Crude Cytosol Research Articles

Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax.

Mitofusins are members of the dynamin-related protein family of large GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. Mitofusins possess four structural domains, including a GTPase domain, two extended helical bundles (HB1 and HB2), and a transmembrane region. We have characterized four Charcot-Marie-Tooth type 2A-associated variants with amino acid substitutions in Mfn2 that are proximal to the hinge that connects HB1 and HB2. A functional defect was not apparent in cells as the mitochondrial morphology of Mfn2-null cells was restored by expression of any of these variants. However, a significant fusion deficiency was observed in vitro, which was improved by the addition of crude cytosol extract or soluble Bax. All four variants had reduced nucleotide-dependent assembly in cis, but not trans, and this was also improved by the addition of Bax. Together, our data demonstrate an important role for this region in Mfn2 GTP-dependent oligomerization and membrane fusion and is consistent with a model where cytosolic factors such as Bax are masking molecular defects associated with Mfn2 disease variants in cells.

Experimental and mathematical analysis of cAMP nanodomains

In their role as second messengers, cyclic nucleotides such as cAMP have a variety of intracellular effects. These complex tasks demand a highly organized orchestration of spatially and temporally confined cAMP action which should be best achieved by compartmentalization of the latter. A great body of evidence suggests that cAMP compartments may be established and maintained by cAMP degrading enzymes, e.g. phosphodiesterases (PDEs). However, the molecular and biophysical details of how PDEs can orchestrate cAMP gradients are entirely unclear. In this paper, using fusion proteins of cAMP FRET-sensors and PDEs in living cells, we provide direct experimental evidence that the cAMP concentration in the vicinity of an individual PDE molecule is below the detection limit of our FRET sensors (<100nM). This cAMP gradient persists in crude cytosol preparations. We developed mathematical models based on diffusion-reaction equations which describe the creation of nanocompartments around a single PDE molecule and more complex spatial PDE arrangements. The analytically solvable equations derived here explicitly determine how the capability of a single PDE, or PDE complexes, to create a nanocompartment depend on the cAMP degradation rate, the diffusive mobility of cAMP, and geometrical and topological parameters. We apply these generic models to our experimental data and determine the diffusive mobility and degradation rate of cAMP. The results obtained for these parameters differ by far from data in literature for free soluble cAMP interacting with PDE. Hence, restricted cAMP diffusion in the vincinity of PDE is necessary to create cAMP nanocompartments in cells.

Characterization of 2,4-Dinitrotoluene Dioxygenase from Recombinant Esherichia coli Strain PFJS39: Its Direct Interaction with Vitreoscilla Hemoglobin

ABSTRACT Burkholderia dinitrotoluene (DNT) dioxygenase in this study (from recombinant Esherichia coli strain PFJS39) is probably a multicomponent enzyme system that oxidizes 2,4-dinitrotoluene (DNT) to 4-methyl-5-nitrocatechol (MNC). DNT dioxygenase was purified by a four-step procedure that utilized consecutive gel filtration chromatography and a nondenaturing gel system. The purified enzyme oxidized DNT only in the presence of NADH and its yield increased by lipase pretreatment of crude cytosol. An estimated molecular weight of 100,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) revealed the presence of three subunits for the samples from consecutive gel filtration chromatography and nondenaturing PAGE. Their molecular weights were 52,000–71,000, 23,000–25,500, and 12,000–16,500. These results suggest that DNT dioxygenase exists as a heterotrimer. The K M of DNT dioxygenase for O2 is 50 μ M, consistent with inhibition results of DNT dioxygenase by Vitreoscilla hemoglobin (its K M for O2 is 7 μ M). The K M for DNT is 180 μ M. The purified enzyme is relatively stable below 40°C, retains activity over a broad pH range, and is stimulated by several cofactors in addition to NADH.

Tuberous Sclerosis Tumor Suppressor Complex-like Complexes Act as GTPase-activating Proteins for Ral GTPases

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

Binding of cytoplasmic testosterone-receptor complex to nucleic from male rat livers.

