Crude Cytosolic Fractions Research Articles

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

A Proteomic Strategy for Global Analysis of Plant Protein Complexes

Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

Flight restriction prevents associative learning deficits but not changes in brain protein-adduct formation during honeybee ageing

Honeybees (Apis mellifera) senesce within 2 weeks after they discontinue nest tasks in favour of foraging. Foraging involves metabolically demanding flight, which in houseflies (Musca domestica) and fruit flies (Drosophila melanogaster) is associated with markers of ageing such as increased mortality and accumulation of oxidative damage. The role of flight in honeybee ageing is incompletely understood. We assessed relationships between honeybee flight activity and ageing by simulating rain that confined foragers to their colonies most of the day. After 15 days on average, flight-restricted foragers were compared with bees with normal (free) flight: one group that foraged for ∼15 days and two additional control groups, for flight duration and chronological age, that foraged for ∼5 days. Free flight over 15 days on average resulted in impaired associative learning ability. In contrast, flight-restricted foragers did as well in learning as bees that foraged for 5 days on average. This negative effect of flight activity was not influenced by chronological age or gustatory responsiveness, a measure of the bees' motivation to learn. Contrasting their intact learning ability, flight-restricted bees accrued the most oxidative brain damage as indicated by malondialdehyde protein adduct levels in crude cytosolic fractions. Concentrations of mono- and poly-ubiquitinated brain proteins were equal between the groups, whereas differences in total protein amounts suggested changes in brain protein metabolism connected to forager age, but not flight. We propose that intense flight is causal to brain deficits in aged bees, and that oxidative protein damage is unlikely to be the underlying mechanism.

Extensive autolytic fragmentation of membranous versus cytosolic calpain following myocardial ischemia–reperfusion

We investigated calpain activation in the heart during ischemia-reperfusion (I-R) by immunologically mapping the fragmentation patterns of calpain and selected calpain substrates. Western blots showed the intact 78 kDa large subunit of membrane-associated calpain was autolytically fragmented to 56 and 43 kDa signature immunopeptides following I-R. Under these conditions, the 78 kDa calpain large subunit from crude cytosolic fractions was markedly less fragmented, with only weakly stained autolytic peptides detected at higher molecular weights (70 and 64 kDa). Western blots also showed corresponding calpain-like degradation products (150 and 145 kDa) of membrane-associated alpha-fodrin (240 kDa) following I-R, but in crude myofibrils alpha-fodrin degradation occurred in a manner uncharacteristic of calpain. For control hearts perfused in the absence of ischemia, autolytic fragmentation of calpain and calpain-like alpha-fodrin degradation were completely absent from most subcellular fractions. The exception was sarcolemma-enriched membranes, where significant calpain autolysis and calpain-like alpha-fodrin degradation were detected. In purified sarcoplasmic reticulum membranes, RyR2 and SERCA2 proteins were also highly degraded, but for RyR2 this did not occur in a manner characteristic of calpain. When I-R-treated hearts were perfused with peptidyl calpain inhibitors (ALLN or ALLM; 25 micromol/L), calpain autolysis and calpain-like degradation of alpha-fodrin were equally attenuated by each inhibitor. However, only ALLN protected against early loss of developed pressure in hearts following I-R, with no functionally protective effect of ALLM observed. Our studies suggest calpain is preferentially activated at membranes following I-R, possibly contributing to impaired ion channel function implicated by others in I-R injury.

Role of Spinal Cord Alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptors in Complete Freund's Adjuvant-Induced Inflammatory Pain

