Postmitochondrial Fraction Research Articles

Pro and antioxidant responses to repeated administration of diazepam in rat brain.

The role of pro/antioxidative processes during a low, subtoxic dose schedule of diazepam (3 mg/kg/day i.p.) for 7 days and its withdrawal in subcellular preparations of rat brain regions was studied in detail. The results indicated heterogeneity in the regional responses as well as in subcellular compartments. After 7 days of exposure to the drug, a decrease in the Mn-SOD activity was observed in the 3 regions studied while a significant increase in Cu/Zn-SOD activity was seen in cerebellum (CBL) and brain stem (BS) along with that of mitochondrial glutathione reductase. The post-mitochondrial fraction (PMF) showed a significant increase in GR activity in cerebrum. Enhancement of total and free thiol levels was observed in cerebrum and cerebellum whereas in BS free thiols were not enhanced. It was interesting to note that in the animals withdrawn from the drug and sacrificed after an interval of 7 days, the level of TBARS showed a highly significant increase in mitochondria of CB and CBL and 89% increase in BS. Similar trend was observed in the post-mitochondrial fractions of all the 3 regions whereas the activity of isozymes of SOD decreased (p < 0.001) in CBL and BS and to a lesser extent in CB. The GR activity was significantly decreased only in the mitochondria of cerebrum with a 34% rise in cerebellum and no change in BS. The PMFs showed a decrease in CB and CBL but a 20% rise in BS. Thus, the data show modulation of antioxidant responses during short-term administration of diazepam, and a lowering of peroxidative decomposition of polyunsaturated fatty acids of membranes. However, after withdrawal of the drug, PUFAs were found to be more vulnerable to peroxidative decomposition and changes in the antioxidant defenses were also observed, which did not come back to normal level during the study.

In vitro endosome-lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver

We have studied the endosome-lysosome transfer of internalized epidermal growth factor receptor (EGFR) complexes in a cell-free system from rat liver. Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [ 125I]EGF was largely associated with a light endosomal fraction devoid of lysosomal markers. After an additional 30 min incubation in vitro in the presence of an ATP-regenerating system, the amount of [ 125I]EGF in this compartment decreased by 39%, with an increase in [ 125I]EGF in lysosomes. No transfer of [ 125I]EGF to the cytosol was detected. To assess the fate of the internalized EGFR protein over the time course of the endo-lysosomal transfer of the ligand, the effect of a saturating dose of native EGF on subsequent lysosomal targeting of the EGFR was evaluated by immunoblotting. A massive translocation of the EGFR to the endosomal compartment was observed in response to ligand injection coincident with its tyrosine phosphorylation and receptor recruitment of the tyrosine-phosphorylated adaptor protein Shc. During cell-free endosome-lysosome fusion, a time-dependent increase in the content of the EGFR and the two 55- and 46-kDa Shc isoforms was observed in lysosomal fractions with a time course superimposable with the lysosomal transfer of the ligand; no transfer of the 66-kDa Shc isoform was detected. The relationship between EGFR tyrosine kinase activity and EGFR sorting in endosomes investigated by immunoblot studies with anti-phosphotyrosine antibodies revealed that endosomal dephosphorylation of EGFR and Shc preceded lysosomal transfer. These results support the view that a lysosomal targeting machinery distinct from the endosomal receptor kinase activity, such as the recruitment of the signaling molecule Shc, may regulate this sorting event in the endosome.