A crude cytoplasmic androgen receptor preparation of rat livers or its partially purified one by a 30% ammonium sulfate precipitation was prelabeled with [3H] testosterone ([3H]T) and then incubated with nuclei from rat livers at 30 degrees C for 30 min. The formations of the [3H]T-cytosol receptor complex and its nuclear bound complex were determined. The formation of testosterone-cytosol receptor complex was a prerequisite for the nuclear binding of testosterone. Nuclear fraction, when incubated with the prelabeled crude cytosol, bound [3H]T-receptor complex as much as when incubated with the prelabeled partially purified cytosol. The addition of unlabeled crude cytosol to the prelabeled partially purified cytosol increased the nuclear binding of the receptor. The presence of a thermostable, low molecular inhibitor and a thermolabile, macromolecular activator in the crude cytosol preparation was suggested by dialysis, heating and gel chromatography.

Protein fingerprints of cultured CA3-CA1 hippocampal neurons: comparative analysis of the distribution of synaptosomal and cytosolic proteins

BackgroundAll studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. This is highly desirable when synaptic plasticity processes are analyzed since the structure and the biochemistry of neurons and synapses get completely reshaped. Because most molecular studies of synapses are nowadays mainly or at least in part based on protein extracts from neuronal cultures, this is not a feasible option: these simplified versions of the brain tissue on one hand provide an homogeneous pure population of neurons but on the other yield only tiny amounts of proteins, many orders of magnitude smaller than conventional brain tissue. As a way to overcome this limitation and to find a simple way to screen for protein changes at cultured synapses, we have produced and characterized two dimensional electrophoresis (2DE) maps of the synaptic proteome of CA3-CA1 hippocampal neurons in culture.ResultsTo obtain 2D maps, hippocampal cultures were mass produced and after synaptic maturation, proteins were extracted following subfractionation procedures and separated by 2D gel electrophoresis. Similar maps were obtained for the crude cytosol of cultured neurons and for synaptosomes purified from CA3-CA1 hippocampal tissue. To efficiently compare these different maps some clearly identifiable reference points were molecularly identified by mass spectrometry and immunolabeling methods. This information was used to run a differential analysis and establish homologies and dissimilarities in these 2D protein profiles.ConclusionBecause reproducible fingerprints of cultured synapses were clearly obtained, we believe that our mapping effort could represent a simple tool to screen for protein expression and/or protein localization changes in CA3-CA1 hippocampal neurons following plasticity.

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%.

E2-25K mediates US11-triggered retro-translocation of MHC class I heavy chains in a permeabilized cell system

In cells expressing human cytomegalovirus US11 protein, newly synthesized MHC class I heavy chains (HCs) are rapidly dislocated from the endoplasmic reticulum (ER) and degraded in the cytosol, a process that is similar to ER-associated degradation (ERAD), the pathway used for degradation of misfolded ER proteins. US11-triggered movement of HCs into the cytosol requires polyubiquitination, but it is unknown which ubiquitin-conjugating and ubiquitin-ligase enzymes are involved. To identify the ubiquitin-conjugating enzyme (E2) required for dislocation, we used a permeabilized cell system, in which endogenous cytosol can be replaced by cow liver cytosol. By fractionating the cytosol, we show that E2-25K can serve as the sole E2 required for dislocation of HCs in vitro. Purified recombinant E2-25K, together with components that convert this E2 to the active E2-ubiquitin thiolester form, can substitute for crude cytosol. E2-25K cannot be replaced by the conjugating enzymes HsUbc7/Ube2G2 or Ube2G1, even though HsUbc7/Ube2G2 and its yeast homolog Ubc7p are known to participate in ERAD. The activity of E2-25K, as measured by ubiquitin dimer formation, is strikingly enhanced when added to permeabilized cells, likely by membrane-bound ubiquitin protein ligases. To identify these ligases, we tested RING domains of various ligases for their activation of E2-25K in vitro. We found that RING domains of gp78/AMFR, a ligase previously implicated in ERAD, and MARCHVII/axotrophin, a ligase of unknown function, greatly enhanced the activity of E2-25K. We conclude that in permeabilized, US11-expressing cells polyubiquitination of the HC substrate can be catalyzed by E2-25K, perhaps in cooperation with the ligase MARCHVII/axotrophin.