Spinal cord α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate acute spinal processing of nociceptive and non-nociceptive information, but whether and how their activation contributes to the central sensitization that underlies persistent inflammatory pain are still unclear. Here, we examined the role of spinal AMPARs in the development and maintenance of complete Freund's adjuvant (CFA)-induced persistent inflammatory pain. Intrathecal application of two selective non-competitive AMPAR antagonists, CFM-2 (25 and 50 μg) and GYKI 52466 (50 μg), significantly attenuated mechanical and thermal hypersensitivities on the ipsilateral hind paw at 2 and 24 h post-CFA injection. Neither CFM-2 nor GYKI 52466 affected the contralateral basal responses to thermal and mechanical stimuli. Locomotor activity was not altered in any of the drug-treated animals. CFA-induced inflammation did not change total expression or distribution of AMPAR subunits GluR1 and GluR2 in dorsal horn but did alter their subcellular distribution. The amount of GluR2 was markedly increased in the crude cytosolic fraction and decreased in the crude membrane fraction from the ipsilateral L4–5 dorsal horn at 24 h (but not at 2 h) post-CFA injection. Conversely, the level of GluR1 was significantly decreased in the crude cytosolic fraction and increased in the crude membrane fraction from the ipsilateral L4–5 dorsal horn at 24 h (but not at 2 h) post-CFA injection. These findings suggest that spinal AMPARs might participate in the central spinal mechanism of persistent inflammatory pain.

High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C

A nonradioactive spectrometric assay for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C (PI–PLC) is described. l-α-Phosphatidylinositol from bovine liver was used as substrate in the presence of the micelle-forming detergent deoxycholic acid. PI–PLC isolated from Bacillus cereus and crude cytosol fractions from porcine brain were used as enzyme sources. PI–PLC activity was determined by measuring the release of 1-stearoyl-2-arachidonoyl-sn-glycerol with reversed-phase HPLC and UV detection at 200nm. PI–PLC from B. cereus was not inhibited by the putative PI–PLC inhibitors U-73122 and ET-18-OCH3 at 100μM, whereas the isobenzofuranone derivative 5 blocked the enzyme with an IC50 of 75μM. PI–PLC activity present in porcine brain cytosol was decreased by all three test compounds at 100μM to approximately 30 to 50%.

A calcium/calmodulin kinase pathway connects brain-derived neurotrophic factor to the cyclic amp-responsive transcription factor in the rat hippocampus

Brain-derived neurotrophic factor (BDNF) plays fundamental roles in synaptic plasticity in rat hippocampus. Recently, using rat hippocampal slices, we found that BDNF induces activation of calcium/calmodulin-dependent protein kinase 2 (CaMKII), a critical mediator of synaptic plasticity. CaMKII in turn activates the p38 subfamily of mitogen-activated protein kinases (MAPK) and its downstream effector, MAPK-activated protein kinase 2 (MAPKAPK-2). Herein, we determined whether some kinases of this pathway connect BDNF to the cyclic AMP response element -binding protein (CREB), a transcription factor also involved in plasticity and survival. Crude cytosolic and nuclear fractions were prepared from hippocampal slices of adult rat, and then kinase involvement in CREB phosphorylation was studied with a combination of pharmacologic inhibition and antibody depletion. In addition, the regional localization of this signaling pathway was immunohistochemically investigated. We show that: (i) the BDNF-stimulated CaMKII cascade phosphorylates the key positive regulatory site of CREB via its end MAPKAPK-2 component; (ii) this process appears to be highly localized in the outermost cell layer of the dentate gyrus. The present findings suggest that CaMKII is involved in neurotrophic-dependent activation of CREB in the dentate gyrus. Such a signaling process could be important for controlling synaptic plasticity in this major area for the afferent inputs to the hippocampal formation.

Loose ligation of the rat sciatic nerve is accompanied by changes in the subcellular content of protein kinase C beta II and gamma in the spinal dorsal horn

This study examined whether loose ligation of the sciatic nerve was accompanied by specific changes in protein kinase C (PKC) βII and γ isozymes in the spinal dorsal horn. The isozyme staining pattern was visualized with immunocytochemistry. Their content in subcellular fractions was estimated from Western immunoblots. In control animals, PKC βII immunoreactivity extended from lamina I into lamina III, while PKC γ immunoreactivity was concentrated within laminae II and III. In ligated animals exhibiting thermal hyperalgesia, the content of both PKC βII and γ in the synaptosomal membrane fraction, but not crude cytosolic fraction, was significantly greater by an average of 40% from their respective controls. These data support suggestions that peripheral nerve injury engenders plastic changes in the dorsal horn to contribute to the development of persistent pain.

Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential.

Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress-induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H(2)O(2) induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H(2)O(2) stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from approximately 30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H(2)O(2) also induced translocation of cytochrome c from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H(2)O(2) showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by approximately 1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress-induced cardiac myocyte apoptosis.