Localization of a Portion of Extranuclear ATM to Peroxisomes

The gene mutated in the human genetic disorder ataxia-telangiectasia codes for a protein, ATM, the known functions of which include response to DNA damage, cell cycle control, and meiotic recombination. Consistent with these functions, ATM is predominantly present in the nucleus of proliferating cells; however, a significant proportion of the protein has also been detected outside the nucleus in cytoplasmic vesicles. To understand the possible role of extra-nuclear ATM, we initially investigated the nature of these vesicles. In this report we demonstrate that a portion of ATM co-localizes with catalase, that ATM is present in purified mouse peroxisomes, and that there are reduced levels of ATM in the post-mitochondrial membrane fraction of cells from a patient with a peroxisome biogenesis disorder. Furthermore the use of the yeast two-hybrid system demonstrated that ATM interacts directly with a protein involved in the import of proteins into the peroxisome matrix. Because peroxisomes are major sites of oxidative metabolism, we investigated catalase activity and lipid hydroperoxide levels in normal and A-T fibroblasts. Significantly decreased catalase activity and increased lipid peroxidation was observed in several A-T cell lines. The localization of ATM to peroxisomes may contribute to the pleiotropic nature of A-T.

The effect of inhibitors of multixenobiotic resistance mechanism on the production of mutagens by Dreissena polymorpha in waters spiked with premutagens

It has been shown previously that accumulation of 2-aminofluorene (AF) or benzo( a)pyrene (BaP) in aquatic invertebrates enhanced in the presence of inhibitors of multi-xenobiotic resistance (MXR) mechanism. In this work we investigated the consequence of the enhanced accumulation of AF or BaP on the production of their direct mutagenic metabolites. The production of mutagens has been measured by the detection of mutagens excreted into the exposure-water. The exposure of the freshwater mussel Dreissena polymorpha to water spiked with AF (55 μM) for 1 and 2 h resulted in a time-dependent production of direct mutagenic metabolites in the hexane extracts of the water, as detected by Salmonella typhimurium TA 98 in the Ames test without activation. The exposure of mussels to AF at concentrations from 5.5 to 55.0 μM (the maximal solubility) resulted in a dose-dependent increase of AF mutagens excreted into the exposure-water. When mussels were exposed to water spiked with 55.0 μM of AF in the presence of model MXR-inhibitors verapamil (20 μM) or cyclosporin A (10 μM), the excretion of direct AF-mutagens into the exposure-waters was increased by 2- or 5-fold, respectively. Such increase in the excretion of direct mutagens could not be demonstrated after exposure of D. polymorpha to BaP (15 μM), either alone or in the presence of verapamil or cyclosporin A. The explanation of this selectivity was found to be based on the potential of mussel postmitochondrial fraction (PMF) to bioactivate exclusively the aromatic amines (AF), but not polycyclic hydrocarbons (BaP), by its methimazole-sensitive FAD-dependent monooxygenase. Thus, the D. polymorpha PMF failed to bioactivate BaP into its mutagenic products because it lacks the cytochrome P450 α-naphthoflavone-sensitive monooxygenase activity. The number of direct mutagens produced by mussel from AF spiked in Sava River water (collected downstream from the inlet of municipal waste waters, known to possess high concentration of chemosensitizers), was 2-fold higher than was the number of mutagens produced from AF spiked in waters known to possess low concentration of chemosensitizers (the Sava River water collected upstream from the inlet of waste waters, or the water from the Lake Jarun, city of Zagreb). Thus, the inhibitors of MXR-defence mechanism present in environment reveal the genotoxicating potential.

Characterization of superoxide dismutase activity in Chironomus riparius Mg. (Diptera, Chironomidae) larvae — a potential biomarker

The activities of superoxide dismutase (SOD) isoenzymes were measured in fourth instar larvae of Chironomus riparius Mg. Three types of superoxide dismutase were identified: Cu,Zn-SOD in hemolymph and postmitochondrial fraction; Mn-SOD in mitochondrial fraction and presumably Fe-SOD in postmitochondrial fraction. The latter could have an endosymbiotic or a parasitic origin. Extracellular and cytosolic SOD activities, especially Cu,Zn-SOD, tended to increase in the last phase of larval development, independently of protein or hemoglobin contents. This supposes that SOD activity in Ch. riparius larvae is probably activated at the end of fourth instar stage. Cu,Zn-SOD and Mn-SOD activities showed a significant increase under severe hypoxia and slight hyperoxia. Oxygen radical scavengers such as SOD may play a role in the increased tolerance of Ch. riparius to oxidative stress. These results suggest that the specific induction of some SOD isoenzymes could be used as a biomarker of environmental disturbance such as oxidative stress initiated by xenobiotics.