A Protein in Crude Cytosol Regulates Glucose-6-phosphatase Activity in Crude Microsomes to Regulate Group Size in Dictyostelium

Dictyostelium discoideum form groups of approximately 2 x 10(4) cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multipolypeptide complex called counting factor (CF). The CF signal transduction pathway involves CF-repressing internal glucose levels by increasing the K(m) of glucose-6-phosphatase. Little is known about how this enzyme is regulated. Glucose-6-phosphatase is associated with microsomes in both Dictyostelium and mammals. We find that the activity of glucose-6-phosphatase in crude microsomes from cells with high, normal, or low CF activity had a negative correlation with the amount of CF present in these cell lines. In crude cytosols (supernatants from ultracentrifugation of cell lysates), the glucose-6-phosphatase activity had a positive correlation with CF accumulation. The crude cytosols were further fractionated into a fraction containing molecules greater than 10 kDa (S>10K) and molecules less than 10 KDa (S<10K). S>10K from wild-type cells strongly repressed the activity of glucose-6-phosphatase in wild-type microsomes, whereas S>10K from countin(-) cells (cells with low CF activity) significantly increased the activity of glucose-6-phosphatase in wild-type microsomes by decreasing K(m). The regulatory activities in the wild-type and countin(-) S>10Ks are heat-labile and protease-sensitive, suggesting that they are proteins. S<10K from both wild-type and countin(-) cells did not significantly change glucose-6-phosphatase activity. Together, the data suggest that, as a part of a pathway modulating multicellular group size, CF regulates one or more proteins greater than 10 KDa in crude cytosol that affect microsome-associated glucose-6-phosphatase activity.

Alteration of glucocorticoid receptor subcellular distribution by hyperthermic stress

The aim of the present study was to examine intracellular redistribution of the glucocorticoid receptor (GR) in rat liver cells during a 24-h time period after exposure of the animals to 41?C whole body hyperthermic stress. The level of the receptor protein in the cytoplasmic and nuclear compartments was measured by immunoblotting procedures applied to both crude cytosol and immunopurified GR, as well as by immunocytochemical analyses applied to both paraffin-embedded liver sections and unfixed nuclear smears. All the experimental approaches employed in the study provided similar results, demonstrating that the transient stress-related decline of the cytoplasmic GR observed during the first five hours after exposure of the animals to whole-body hyperthermic stress is accompanied by enhanced nuclear accumulation of the receptor. The study can contribute to a better understanding of the influence of stress on the glucocorticoid signal transduction pathway. .

Investigation of the role of lipoxygenase in bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human lung.

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen believed to play a role in human lung cancer. Bioactivation of NNK involves alpha-carbon hydroxylation that could be catalyzed by cytochrome P450, hemoglobin, and lipoxygenases (LOX). In the present study, the role of LOX in NNK bioactivation was investigated. Formation of keto acid, the endpoint metabolite of alpha-methylene NNK hydroxylation, was observed in human lung cytosols incubated with 4.2 microM [5-(3)H]NNK (N = 6). Following concanavalin A affinity chromatography to enrich human lung lipoxygenase (HLLO), the fraction containing cytosolic components less LOX (fraction 1) retained the ability to bioactivate NNK. Although enriched HLLO exhibited the characteristic dioxygenase and hydroperoxidase activities, it did not bioactivate NNK. The LOX inhibitor nordihydroguaiaretic acid inhibited dioxygenase activity of HLLO by 83 +/- 19% (P < 0.05, N = 6), but did not inhibit keto acid formation in the crude cytosols (N = 6, P > 0.05). Failure of soybean LOX to catalyze NNK bioactivation supported the results observed in human lung cytosols, and failure of chemically generated alkylperoxyl radicals to bioactivate NNK further suggested that the dioxygenase activity of LOX is not likely to be involved in NNK bioactivation. Horseradish peroxidase and myeloperoxidase catalyzed NNK bioactivation were also nondetectable. Our results demonstrate that, although human lung cytosols can bioactivate NNK to form keto acid, LOX is not involved. We have attributed the ability of crude human lung cytosols to bioactivate NNK to hemoglobin. The inhibitory effect of 1-aminobenzotriazole and arachidonic acid on keto acid formation in the crude cytosols and in fraction 1, respectively (P < 0.05, N = 6), is consistent with hemoglobin-catalyzed NNK bioactivation.

