from a microsomal fraction of rat hepatocytes, and a standard Golgi enzyme marker assay is given in a support protocol.

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of ‘small peroxisomes’, containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.

Import of a Cytosolic Protein into Lysosomes by Chaperone-mediated Autophagy Depends on Its Folding State

Import of a Cytosolic Protein into Lysosomes by Chaperone-mediated Autophagy Depends on Its Folding State

Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient Centrifugation

This unit covers the use of Percoll, Nycodenz, and iodixanol for purification of lysosomes from a light mitochondrial pellet or postnuclear supernatant. The first protocol describes isolation of lysosomes from rat liver using a Percoll gradient; it includes some comments on using brain and kidney tissue as source material. Alternatively, this protocol can be modified to enhance the separation of lysosomes by organelle density perturbation. Another alternative uses a discontinuous gradient of Nycodenz to purify lysosomes from a rat liver light mitochondrial pellet. A continuous iodixanol gradient, which can be used to purify other organelles in the light mitochondrial fraction, is yet another alternative for purifying lysosomes. Lysosomes can also be isolated from some of the more commonly used cultured cells. The unit also includes assays for common lysosome marker enzymes.

Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient Centrifugation

Peroxisome purification depends on a two-step procedure: differential centrifugation to prepare a light mitochondrial fraction and fractionation on a density-gradient medium preferably iodixanol or Nycodenz, to isolate the peroxisome enriched fraction. The iodixanol gradient may be a preformed continuous gradient or a self-generating gradient. Alternatively a continuous Nycodenz gradient or a simple Nycodenz barrier may be used for the second step. The unit contains protocols for peroxisome isolation from rat liver, tissue culture cells (HepG2 cells), and yeast spheroplasts. The extent of endoplasmic reticulum contamination of the prep can be assessed using an assay for the marker enzyme NADPH-cytochrome creductase.

The Existence of a Lysosomal Redox Chain and the Role of Ubiquinone

Several studies concerning the distribution of ubiquinone (UQ) in the cell report a preferential accumulation of this biogenic quinone in mitochondria, plasma membranes, Golgi vesicles, and lysosomes. Except for mitochondria, no recent comprehensive experimental evidence exists on the particular function of UQ in these subcellular organelles. The aim of a recent study was to elucidate whether UQ is an active part of an electron-transfer system in lysosomes. In the present work, a lysosomal fraction was prepared from a light mitochondrial fraction of rat liver by isopycnic centrifugation. The purity of our preparation was verified by estimation of the respective marker enzymes. Analysis of lysosomes for putative redox carriers and redox processes in lysosomes was carried out by optical spectroscopy, HPLC, oxymetry, and ESR techniques. UQ was detected in an amount of 2.2 nmol/mg of protein in lysosomes. Furthermore, a b-type cytochrome and a flavin–adenine dinucleotide (FAD) were identified as other potential electron carriers. Since NADH was reported to serve as a substrate of UQ redox chains in plasma membranes, we also tested this reductant in lysosomes. Our experiments demonstrate a NADH-dependent reduction of UQ by two subsequent one-electron-transfer steps giving rise to the presence of ubisemiquinone and an increase of the ubiquinol pool in lysosomes. Lysosomal NADH oxidation was accompanied by an approximately equimolar oxygen consumption, suggesting that O2 acts as a terminal acceptor of this redox chain. DMPO/•OH spin adducts were detected by ESR in NADH-supplemented lysosomes, suggesting a univalent reduction of oxygen. The kinetic analysis of redox changes in lysosomes revealed that electron carriers operate in the sequence NADH > FAD > cytochrome b > ubiquinone > oxygen. By using the basic spin label TEMPAMINE, we showed that the NADH-related redox chain in lysosomes supports proton accumulation in lysosomes. In contrast to the hypothesis that UQ in lysosomes is simply a waste product of autophagy in the cell, we demonstrated that this lipophilic electron carrier is a native constituent of a lysosomal electron transport chain, which promotes proton translocation across the lysosomal membrane.