The association of metalloendopeptidase EC 3.4.24.15 at the extracellular surface of the AtT-20 cell plasma membrane

Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble, neuropeptide-degrading metalloenzyme, widely expressed in the brain, pituitary and gonads. For the physiological metabolism of neuropeptides, the enzyme should be located extracellularly, either associated with the plasma membrane or in the extracellular milieu. Western immunoblot analyses of crude cytosolic and post-nuclear membrane fractions prepared by differential centrifugation revealed a slightly smaller molecular mass (∼2 kDa) for EP24.15 in the post-nuclear membrane fraction. This smaller EP24.15 species was also present in an enriched fraction of plasma membrane prepared by Percoll gradient centrifugation. To ascertain whether EP24.15 is associated with the extracellular surface of plasma membrane, two sets of experiments were carried out. First, Western immunoblot analysis of AtT-20 cells treated with the membrane-impermeable, thiol-cleavable cross-linker, 3,3′-dithio-bis(sulpho-succinimidyl-propionate) (DTSSP), indicated an extracellular membrane association. After cross-linking and thiol-reduction, a distinct band corresponding to EP24.15 was significantly diminished under non-reducing conditions. Second, immunocytochemical studies performed at 4°C on non-permeabilized AtT-20 cells (i.e., non-fixed to prevent antibody internalization), indicated that EP24.15 was expressed on the surface of the AtT-20 cells. We furthermore determined that EP24.15 enzymatic activity is present on the extracellular surface of the cell discernable from the secreted enzyme. These results suggest that the EP24.15 is associated with the extracellular surface of the AtT-20 cell plasma membrane and is enzymatically active. Taken together, the results are consistent with a putative role in the degradation of neuropeptides acting at the external cell surface.

Altered protein kinase C activation of Na+/Ca2+ exchange in mesangial cells from salt-sensitive rats.

The purpose of these studies was to determine whether there is a defect in protein kinase C (PKC) regulation of the Na+/Ca2+ exchanger in cultured mesangial cells (MC) from Dahl/Rapp salt-sensitive (S) and salt-resistant (R) rats. R and S MCs were cultured, grown on coverslips, and loaded with fura 2 for measurement of single cell cytosolic calcium concentration ([Ca2+]i) in a microscope-based photometry system. Studies were performed in cells that were exposed to serum (serum fed) and in cells that were serum deprived for 24 h. Baseline [Ca2+]i values measured in a Ringer solution containing 150 mM NaCl were similar between R and S MCs in both serum-fed and serum-deprived groups, although baseline [Ca2+]i values were uniformly higher in the serum-deprived groups. Exchanger activity was assessed by reducing extracellular Na (Nae) from 150 to 2 mM, which resulted in movement of Na+ out of and Ca2+ into these cells (reverse-mode Na+/Ca2+ exchange). PKC was activated in these cells with 15-min exposure to 100 nM phorbol 12-myristate 13-acetate (PMA). In the absence of PMA, the change in [Ca2+]i (Delta[Ca2+]i) with reduction in Nae was similar between R and S MCs in both serum-fed and serum-deprived groups, although the magnitude of Delta[Ca2+]i was enhanced by serum deprivation. In both serum-fed and serum-deprived groups, PMA significantly increased Delta[Ca2+]i in R but not S MCs. Upregulation of exchanger activity in R MCs could be abolished by prior 24-h exposure to PMA, a maneuver that downregulates PKC activity. Other studies were performed to evaluate exchanger protein expression using monoclonal and polyclonal antibodies. Immunoblots of PMA-treated cells revealed an increase in the levels of 70- and 120-kDa proteins in the crude membrane fraction of R but not S MCs, an increase which was abrogated by prior 24-h PMA pretreatment and corresponded to reduction in the 70-kDa protein in the crude cytosolic fraction. These data demonstrate that PKC enhances Na+/Ca2+ exchange activity in MCs from R but not from S rats, suggesting that there may be a defect in the PKC-Na+/Ca2+ exchange regulation pathway in MCs of S rats.