Distinct roles for the cytoplasmic tail sequences of Emp24p and Erv25p in transport between the endoplasmic reticulum and Golgi complex.

Heteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting. Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast. Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable. Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p. To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed. The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K(d) approximately 100 microm), and binding depended on a pair of aromatic residues found in both tail sequences. COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently. These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER. The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER.

Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells

Toxic medium chain length alkanals, alkenals, and 4-hydroxyalkenals that are generated during lipid peroxidation are potential substrates for aldehyde dehydrogenase (ALDH) isoforms. We have developed transgenic cell lines to examine the potential for either human ALDH1A1 or ALDH3A1 to protect against damage mediated by these toxic aldehydes. Using crude cytosols from stably transfected cell lines, these aldehydes were confirmed to be excellent substrates for ALDH3A1, but were poorly oxidized by ALDH1A1. Expression of ALDH3A1 by stable transfection in V79 cells conferred a high level of protection against growth inhibition by the medium-chain length aldehyde substrates with highest substrate activity, including hexanal, trans-2-hexenal, trans-2-octenal, trans-2-nonenal, and 4-hydroxy-2-nonenal (HNE). This was reflected in a parallel ability of ALDH3A1 to prevent depletion of glutathione by these aldehydes. Expression of hALDH3 completely blocked the potent induction of apoptosis by HNE in both V79 cells and in a RAW 264.7 murine macrophage cell line, consistent with the observed total prevention of HNE-protein adduct formation. Structure–activity studies indicated that the rank order of potency for the contributions of HNE functional groups to toxicity was aldehyde ≥C2=C3 double bond>>C4-hydroxyl group. Oxidation of the aldehyde moiety of HNE to a carboxyl by ALDH3A1 expressed in stably transfected cell lines drastically reduced its potency for growth inhibition and apoptosis induction. In contrast, ALDH1A1 expression provided only moderate protection against trans-2-nonenal (t2NE), and none against the other six–nine carbon aldehydes. Neither ALDH1A1 nor ALDH3A1 conferred any protection against acrolein, acetaldehyde, or chloroacetaldehyde. A small degree of protection against malondialdehyde was afforded by ALDH1A1, but not ALDH3A1. Paradoxically, cells expressing ALDH3A1 were 1.5-fold more sensitive to benzaldehyde toxicity than control V79 cells. These studies demonstrate that expression of class 3 ALDH, but not class 1 ALDH, can be an important determinant of cellular resistance to toxicity mediated by aldehydes of intermediate chain length that are produced during lipid peroxidation.

Distinct mechanisms of activation of Stat1 and Stat3 by platelet-derived growth factor receptor in a cell-free system.

Ligand-dependent activation of the platelet-derived growth factor receptor (PDGFR) in fibroblasts in culture leads to the activation of the JAK family of protein-tyrosine kinases and of the transcription factors Stat1 and Stat3. To determine the biochemical mechanism of STAT activation by PDGFR, we devised a cell-free system composed of a membrane fraction from cells overexpressing PDGFR. When supplemented with crude cytosol, the membrane fraction supported PDGF- and ATP-dependent activation of both Stat1 and Stat3. However, the extent of Stat3 activation differed depending on the source of the cytosolic fraction. Using purified recombinant STAT proteins produced in Escherichia coli, we found that Stat1 could be activated by immunopurified PDGFR and showed no additional requirement for membrane- or cytosol-derived proteins. In contrast, activation of Stat3 exhibited a strong requirement for the cytosolic fraction. The activity present in the cytosolic fraction could be depleted with antibodies to JAK proteins. We conclude that the mechanisms of activation of Stat1 and Stat3 by PDGFR are distinct. Stat1 activation appears to result from a direct interaction with the receptor, whereas Stat3 activation additionally requires JAK proteins.

