Subcellular distribution of farnesyl protein transferase in rat liver.

Farnesyl protein transferase (FPT) activity was measured in rat liver subcellular fractions by using an unspecific acceptor for the farnesyl groups. The highest specific activity was found in mitochondria and it exceeded that of the microsomes three-fold. Considerably lower specific activities were found in the nuclei and cytosol. Further subfractionation revealed that the mitochondrial FPT activity is located in the matrix. The beta-subunit of the mitochondrial enzyme has an apparent molecular mass of 46 kDa, which is similar to its cytosolic counterpart. The results suggest that protein farnesylation can take place in a number of subcellular organelles.     

Intracellular distribution of fumarase in various animals

The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.     

Cellular localization of α-ketoglutarate: Glyoxylate carboligase in rat tissues

α-Ketoglutarate : glyoxylate carboligase activity has been reported by other laboratories to be present in mitochondria and in the cytosol of mammalian tissues; the mitochondrial activity is associated with the α-ketoglutarate decarboxylase moiety of the α-ketoglutarate dehydrogenase complex. The cellular distribution of the carboligase has been re-examined here using marker enzymes of known localization in order to monitor the composition of subcellular fractions prepared by differential centrifugation. Carboligase activity paralleled the activity of the mitochondrial matrix enzyme citrate synthase in subcellular fractions prepared from rat liver, heart and brain as well as from rabbit liver. Whole rat liver mitochondria upon lysis released both carboligase and citrate synthase. The activity patterns of several other extramitochondrial marker enzymes differed significantly from that of carboligase in rat liver. In addition, the distribution pattern of carboligase was similar to that of α-ketoglutarate decarboxylase and of α-ketoglutarate dehydrogenase complex.The data indicate that α-ketoglutarate : gloxylate carboligase activity is located exclusively within the mitochondria of the rat and rabbit tissues investigated. There is no evidence for a cytosolic form of the enzyme. Thus the report from another laboratory that the molecular etiology of the human genetic disorder hyperoxaluria type I is a deficiency of cytosolic carboligase must be questioned.     

Subcellular distribution and properties of aldehyde dehydrogenase from 2-acetylaminofluorine-induced rat hepatomas

The subcellular distribution and properties of four aldehyde dehydrogenase isoenzymes (I-IV) identified in 2-acetylaminofluorene-induced rat hepatomas and three aldehyde dehydrogenases (I-III) identified in normal rat liver are compared. In normal liver, mitochondria (50%) and microsomal fraction (27%) possess the majority of the aldehyde dehydrogenase, with cytosol possessing little, if any, activity. Isoenzymes I-III can be identified in both fractions and differ from each other on the basis of substrate and coenzyme specificity, substrate K(m), inhibition by disulfiram and anti-(hepatoma aldehyde dehydrogenase) sera, and/or isoelectric point. Hepatomas possess considerable cytosolic aldehyde dehydrogenase (20%), in addition to mitochondrial (23%) and microsomal (35%) activity. Although isoenzymes I-III are present in tumour mitochondrial and microsomal fractions, little isoenzyme I or II is found in cytosol. Of hepatoma cytosolic aldehyde dehydrogenase activity, 50% is a hepatoma-specific isoenzyme (IV), differing in several properties from isoenzymes I-III; the remainder of the tumour cytosolic activity is due to isoenzyme III (48%). The data indicate that the tumour-specific aldehyde dehydrogenase phenotype is explainable by qualitative and quantitative changes involving primarily cytosolic and microsomal aldehyde dehydrogenase. The qualitative change requires the derepression of a gene for an aldehyde dehydrogenase expressed in normal liver only after exposure to potentially harmful xenobiotics. The quantitative change involves both an increase in activity and a change in subcellular location of a basal normal-liver aldehyde dehydrogenase isoenzyme.     