Peroxisome subpopulations of the rat liver. Isolation by immune free flow electrophoresis.

Peroxisomes (POs) are a heterogenous population of cell organelles which, in mammals, are most abundant in liver and kidney. Although they are usually isolated by differential and density gradient centrifugation, isolation is hampered by their high fragility, sensitivity to mechanical stress, and their sedimentation characteristics, which are close to those of other major organelles, particularly microsomes. Consequently, until now only the so-called "heavy" POs with a buoyant density of 1.22-1.24 g/cm(3) have been highly purified from rat liver, whereas the other subpopulations also present in that tissue have escaped adequate characterization. The purification of these subpopulations has become an essential task in view of the functional significance of POs in humans, and the putative importance of peroxisomal subpopulations in the biogenesis of this organelle. Here we used an alternative novel approach to density gradient centrifugation, called immune free flow electrophoresis (IFFE). IFFE combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle complexed to an antibody against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided that the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of IgG molecules. Because of this reduced electrophoretic mobility, IgG-coupled particles can be separated in an electric field from those that are noncoupled and hence more mobile. The IFFE technique has been recently applied for isolation of regular POs (rho = 1.22-1.24 g/cm(3)) from a light mitochondrial fraction of rat liver. We succeeded in isolating different subpopulations of POs by applying IFFE to heavy, light, and postmitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The PO subfractions obtained differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic POs.

Subcellular location of enzymes involved in oxidation of n -alkane by Cladosporium resinae

More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chitinase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 μM Bafilomycin A1, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density-gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively.

Activity and localization of cerebral cysteinic cathepsins after the action of ionizing radiation

The effect of total X-ray irradiation (0.155 C/kg) on the activity of cathepsin L (KF 3.4.22.15) from the light mitochondrial fraction and microsomal soluble fraction of the gray substance of the neocortex was studied in rats. The estimations were performed 1, 24, 48, and 120 h after irradiation. The changes in the cathepsin L activity observed in acute radiation pathology were complex and multiphasic.

Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid CoA thioester

The substrate specificity of rat liver peroxisomal 3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24 E)-3α,7α,12α-trihydroxy-5β-cholest-24-enoic acid (Δ 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12- 18O 3]-(25 R)- and (25 S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5β-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25 R)- and 18O-labeled (25 S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed Δ 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The Δ 24-THCA was derived only from (25 S)-THCA CoA thioester, indicating that the 25 S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.

Incubation of superoxide dismutase with malondialdehyde and 4-hydroxy-2-nonenal forms new active isoforms and adducts. An evaluation of xenobiotics in fish

The effects in fish ( Sparus aurata) of dieldrin, previously reported to be an inducer of peroxisomal enzymes (Pedrajas et al., Comp. Biochem. Physiol. 115C (1996) 125–131), were compared with those of clofibrate. Although dieldrin provoked the more severe peroxisomal changes, both compounds induced oxidative stress as detected by the increased levels of microsomal thiobarbituric acid reactive substances; however the malondialdehyde (MDA) content, determined after HPLC separation of the MDA–TBA complex, was not significantly altered. These results suggest that, besides MDA, other aldehydes were formed in xenobiotic-injected fish, leading us to assess the oxidative effects of such xenobiotics by following changes in superoxide dismutase (SOD) pattern. New active SOD isoforms were detected by isoelectrofocusing in the light mitochondrial (LMF) and cytosolic (CF) fractions. Most of the new SOD bands could be reproduced in vitro by incubation of fish liver cell-free extracts with MDA. To clarify the effects of aldehydes, Cu,Zn- and Mn-SOD isoforms were purified and amino acid analysis was carried out. The new bands found in LMF and CF fractions were reproduced in vitro after incubation of pure SODs with MDA and 4-hydroxy-2-nonenal (HNE), the new SOD bands formed being coincident with the loss of Lys or His residues. Lysine residues were preferentially derivatized after treatment of Cu,Zn-SOD with MDA, but in Mn-SOD the lysine residues were modified only after treatment with MDA, while the histidine residues were modified only by HNE. No change of SOD activity was detected after MDA or HNE exposure, although at the higher aldehyde concentrations used protein aggregates were formed. Therefore, the appearance of new active SOD bands, after isoelectrofocusing separation, can be proposed as a biomarker of oxidative stress.