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.

Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450: Comparison of three types of expression systems

Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs). Each of these use a single expression vector. In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors. The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED. This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2). This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2. The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED. The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and the highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system.

Promotion of transition metal-induced reactive oxygen species formation by β-amyloid

β-amyloid protein appears to be involved in the neural degeneration associated with Alzheimer's disease. However, its mechanism of action is poorly understood. The ability of the neurotoxic peptide fragment (25–35) derived from β-amyloid, to promote the generation of reactive oxygen species (ROS) by a postmitochondrial fraction (P2) derived from rat cerebrocortex, has been examined. The peptide fragment, when incubated together with P2, did not cause excess ROS formation. However, 10 μM FeSO 4 or 10 μM CuSO 4 were able to enhance ROS production in the P2 fraction and this was increased further in the concurrent presence of the 25–35 fragment. The corresponding inverse sequence non-neurotoxic peptide (35–25) had no parallel ability to augment iron-stimulated ROS production suggesting a degree of specificity for the observed effect. There was no formation of excess ROS when the 25–35 peptide and 0.5 mM Al 2(SO 4) 3 were incubated with the P2 fraction. However in the presence of both aluminum and iron salts together with the 25–35 peptide, ROS production was augmented to a level significantly higher than that in the absence of aluminum. Polyglutamate, a peptide reported to mitigate aluminum toxicity had no effect on iron-related ROS generation but completely prevented its further potentiation by aluminum. The results indicate that β-amyloid is able to potentiate the free-radical promoting capacity of metal ions such as iron, copper and aluminum. Such potentiation may be a relevant mechanism underlying β-amyloid-induced degeneration of nerve cells.

Isolation of peroxisome subpopulations from rat liver by means of immune free-flow electrophoresis.

Immune free-flow electrophoresis (IFFE) has been applied to the separation of peroxisomes (PO). IFFE is a modification of antigen-specific electrophoretic cell separation (ASECS), and combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It differs from the latter in the pH of the electrophoresis buffer, which was shifted from the physiological range (ASECS) to the pI of IgG molecules (pH approximately 8.0), thus further decreasing the mobility produced by the binding of a specific antibody. This enhances the mobility differences between IgG-coupled particles and those nondecorated, with resultant improved separation. We have now succeeded in isolating different subpopulations of PO by applying IFFE to heavy, light, and post-mitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The obtained PO subfractions differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic PO.

Purification, Characterization and Differentiation-Dependent Expression of a Perchloric Acid Soluble Protein from Rat Kidney

We have recently reported the presence of a novel perchloric acid soluble protein in rat liver (PSP1) that inhibits cell-free protein synthesis in a rabbit reticulocyte system. While studying the perchloric acid soluble proteins from different tissues of rats, we found that the kidney protein cross-reacted with antibody against the PSP1. In this investigation, we have purified a perchloric acid soluble protein from the rat kidney and studied its characterization and expression. The protein extracted from the postmitochondrial supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. By immunoscreening with the rabbit antisera against the PSP1, we detected a cDNA that contained an open reading frame of 411 bp, encoding a 137 amino-acid protein with a molecular mass of 14,149 daltons. The deduced amino acid sequence was completely identical with that of PSP1 from rat liver. The perchloric acid soluble protein from rat kidney (K-PSP1) also inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner than RNase A. Immunohistochemistry showed that the expression of K-PSP1 increased from fetal 17th day to postnatal 4th week, and it remained almost the same until the 7th week of postnatal age. Furthermore, the expression of K-PSP1 in the kidney of the nephrotic rat model was shown to be differentiation dependent. On the other hand, the expression of K-PSP1 in renal tumor cells was downregulated as compared with intact tissue. These results suggest that the expression of K-PSP1 is regulated in a differentiation-dependent manner in the kidney.