Separation of metallothionein isoforms of mouse liver cytosol by capillary zone electrophoresis

Metallothionein (MT) isoforms of mouse liver cytosol were separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH, samples prepared from non-treated, heat-treated, and ethanol-precipitated specimens were compared. The liver was homogenized in three kinds of media, 0.25 M sucrose containing 100 mM Tris–HCl buffer at pH 7.4 (BS), BS containing 1% ascorbic acid (BS-C), and BS containing 5 mM β-mercaptoethanol (BS-M). Mouse liver was used 24 h after subcutaneous injection of 50 mg Zn kg −1. In the non-treated specimen of the cytosol fraction, the MT-2 isoform was separated in all three media, while the MT-1 isoform was difficult to identify. In the ethanol-precipitated specimen, MT isoforms were separated well using either BS or BS-C. However, when BS-M was used, a small MT-2 peak was obtained the MT-1 peak could not be identified. MT-1 isoform in the heat-treated specimen was difficult to identify. In contrast, MT-2 isoform was separated well in all three kinds of media. In the non-treated specimen of the control liver cytosol, the MT-2 isoform was detected using all three media, the MT-1 peak was undetected. Based on these results, MT isoforms can be detected in the crude cytosol fraction of liver using CZE combined with a polyacrylamide-coated tube at neutral pH.

Demonstration and purification of a myristoyl-CoA binding protein from bovine cardiac muscle

Protein myristoylation refers to the co-translational addition of myristoyl group to an amino-terminal glycine residue of a protein by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). The myristoylation reaction depends on the availability of the cellular pools of coenzyme A and myristate and their subsequent formation of myristoyl-CoA, the substrate of NMT. In the present study a myristoyl-CoA binding protein (MCBP) was purified using various column chromatographies: hydroxylapatite, DEAE Sepharose CL-6B and Sephacryl S-300 gel filtration. The purified protein exhibited an apparent molecular mass of 50 kDa on SDS-polyacrylamide gel electrophoresis. Incubation of protein with [1- 14C]myristoyl-CoA followed by denaturing gel electrophoresis, fluorography and treatment with hydroxylamine yielded results that are highly suggestive of a covalent ester-linked acyl-protein complex. This complex formation was not observed in the crude cytosolic fractions. The addition of cytosolic fraction to a progressing acyl-protein complex, resulted in deacylation suggesting a role for thioesterase or/proteinases in the regulation of the acylation reaction in bovine cardiac muscle. The acyl-protein complex formation was not inhibited by NIP 71, a 71 kDa NMT inhibitory protein from bovine brain.

Evidence of an association between functional abnormalities and defective diacylglycerol kinase activity in peripheral blood neutrophils from patients with localized juvenile periodontitis.

Peripheral neutrophils from patients with localized juvenile periodontitis (LJP) show functional abnormalities, such as impaired locomotion and enhanced respiratory burst activity. A defect in intracellular signalling mechanism has been proposed to be responsible for some changes, but direct evidence is lacking. In this study we have determined the activity of diacylglycerol (DAG) kinase, an enzyme controlling the DAG/protein kinase C (PKC) pathway, in crude cytosolic and membrane fractions of neutrophils from 5L JP patients and age and gender-matched normal individuals. No difference was observed in the DAG kinase activity in subcellular fractions from unstimulated cells between the 2 groups. When normal neutrophils were stimulated with N formyl-methionyl-leucyl-phenylalanine (FMLP), the enzyme activity was markedly increased in both subcellular fractions. In contrast, neutrophils from 3 of the 5 LJP patients tested completely failed to rise the DAG kinase activity upon chemoattractant stimulation. These data indicate that in some LJP patients the neutrophil DAG kinase may be defective. To examine whether a decrease in DAG kinase activity could account for some neutrophil abnormalities seen in LJP, normal neutrophils were treated with R59022, a DAG kinase inhibitor, that has been shown to reduce DAG kinase activity in human neutrophils. Upon stimulation with FMLP, R59022-treated normal neutrophils showed significantly reduced chemotactic response and enhanced respiratory burst activity, two typical functional abnormalities featured by LJP cells. It is concluded that a defect in DAG kinase may cause, through an abnormal accumulation of the endogenous PKC activator DAG some of the functional changes observed in neutrophils from LJP patients.