Pharmacology and subcellular distribution of [ [formula omitted]]rilmenidine binding sites in rat brain

We have previously reported that in rat brain membranes, [ 3 H ]rilmenidine, in addition to labelling α 2-adrenoceptors and the I 2B-subtype of imidazoline receptor binding site (I 2B-RBS), may label an additional I-RBS population, distinct from previously classified I 1-RBS and I 2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [ 3 H ]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [ 3 H ]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density ( B max) and subcellular distribution of [ 3 H ]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [ 3 H ]RO41-1049 and [ 3 H ]RO19-6327 and I 2-RBS labelled by [ 3 H ]2-BFI. Inhibition of [ 3 H ]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29–0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60–140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (>2 μM) for 50–75% of [ 3 H ]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the B max of [ 3 H ]rilmenidine-labelled I-RBS (1.45±0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10±0.15 pmol/mg) and MAO-B (10.35±0.50 pmol/mg) sites. These results suggest that [ 3 H ]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates—nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions—revealed that 45% of total [ 3 H ]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [ 3 H ]RO19-6327 and [ 3 H ]2-BFI which bound 11–13% in the P fraction and 36–38% and 35–44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6–15% of total. These studies reveal that [ 3 H ]rilmenidine-labelled I-RBS, unlike the I 2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.

Immuno‐isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting

Immuno-isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic properties. We have established a method for immuno-isolation of peroxisomes (PO) from both rat liver and the human hepatoblastoma cell line HepG2 using magnetic beads as solid support. A polyclonal antibody raised against the cytoplasmic C-terminal 10 amino acids of the rat 70 kDa peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M-450). The coated beads were incubated with a light mitochondrial fraction and the organelle-bead complexes formed were separated by magnetic sorting in a free-flow system without pelleting the complexes during the isolation procedure. Scanning electron microscopy revealed decoration of beads with particles measuring 150-400 nm in diameter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existence of inherited diseases such as the Zellweger syndrome in which intact PO are lacking, but peroxisomal remnants called "ghosts" are observed instead. Peroxisomal disorders are usually studied using skin fibroblast cell lines derived from afflicted patients and immuno-magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fatal peroxisomal disorders.

Bio-Analytical Studies of Bile Acids.

Bile acids are synthesized from cholesterol in the liver excreted into duodenum via the bile duct as their glycine and taurine conjugates, and converted in part into secondary bile acids by intestinal bacteria. It is well-known that further hydroxylation at C-1, -2, -4, -6 and -19, and conjugation at C-3 with glucuronic acid or sulfuric acid take place in the liver. In this paper bio-analytical studies on bile acids by hyphenated mass spectrometry related to drug metabolism will be discussed. In the first, to clarify an extrahepatic reduction system, 18O labeled 3-oxo bile acids were synthesized and after incubation with human blood, biotransformed products were separated and characterized by GC-MS. The 3-oxo group was reduced into the 3α- and 3β-hydroxyl function catalyzed by the enzyme (s) in human red blood cells. This result strongly implies that biologically active compounds such as drugs with the oxo group are also transformed into hydroxylated metabolites in human blood. On the biosynthesis of bile acids, the final step involving the oxidative cleavage of a side chain of 5β-cholestanoic acid, which has a chiral center at C-25, to form primary bile acids takes place in the peroxisomes. From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 5β-cholestanoic acid into (24E)-5β-cholest-24-enoic acid was then studies with GC-MS. Substrates labeled with 2H at C 24 and C-25 stereoselectively were incubated with a rat liver light mitochondrial fraction and the stereospecific elimination of a pro-R hydrogen at C-24 in both (25R) and (25S)-cholestanoic acids indicating syn-elimination for the former, whereas anti-elimination for the letter was observed. When CoA thioesters of (25R)-and (25S)-5β-cholanoic acids were incubated individually with a rat liver peroxisomal fraction, rapid epimerization to form an equal mixture of stereoisomers from either direction was occurred. Moreover only the 25S antipode was easily transformed into dehydrogenated 5β-cholestanoic acid by peroxisomal acyl-CoA oxidase. The conjugation of carboxylic acids with n-glucuronic acid to form acyl glucuronides plays a significant role in the metabolism and disposition of drugs. The formation of 24-acyl glucuronides of bile acids in the rat liver are also discussed.