Thimet Oligopeptidase and the Stability of MHC Class I Epitopes in Macrophage Cytosol

In this study we investigated the fate of a class of proteasome-generated oligopeptides, exposing them to the crude cytosol of macrophages or to the purified recombinant thimet oligopeptidase. Among the proteasome products of known sequences are MHC class I epitopes, 13 of which were randomly chosen to be used as putative substrates. Surprisingly, our results clearly showed that the majority of the peptides were poorly or not degraded, either by the purified enzyme or by the crude macrophage cytosol. The peptides, which were resistant to hydrolysis, displayed high affinity for the thimet oligopeptidase as competitive inhibitors. Regardless of the fact that our data do not allow prediction of whether or not a specific peptide would be degraded, it seems very likely that the structural features, which rule out the stability of the MHC class I peptides in the cytosol, may have implications in an optimized repertoire selection for antigen presentation.

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.

Isolation and Characterization of a Novel Low Molecular Weight Protein Involved in Intra-Golgi Traffic

Analysis of the cytosolic requirements for in vitro intra-Golgi transport led to the characterization of three proteins: N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment protein (SNAP), and p115, all involved in the docking and fusion of transport vesicles to their target membranes. In the course of determining the minimal cytosolic requirements for intra-Golgi transport in vitro, we identified three additional factors that are sufficient to replace crude cytosol. We describe here the purification and characterization of one of these factors, a novel 16-kDa protein, p16, an essential factor for intra-Golgi protein transport. Based on transport activity, this purification procedure resulted in approximately 1,400-fold enrichment of p16 to apparent homogeneity. The activity of p16 could be observed in the absence of vesicle formation, suggesting that it may participate in the docking and fusion processes.

Differential Modes of Nuclear Localization Signal (NLS) Recognition by Three Distinct Classes of NLS Receptors

The targeting of karyophilic proteins to nuclear pores is mediated via the formation of a nuclear pore-targeting complex, through the interaction of nuclear localization signal (NLS) with its NLS receptor. Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1. This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1. Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS. By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system. Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs. These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS.

Regulation of Protein Kinase B in Rat Adipocytes by Insulin, Vanadate, and Peroxovanadate: MEMBRANE TRANSLOCATION IN RESPONSE TO PEROXOVANADATE

Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.

Biosynthesis of α-Farnesene in Apple Fruit

α-Farnesene is an acyclic sesquiterpene hydrocarbon that is a constituent of the surface wax of apples (Malus domestica Borkh.). Although, oxidation products of α-farnesene have been implicated in the development of the physiological disorder superficial scald in apple, the mechanism of α-farnesene biosynthesis has not been studied in detail. We are currently investigating α-farnesene biosynthesis in relation to superficial scald development in apples. Radiolabelled feeding experiments using isolated tissue segments indicated that α-farnesene is derived from trans,trans-farnesyl pyrophosphate (FPP), mainly in the skin rather than cortex. Among the other labeled products detected, farnesol level was over a hundred-fold higher compared to α-farnesene. However, [1-3H] trans,trans-Farnesol was not incorporated into α-farnesene. Feeding radiolabelled FPP to skin tissue segments of scald-developing and normal apples showed differential incorporation of radiolabel into various products. Though the incorporation into α-farnesene was nearly the same, there was higher levels of incorporation into farnesyl esters in normal apples. As well, the levels of radiolabelled in the farnesol fraction was three times higher in scald-developing regions. These results indicate that there are potential difference in the biosynthesis and metabolism of farnesyl components between scald-developing and normal apples. In studies using cell-free extracts, farnesol formation was observed from labeled FPP and was two-fold higher in crude membrane extract compared to crude cytosol. Our results indicate that α-farnesene formation in apple fruit tissue is through FPP and is possibly catalyzed by a single sesquiterpene synthase enzyme. Purification and characterization of this enzyme are in progress.