UDP-glucose:solasodine glucosyltransferase from eggplant (Solanum melongena L.) leaves: partial purification and characterization.

Uridine 5'-diphosphoglucose-dependent glucosyltransferase which catalyzes the glycosylation of solasodine i.e. UDP-glucose:solasodine glucosyltransferase, is present in leaves, roots, unripe fruits and unripe seeds of eggplant (Solanum melongena L.). The glucosylation product is chromatographically identical with authentic solasodine 3 beta-D-monoglucoside, a putative intermediate in the biosynthesis of solasodine-based glycoalkaloids characteristic of the eggplant. The enzyme was purified about 50-fold from crude cytosol fraction of eggplant leaves by ammonium sulphate precipitation and column chromatography on Q-Sepharose and Sephadex G-100. The native enzyme has a molecular mass of approx. 55 kDa and pH optimum of 8.5. Divalent metal ions are not required for its activity but the presence of free-SH groups is essential. Besides solasodine (Km = 0.04 microM), the enzyme effectively glucosylates tomatidine, another steroidal alkaloid of the spirosolane type, but it is virtually inactive towards the solanidane-type steroidal alkaloids such as solanidine or demissidine. The enzyme is specific for UDP-glucose (Km = 2.1 microM) since unlabelled ADP-, GDP-, CDP- or TDP-glucose could not effectively compete with UDP-[14C]glucose used as the sugar donor for solasodine glucosylation. Moreover, no synthesis of labelled solasodine galactoside was observed when UDP-[14C]glucose was replaced with UDP-[14C]galactose.

Subcellular localization of diacylglycerol kinase activity in stimulated and unstimulated human peripheral blood lymphocytes and neutrophils.

The diacylglycerol (DAG) kinase activity was determined in crude cytosol and membrane fractions in human peripheral blood lymphocytes and neutrophils. In neutrophils the basal lipid kinase activity was identical in the two subcellular fractions whereas in lymphocytes the basal enzyme activity was 1.6-fold higher in the membrane fraction. In general, the DAG kinase activity in lymphocyte fractions was 10- to 20-fold higher than in neutrophil fractions. When lymphocytes were stimulated with phytohaemagglutinin for 4 hours, a significant decrease in the DAG kinase activity in the membrane fraction was detected. By contrast, a 30-min activation with N-formyl-methionyl-leucyl-phenylalanine markedly increased the lipid kinase activity in both subcellular fractions in neutrophils. This activity was partially inhibited by the compound R59022. These results suggest that the DAG kinase is widely distributed in human leukocytes, but the enzyme's activity and regulation may vary in different leukocyte types.

Effects of Ethanol Administration on Components of the Ubiquitin Proteolytic Pathway in Rat Liver

Hepatic protein accumulation during ethanol administration may result partly from an ethanol-elicited decline in hepatic protein degradation, which we have previously shown. We conducted the current studies to examine the effects of ethanol administration on the levels of hepatic ubiquitin, an 8.5-kd protein which is an important mediator of extralysosomal protein catabolism. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1 to 5 weeks. Ubiquitin was immunochemically quantified by competitive enzyme-linked immunosorbent assay (ELISA) in crude cytosol fractions from whole liver and in 12,000g supernatants of hepatocyte lysates. Ubiquitin levels in hepatic cytosol fractions of ethanol-fed rats exceeded those of controls by about 30%. Isolated hepatocytes from ethanol-fed animals also showed a 40% to 75% elevation of ubiquitin above that in cells of pair-fed controls and this difference exceeded the relative rise in hepatocellular protein. In hepatocyte lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, we detected monomeric ubiquitin and higher molecular mass ubiquitin-protein conjugates. However, the immunoblot analyses revealed no quantitative changes in the level of either free or conjugated ubiquitin. The ubiquitin conjugating activity of crude and diethyl aminoethyl-fractionated liver cytosols of ethanol-fed rats had equal capacities to those from controls in catalyzing the formation of ubiquitin-protein conjugates. Our findings indicate that chronic ethanol consumption increased the level of immunoreactive ubiquitin in rat liver. This may have resulted from enhanced ubiquitin production because of an ethanol-elicited stress response and/or decreased catabolism of ubiquitin and its conjugates. Our findings also provide no indication that the ethanol-elicited reduction in hepatic proteolysis is because of a ubiquitin-mediated mechanisms.