γ-Glutamylcysteinylethyl ester attenuates progression of carbon tetrachloride-induced acute liver injury in mice

We examined the effect of γ-glutamylcysteinylethyl ester ( γ-GCE), which is readily transported into hepatocytes and increases hepatocellular reduced glutathione (GSH) levels, on the progression of carbon tetrachloride (CCl 4)-induced liver injury in mice in comparison with that of GSH. Administration of more than 160 μmol/kg of γ-GCE, but not GSH, to mice at 3 h after intraperitoneal injection of CCl 4 (1 ml/kg) significantly attenuated increases in serum asparatate aminotransferase and alanine aminotransferase activities at 24 h after the CCl 4 injection. Increases in hepatic lipid peroxide (LPO) concentrations and decreases in hepatic GSH concentrations after the CCl 4 injection were significantly diminished by the γ-GCE (160 μmol/kg) administration, but not by the same dose of GSH. γ-GCE, γ-glutamylcysteine, and cysteine acted as substrates for glutathione peroxidases much less efficiently than GSH in the post-mitochondrial fraction of normal mouse liver cells. These results indicate that γ-GCE attenuates the progression of CCl 4-induced acute liver injury in mice through the maintenance of hepatic GSH levels, leading to inhibition of hepatic LPO formation, which could be due to an efficient utilization of GSH converted from γ-GCE in the liver cells.

Diazepam induced early oxidative changes at the subcellular level in rat brain.

Studies were conducted to ascertain any involvement of free radical mediated prooxidative processes in different brain regions following diazepam administration. A significant decrease in TBA reactive substance formation was observed in cerebral cortex, cerebellum and brain stem regions after single doses of 1.5, 3 and 6 mg/kg b.wt. For further studies rats were given diazepam (i.p.) at 3 mg/kg body weight dose and sacrificed after 1 h to follow changes in the pro/antioxidant status. An enhancement in the TBARS formation was found in the mitochondrial fractions from cerebral cortex and brain stem. This effect was highest in brain stem being 107% as compared to controls. In the post mitochondrial fraction, cerebellum showed 49% enhancement whereas decreased formation of thiobarbituric acid reactive substances was observed in cerebral cortex and brain stem. Isozymes of superoxide dismutase showed a decrease in activity which was region dependent. Even though, total thiols were not significantly altered, free thiols showed depletion in cerebellum (39.8%) and brain stem (50%). Glutathione reductase activity was also decreased in cerebellum and brain stem. The results indicate that a single dose of diazepam causes free radical mediated changes and the modulatory response of antioxidant defences appears to be region specific.

Differential activation of mRNA during maturation of bovine oocytes is correlated with changes in poly(A)-tail length

Some aspects of the state of aggregation and functioning of polysomes have been investigated in preparations obtained from rat liver lobes subjected to ischemia. The incroporation of amino acid into protein is reduced in postmitochondrial supernatant fractions, microsomes, and C-ribosomes from ischemic livers. In the presence of cell sap from ischemic livers, normal C-ribosomes, but not normal microsomes, show a reduced incorporating capacity, which is not affected by a fivefold increase of the amount of cell sap in the incubation mixture. The analysis of the polysomal patterns shows that ischemia causes a progressive disaggregation of polyribosomes, with a reduction in the number of heavy aggregates and an increase of dimers and monomers. This disaggregation is best seen in the ribosomes recovered from the fraction bound to the membranes of the endoplasmic reticulum. The determination of RNA -used as a reference basis of the various subcellular fractions—shows that ribosomes are also reduced in number in preparations from ischemic livers. Bound ribosomes are more affected than the free forms. On the whole, ribosomes obtained from ischemic livers seem to be reduced in number, less aggregated, and less efficient in carrying out protein synthesis.