Substrate specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates.

The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (Km and Vmax) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol.

Effect of the hydroxyl group on the oxidative cleavage (β-oxidation) of steroidal side chain for bile acid biosynthesis in rat liver homogenate

Mono-, di-, tri-, and tetrahydroxy-5β-cholestan-26-oic acids were incubated with rat liver homogenate (800 × g supernatant and light mitochondrial fraction) to study substrate specificity in the side-chain cleavage reaction (β-oxidation) of bile acid biosynthesis. The C 27-intermediates (5β-cholest-24-en-26-oic acids and 24-hydroxy-5β-cholestan-26-oic acids) in β-oxidation and the corresponding C 24-bile acids were quantitatively determined by capillary gas chromatography. Monohydroxy-5β-cholestan-26-oic acid was not converted into C 24-bile acid. Di- and trihydroxy-5β-cholestan-26-oic acids were effectively transformed into the C 27-intermediates and C 24-bile acids. Tetrahydroxy-5β-cholestan-26-oic acids were also converted into C 27-intermediates and corresponding C 24-bile acids. The intermediate 24-hydroxy-5β-cholestan-26-oic acids could not be detected in the products by incubation with the light mitochondrial fraction. The total specific activity of protein in the light mitochondrial fraction for the production of C 27-intermediates and C 24-bile acids was higher than that of 800 × g supernatant solution. The effects of the number and the position of hydroxyl groups on the side-chain degradation are discussed.

Subcellular localization of dihydropyrimidine dehydrogenase

Although dihydropyrimidine dehydrogenase (DPD) has been purified and characterized from liver tissues of various mammals conflicting data exist on its subcellular localization. To determine the localization of DPD we prepared crude subcellular fractions of a rat liver homogenate by means of differential centrifugation. In the fractions obtained (heavy mitochondrial, light mitochondrial, microsomal and cytosolic) the activities of different marker enzymes were measured as well as the activity of DPD. These results showed that almost all of the activity of DPD was located in the cytosolic fraction. To exclude any particulate-associated DPD, a light mitochondrial fraction was subsequently subjected to equilibrium density gradient centrifugation. The distribution profile of the activity of DPD and the various marker enzymes indicated that DPD from rat liver was exclusively located in the cytosol since no significant activity of DPD could be detected in any subcellular organelle.

Subcellular localization of dihydropyrimidine dehydrogenase.

Although dihydropyrimidine dehydrogenase (DPD) has been purified and characterized from liver tissues of various mammals conflicting data exist on its subcellular localization. To determine the localization of DPD we prepared crude subcellular fractions of a rat liver homogenate by means of differential centrifugation. In the fractions obtained (heavy mitochondrial, light mitochondrial, microsomal and cytosolic) the activities of different marker enzymes were measured as well as the activity of DPD. These results showed that almost all of the activity of DPD was located in the cytosolic fraction. To exclude any particulate-associated DPD, a light mitochondrial fraction was subsequently subjected to equilibrium density gradient centrifugation. The distribution profile of the activity of DPD and the various marker enzymes indicated that DPD from rat liver was exclusively located in the cytosol since no significant activity of DPD could be detected in any subcellular organelle.