Effects of ethanol administration on components of the ubiquitin proteolytic pathway in rat liver

Hepatic protein accumulation during ethanol administration may result partly from an ethanol-elicited decline in hepatic protein degradation, which we have previously shown. We conducted the current studies to examine the effects of ethanol administration on the levels of hepatic ubiquitin, an 8.5-kd protein which is an important mediator of extralysosomal protein catabolism. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1 to 5 weeks. Ubiquitin was immunochemically quantified by competitive enzyme-linked immunosorbent assay (ELISA) in crude cytosol fractions from whole liver and in 12,000g supernatants of hepatocyte lysates. Ubiquitin levels in hepatic cytosol fractions of ethanol-fed rats exceeded those of controls by about 30%. Isolated hepatocytes from ethanol-fed animals also showed a 40% to 75% elevation of ubiquitin above that in cells of pair-fed controls and this difference exceeded the relative rise in hepatocellular protein. In hepatocyte lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, we detected monomeric ubiquitin and higher molecular mass ubiquitin-protein conjugates. However, the immunoblot analyses revealed no quantitative changes in the level of either free or conjugated ubiquitin. The ubiquitin conjugating activity of crude and diethyl aminoethyl-fractionated liver cytosols of ethanol-fed rats had equal capacities to those from controls in catalyzing the formation of ubiquitin-protein conjugates. Our findings indicate that chronic ethanol consumption increased the level of immunoreactive ubiquitin in rat liver. This may have resulted from enhanced ubiquitin production because of an ethanol-elicited stress response and/or decreased catabolism of ubiquitin and its conjugates. Our findings also provide no indication that the ethanol-elicited reduction in hepatic proteolysis is because of a ubiquitin-mediated mechanisms. (Hepatology 1996 Jun;23(6):1556-63)

Increased protein kinase C isoform γ in the hippocampus of pentylenetetrazol-induced chemoshocked mouse

Protein kinase C (PKC) activity, Western blot analysis of PKCα, -β and -γ, endogenous substrate protein phosphorylation and Western blot analysis of neuromodulin were studied in the cortex, striatum, hippocampus and cerebellum of mouse brain after pentylenetetrazol-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions of these four brain regions were partially purified by DE-52 columns eluted with buffer containing 100 or 200 mM KCI. Almost the same PKC activity in the cortex, striatum, hippocampus and cerebellum was found. This kinase activity was increased in the membrane fractions of hippocampus from chemoshocked mice, while that in other brain regions was not changed. On further analysis by immunoblotting, this increased activity was found to be due to the increase of PKCγ isozyme. The in vitro phosphorylation of neuromodulin was also found to be increased in the hippocampus of chemoshocked mice, while the level of neuromodulin was not changed after chemoshock. Therefore, an increase of PKCγ alone, but not neuromodulin, in the hippocampus contributed to the increased phosphorylation of this substrate in chemoshocked mice.

Heterogeneity of Protein Kinase C Activity in Human U-373 and G-26 Mice Glioma Cells

Protein kinase C (PKC) enzyme activity in a mouse glioma cell line G-26 and a human glioma cell line U-373 were compared at similar cell confluency in-vitro to establish if a G-26 in-vivo mouse model would be useful to examine the role of PKC inhibitors in controlling human glioma growth in-vivo. Original crude cytosolic and membrane PKC fractions of both mouse glioma G-26 and human glioma U-373 cells did not display significant PKC activity compared to partially purified PKC. Partial purification of mouse glioma G-26 and U-373 cytosolic and membrane fractions showed different cytosolic and membrane PKC activity profiles. Total PKC activity was higher (ρ = 0.0001) in human glioma U-373 (7840 picomoles/mg/min) than in mouse glioma G-26 cells (2890 picomoles/mg/min). Thus, results from trials using nude mice human glioma xenografts may be more valid than those obtained from a G-26 in-vivo mouse model for studying the effects of therapeutic drugs on PKC isozymes.