Distribution of proteins in different subcellular fractions of rat brain studied by two-dimensional gel electrophoresis

The subcellular distribution of proteins normally visible on two-dimension gels of rat brain tissue punches and crude brain homogenate was investigated using two-dimensional gel electrophoresis and computerized scanning densitometry. Seven enriched subcellular fractions (cytosol, mitochondria, microsomes, nucleus, crude synaptic vesicles, myelin and synaptic membrane) were generated from a crude extract of rat brain. Fifty microgram samples of the crude homogenate and each fraction were then taken and the proteins within these samples separated by two-dimensional gel electrophoresis. Proteins were stained with silver and the gels then analyzed by computerized scanning densitometry. Of 136 proteins visible on two-dimension gels of the crude homogenate that were quantitatively examined, a total of 73 (54%) were identified as being primarily located in a single subcellular fraction. The majority of these 73 proteins were found to be located primarily in either the cytosolic or mitochondrial fractions, while fewer proteins were identified as being primarily located in the microsomal, nuclear or crude synaptic vesicular subfractions. In contrast, the myelin and synaptic membrane fractions were found to be the primary location for only a single protein each that is clearly visible in the crude homogenate. In addition, gels of four of the subfractions (mitochondria, cytosol, nucleus and myelin) contained proteins that are not normally visible on gels generated using a crude extract. The subcellular location of a number of proteins found previously to be altered by specific experimental manipulations was also determined, providing further information on these proteins in brain. These results should prove useful in future experiments designed towards isolating and characterizing specific proteins of neurochemical interest.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

Tissue distribution of avian pancreatic polypeptide-degrading activity

Degradation of avian pancreatic polypeptide (APP) by subcellular fractions from homogenates of chicken kidney, liver, and brain was characterized in this study. Chicken kidney cytosol exhibited the highest degrading activity of all kidney subcellular fractions studied including nuclear, mitochondrial, and microsomal. The cytosolic kidney APP-degrading activity was inhibited in a dose-dependent manner by bacitracin, serine protease inhibitors, and dithiothreitol, and eluted in the void volume of a Sephadex G-100 column, indicating that it is a soluble, serine protease-like activity with a Mr greater than 100,000 kDa and with some dependence on disulfide bonds. Soluble cytosol fractions from chicken liver, kidney, and brain all exhibited greater APP-degrading activity than that of corresponding membrane fractions and, furthermore, were similar in activity between one another. It is concluded that APP degradation by tissue homogenates occurs via a soluble, cytosolic protease which is inhibited by selected serine protease inhibitors; the activity does not differ among liver, kidney, and brain, three tissues which show different receptivity for APP.     

Adenosine deaminase: physical and chemical properties of partially purified mitochondrial and cytosol enzyme from rat liver.

Adenosine deaminase (ADA) was partially purified 486- and 994-fold from rat liver mitochondria and cytosol, respectively. Relative molecular mass of the enzymes from both fractions was 34,000. Km for adenosine and 2'-deoxy-adenosine were 3.08 x 10(-5) M and 3.03 x 10(-5) M for mitochondrial ADA and 3.12 x 10(-5) M and 2.87 x 10(-5) M for cytosolic ADA. The enzyme from both subcellular fractions had the maximum activity at pH 7.5-8.0, and pI 5.2 and 4.2 for mitochondrial and cytosolic enzyme, respectively. The enzyme was inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine and 2'-deoxycoformycin with Ki 4.4 x 10(-7) M and 3.2 x 10(-7) M for mitochondrial ADA and 4.9 x 10(-7) M 2.8 x 10(-7) M for cytosolic ADA. Among the natural nucleoside and deoxynucleotide derivatives tested, deoxy-GTP and UTP inhibited only cytosolic adenosine deaminase by 60% and 40%, respectively.     

The subcellular distribution of beta-carotene in bovine corpus luteum

The distribution of beta-carotene was determined in various subcellular fractions of bovine corpus luteum. It was found in significant amounts in all subcellular fractions examined including nuclear, mitochondrial, microsomal, cytosolic, and floating lipid. Much of the beta-carotene found in the crude nuclear and mitochondrial fractions was loosely bound and could be removed with repeated washings. In contrast, the microsomal beta-carotene could only be removed by detergent extraction suggesting that it is an integral component of this membrane preparation. In the cytosol fraction beta-carotene was bound to high-molecular-weight protein(s), quite possibly a plasma-derived lipoprotein. The subcellular distribution of beta-carotene in corpus luteum is quite similar to the distribution of its metabolite, retinol, in liver. This finding coupled with other recently published data suggests that beta-carotene could play a distinct role in corpora lutea function.     

Localization of mRNAs encoding human mitochondrial oxidative phosphorylation proteins

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells.     

Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.     

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.     