Rate of pyrene metabolism in rat liver post-mitochondrial fractions

The objective of the present study was to estimate the maximal velocity ( V max) and Michaelis affinity constant ( K m) for the oxidation of pyrene to 1-hydroxypyrene using rat liver post-mitochondrial fractions. The approach involved the determination of the concentrations of 1-hydroxypyrene formed during 5 min incubations of pyrene (initial concentrations: 0.0025–0.5 μM), and correcting for the rate of 1-hydroxypyrene disappearance (2.16×10 −5 per (mg protein/l)/min) during the incubation period. The V max and K m for pyrene metabolism in the rat corresponded to 0.0577±0.0108 μmol/min per g liver and 27.73±13.54 μM, respectively. The intrinsic clearance ( CL int) of pyrene in the rat estimated in the present study (0.041–0.111 l/min per kg) was within the range of the previously reported CL int in humans (0.037–0.125 l/min per kg). The results of this study suggest that CL int of pyrene in humans can be predicted from such data obtained in the rat.

In vitro and in vivo evaluations of the methaemoglobinaemic potential of xylidine isomers in the rat.

The objective of the present study was to evaluate the methaemoglobinaemic potential of the six isomers of xylidine (XYL) by two approaches: in vitro, using rat red blood cells and hepatic post-mitochondrial fractions in a two-compartmental dialysis system; and in vivo, following a single oral dose of 4.8 mmol kg(-1) (p.o.) of each of the six XYL isomers. The in vitro experiments showed that all six XYL isomers at 1 mM concentration induced significant methaemoglobinaemia in the presence of active hepatic fractions, whereas non-bioactivated XYL isomers were totally inactive. At lower incubation concentrations (0.3 mM and 0.06 mM), 3,5-XYL was still active, whereas the other isomers were less potent (0.3 mM) or totally ineffective (0.06 mM). The in vivo experiment revealed that all XYL isomers, except 3,5-XYL, did not induce significant methaemoglobinaemia after a single oral dose of 4.8 mmol kg(-1). The maximal percentage of methaemoglobin was 31.3 +/- 1.5 in the 3,5-XYL-treated rats, whereas it never exceeded 3% in all the other treatment groups, indicating that 4.8 mmol kg(-1) (p.o.) is in fact a no-observable-adverse-effect level for these XYL isomers. The quantitative differences between in vivo and in vitro results may have been due to additional bioactivation pathways (N-hydroxylation or ring hydroxylation) mediated by high Km enzymes operative at the high incubation concentrations used in vitro. The results of the present study suggest that 3,5-XYL is likely to be the only active isomer in the Sprague-Dawley rat at low exposure levels.

EVIDENCE FOR OXIDATIVE STRESS IN THE HEPATIC MITOCHONDRIA OF BILE DUCT LIGATED RATS

Lipid peroxidation increased both in the liver homogenates and in the hepatic mitochondrial fraction of bile duct ligated (BDL)-rats. Although mitochondrial superoxide dismutase (SOD) activity did not change in the liver, glutathione (GSH) levels and glutathione peroxidase (GSH-Px) activity decreased in hepatic mitochondrial fraction of BDL-rats as compared to sham-operated rats. In addition, GSH-Px and glutathione transferase (GST) activities decreased but SOD activity remained unchanged in the post-mitochondrial fraction of the liver from BDL-rats. However, erythrocyte lipid peroxide and GSH levels did not change in BDL-rats. In conclusion, our results show that the disturbance of oxidant–antioxidant balance especially in mitochondria may be responsible for cholestatic liver injury in BDL-rats.

Bcl-2 and the Outer Mitochondrial Membrane in the Inactivation of Cytochrome c during Fas-mediated Apoptosis

Fas-driven apoptosis in Jurkat cells results in the inactivation of cytochrome c with cessation of oxygen consumption. Overexpression of Bcl-2 was found to protect against acidification and apoptosis mediated by Fas ligation in these cells. Bcl-2 is present in the outer mitochondrial membrane, but the molecular mechanism by which it protects cells is unknown. Because Bcl-2 projects into the mitochondrial intermembrane space and cytochrome c is located in the intermembrane space, we considered the possibility that Bcl-2 might protect cytochrome c from inactivation during Fas-mediated apoptosis. The present study shows that 1) in Jurkat cells, cytochrome c inactivation during Fas-driven apoptosis requires the permeabilization of the outer mitochondrial membrane; and 2) the post-mitochondrial fraction from CEM cells that overexpress Bcl-2 both prevents and reverses cytochrome c inactivation.

S-adenosylmethionine decarboxylase activity is decreased in the rat cortex after traumatic brain injury.

S-Adenosyl-L-methionine decarboxylase (SAMdc) and L-ornithine decarboxylase (ODC) are major enzymes regulating polyamine synthesis. Following ischemia, putrescine content increases as a result of posttraumatic activation of ODC and inhibition of SAMdc. These alterations are thought to mediate edema and cell death. The purpose of this study was to quantify SAMdc activity and edema in the brain following controlled cortical impact injury. Anesthetized adult male rats underwent a right parietal craniectomy and were subjected to cortical impact injury. Tissues were obtained from three bilateral regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). SAMdc activity was determined in the postmitochondrial fraction from homogenates of fresh, unfrozen tissues by measuring the decarboxylation of S-adenosyl-L-[carboxyl-14C]methionine. Basal SAMdc activity was determined in unoperated rats, and regional differences were noted: Activity was lower in the CPF than in the CPm and CPs. SAMdc activity decreased to the greatest extent in the ipsilateral CPm (impact site) from 1 to 72 h following traumatic brain injury. Significant edema was found in the ipsilateral CPm 1, 8, 16, 24, and 48 h after injury. Decreased SAMdc activity impairs the conversion of putrescine to polyamines and may contribute to delayed pathological changes in the brain after traumatic injury.

Xenobiotic Metabolism by Rat Thymus: Induction of Thymic P450 Enzymes

We investigated the drug-metabolizing capacity of the thymus, and compared it with that of the liver. An attempt was also made to characterize the activity of different thymic P450 enzymes. Post-mitochondrial and microsomal fractions of thymus were prepared from female rats. The microsomal enzyme inducers were pregnenolone-16α-carbonitrile (PCN), phenobarbital and acetone. The aromatic hydroxylation of zoxazolamine and p-nitrophenol, N-demethylation of ethylmorphine and erythromycin, and O-depentylation of pentoxyresorufin were assayed. It was found that thymic tissue could affect drug biotransformations. PCN, phenobarbital and acetone induced metabolism of both erythromycin and p-nitrophenol in liver and thymus. However, liver resorufin depentylase was induced only by phenobarbital; no activity was detected in thymus. The thymus can metabolize xenobiotics, although to a lesser extent than the liver. Classic microsomal inducers increased the drug-metabolizing activity of the thymus, and CYP3A1 and CYP2E1 activity was identified.

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase: IDENTIFICATION OF TISSUE-SPECIFIC INTERACTIONS

Chromatography on immobilized antibodies specific to nucleoside diphosphate (NDP) kinase was utilized for affinity purification of this enzyme from detergent extracts of frog heart post-mitochondrial fractions. SDS-polyacrylamide gel electrophoresis analysis of eluates from these supports shows that five polypeptides co-purify with nucleoside diphosphate (NDP) kinase. Tryptic digests of each band were analyzed by mass spectrometric microsequencing. Data base searches by peptide mass matching and sequence homology led to the identification of these proteins as glyceraldehyde-3-phosphate dehydrogenase (40 kDa), creatine kinase (45 kDa), vimentin (55 kDa), pyruvate kinase (60 kDa), and a putative member of the antioxidant protein family (28 kDa). Distinct protein compositions were found in eluates of lung and liver extracts processed in a like manner. The 28-kDa band and vimentin were associated with NDP kinase from all tissues, but co-purification of pyruvate kinase was seen only in liver, while creatine kinase and glyceraldehyde-3-phosphate dehydrogenase were absent from eluates from lung and liver. The results suggest that while NDP kinase is associated with vimentin intermediate filaments and an antioxidant protein in most tissues, it interacts with energy metabolism enzymes in a tissue-specific manner.