Study on chemical species of iodine in human liver

The distribution and chemical species of iodine in various subcellular fractions of human liver were studied by using epithermal neutron activation analysis combined with chemical and biochemical separation techniques, such as gradient centrifugation and gel chromatography. It was found that the total iodine content orders in various subcellular fractions is as follows: nuclei > cytosol > mitochondria > lysosome > microsome. In the lysosomal fraction, iodine is mainly bound to macromolecules, whereas in the nuclei and mitochondrial fractions, mainly with lower-molecular-weight organic compounds. In the cytosol fraction, iodine is combined with three proteins, in which iodine is chiefly bound with mid- and high-molecular-weight proteins.     

Procaspase-9 is attached to the mitochondrial outer membrane in the early stages of apoptosis

Procaspase-9 is the zymogen form of one of the apoptosis initiators, caspase-9. Its cellular location may differ depending on the cell type; it is found throughout the cytosol, although some of it may be associated with the mitochondria. Procaspase-9 relocates from the cytosol to the mitochondria shortly after the triggering of apoptosis in rat hepatocytes. We investigated whether the mitochondrial protein import machineries import procaspase-9. The combined results of protein import analyses, mitochondrial fractionation and protease treatments of intact and swollen mitochondria imply that procaspase-9 attaches to the outer surface of the mitochondrial outer membrane.     

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.     

Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.     

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

Apoptosis induces efflux of the mitochondrial matrix enzyme deoxyguanosine kinase

Deoxyguanosine kinase (dGK) initiates the salvage of purine deoxynucleosides in mitochondria and is a key enzyme in mitochondrial DNA precursor synthesis. The active form of the enzyme is a 60-kDa protein normally located in the mitochondrial matrix. Here we describe the subcellular distribution of dGK during apoptosis in human epithelial kidney 293 cells and human lymphoblast Molt-4 cells. Immunological methods were used to monitor dGK as well as other mitochondrial proteins. Surprisingly, dGK was found to relocate to the cytosolic compartment at a similar rate as cytochrome c, a mitochondrial intermembraneous enzyme known to enter the cytosol early in apoptosis. The redistribution of dGK from the mitochondria to the cytosol may be of importance for the activation of apoptotic purine nucleoside cofactors such as dATP and demonstrates that mitochondrial matrix proteins may selectively leak out during apoptosis.     

Subcellular distribution of tissue radiocopper following intravenous administration of 67Cu-labeled Cu-PTSM

The subcellular distribution of radiocopper in the brain and liver of rats has been determined following i.v. administration of Cu-PTSM, pyruvaldehyde bis(N⁴-methylthiosemicarbazonato)copper(II), labeled with copper-67. Homogenized tissue samples were separated by differential centrifugation into four subcellular fractions: (I) cell membrane + nuclei; (II) mitochondria; (III) microsomes; and (IV) cell cytosol. Upon sacrifice at 10 min post-Cu-PTSM injection, brain fractions, I, II, III and IV contain 35 ± 12, 11 ± 3, 2.8 ± 1.3 and 51 ± 7% of brain activity, respectively (n = 4). In animals sacrificed 24 h post-injection the subcellular fractions of brain tissue show little change from the radiocopper distribution seen at 10 min post-injection, although the mitochondrial fraction may contain slightly more tracer and the cytosolic fraction slightly less (I, 40 ± 10%; II, 18 ± 5%; III, 3.4 ± 1.5%; and IV, 38 ± 5%; n = 5). Subcellular fractions I, II, III and IV of liver contain 25 ± 5, 12 ± 3, 17 ± 4 and 46 ± 6% of ⁶⁷Cu tracer in animals sacrificed 10 min post-Cu-PTSM injection. An identical subcellular distribution of ⁶⁷Cu, was found in the liver following i.v. administration of ionic radiocopper (as Cu-citrate). The liver and brain cytosolic fractions at 10 min post-injection were further separated by Sephadex column chromatography. In liver cytosol, three different radiocopper components with molecular weights of about 140,000, 41,000–46,000 and 10,000–16,000 Da were found. In the brain supernatant fraction, most of the radiocopper was bound to a single low molecular weight cytosolic component (14,000–16,000 Da). These results suggest that the intracellular decomposition of tracer Cu-PTSM may result in the radiocopper entering the normal cellular pools for copper ions.