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PubMed1983

Cytoplasmic dynein of the sea urchin egg. II. Purification, characterization and interactions with microtubules and Ca-calmodulin.

Purification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised several chromatographies, including a calmodulin-Sepharose 4B affinity column chromatography. This is the first report of the purification of cytoplasmic dynein to near homogeneity. The purified fraction was composed of a single high molecular weight polypeptide and some minor low molecular weight polypeptides. The high molecular weight polypeptide comigrated with flagellar dynein A beta chain from sperm on SDS-polyacrylamide gels. There was no polypeptide stainable with periodic acid-Schiff reagent (PAS) in the purified cytoplasmic dynein fraction. Cytoplasmic dynein showed characteristics quite similar to those of axonemal dynein, i.e. high substrate specificity for ATP and inhibition by low concentrations of vanadate, though its Ca-ATPase activity showed almost the same dependence on the concentration of either divalent cations or KC1 as the Mg-ATPase activity. The purified enzyme seemed to possess functional form as judged from its properties: 1) pH dependence of the ATPase activity, 2) dependence of the ATPase activity on MgCl2 and KCl concentration, 3) Km for Mg-ATP, and 4) binding to flagellar doublet microtubules. Cytoplasmic dynein bound to calmodulin-Sepharose 4B only in the presence of Ca2+, and was eluted with EGTA. Furthermore, the ATPase activity was enhanced 6-fold by calmodulin in a Ca2+-dependent manner. The activation by calmodulin was prevented by a stoichiometric amount of trifluoperazine.

Adenosine Triphosphatases↗
PubMed2003

Cytoplasmic versus nuclear localization of Fos-related proteins in the frog, Rana esculenta, testis: in vivo and direct in vitro effect of a gonadotropin-releasing hormone agonist.

Evidence has been accumulated indicating that GnRH-like peptides are present in a variety of extrabrain areas of mammalian and nonmammalian vertebrates. A pioneer study carried out in the frog, Rana esculenta, demonstrated that testicular GnRH induced spermatogonial proliferation. Recently, we have shown that in proliferating spermatogonia (SPG) of frogs, a change of localization of the oncoprotein Fos, from the cytoplasm to the nucleus, occurs. This leads to the hypothesis that one or more testicular GnRH peptides may regulate SPG proliferation through Fos family proteins. Therefore, in vivo experiments in intact R. esculenta and in vitro incubations of testis fragments have been carried out using GnRH agonist (GnRHa; buserelin) and GnRH antagonist (D-pGlu(1),D-Phe(2),D-Trp(3,6)-GnRH). Cytoplasmic and nuclear Fos-like protein localization has been found by Western blot analysis in testicular extracts. Immunocytochemistry confirmed that cytoplasmic immunostaining was restricted to SPG; change of localization into the nuclear compartment was observed after GnRHa treatment. Northern blot analysis showed that treatments of testis fragments with GnRHa did not modify testicular c-fos mRNA expression. On the contrary, a Fos-like protein of 52 kDa, while not affected in vivo, disappeared from testicular cytosolic extracts after in vitro treatment with GnRHa. Contemporaneously, a 55-kDa Fos-related signal appeared in nuclear extracts. The GnRH antagonist counteracted the effects of GnRHa. Furthermore, in vivo treatments showed that GnRHa acted negatively on a 43-kDa nuclear Fos-related signal and that gonadotropins caused the decrease of 52-kDa cytoplasmic signal. In conclusion, we show, to our knowledge for the first time, that Fos is regulated by GnRHa directly (not through the pituitary) at the testicular level. The main effect appears to be related to Fos translocation from cytoplasmic to nuclear compartments of SPG.

Animals↗
PubMed2004

Expression of cyclin B1 messenger RNA isoforms and initiation of cytoplasmic polyadenylation in the bovine oocyte.

Oocytes can synthesize and store maternal mRNA in an inactive translational state until the start of in vitro maturation. Cytoplasmic polyadenylation, driven by 3'-untranslated region (UTR) cis-acting cytoplasmic polyadenylation element (CPE), is associated with translational activation of cyclin B1 mRNA during maturation. The main aim of the present study was to investigate if bovine oocyte cyclin B1 mRNA undergoes cytoplasmic polyadenylation/translation during in vitro maturation, as in other species. We have found that cyclin B1 mRNA is present in two isoforms, consisting of the same open reading frame but with different 3'-UTR lengths. Only the longest isoform (cyclin B1L) has a putative CPE sequence and other regulatory sequences, and its mRNA level decreases during early embryo development. The polyadenylation state of cyclin B1L during in vitro maturation was studied. Results demonstrated that cyclin B1L bears a relatively long poly(A) tail in germinal vesicle-stage oocytes, which is further lengthened at 10 h of maturation, before metaphase I. Interestingly, cyclin B1L bears a short poly(A) tail when the ovaries and the oocytes are transported and manipulated on ice to stop the polyadenylation process. Cytoplasmic polyadenylation most probably occurs during ovary transport in warm saline, when oocytes are still in their follicular environment. Our results also show a link between cytoplasmic polyadenylation of cyclin B1 and translation/appearance of cyclin B1 protein before in vitro maturation.

Animals↗
PubMed1984

Phagocytosis of sperm cytoplasmic droplets by a specialized region in the epididymis of the brushtailed possum, Trichosurus vulpecula.

During sperm maturation in the brushtailed possum, Trichosurus vulpecula, cytoplasmic droplets are shed from maturing spermatozoa in the distal regions of the head of the epididymis. Examination of luminal contents from various regions of the epididymis showed that the proportion of detached droplets in the luminal contents was reduced from about 45% in the proximal corpus epididymidis to less than 10% in the distal corpus and cauda epididymides. In contrast, the proportion of droplet-free spermatozoa increased from about 45% to more than 90% in the luminal contents. Disappearance of detached cytoplasmic droplets from the lumen was found to be associated with a region of specialized principal cells lining Regions 6 and 7 of the epididymis which selectively sequester and phagocytose free droplets from the luminal milieu. The luminal surfaces of these cells are characterized by a complex system of interdigitating processes which appear as waves of microfolds . These processes contrast with the stereocilia which cover the luminal surfaces of principal cells in adjacent, nonphagocytic regions of the duct. Cytoplasmic droplets are phagocytosed with their limiting membrane intact and gradually become condensed as they are transported deeper into the cell. Membrane lamellae are gradually compacted, transformed into concentrically arranged membrane stacks and then condensed into small electron-dense vesicles, which are probably degraded by the epithelial cells. The presence of a specific recognition factor on cytoplasmic droplets is suggested by the observation that phagocytic principal cells are able to selectively remove detached cytoplasmic droplets from the lumen in the presence of sperm-associated droplets and spermatozoa.

Animals↗
PubMed2003

Isoflurane reduction of carbachol-evoked cytoplasmic calcium transients is dependent on caffeine-sensitive calcium stores.

BACKGROUND: Many muscarinic functions are relevant to anesthesia, and alterations in muscarinic activity affect the anesthetic/analgesic potency of various drugs. Volatile anesthetics have been shown to depress muscarinic receptor function, and inhibition of the muscarinic signaling pathway alters the minimal alveolar anesthetic concentration of inhaled anesthetics. The purpose of this investigation was to determine in a neuronal cell which source of Ca2+ underlying the carbachol-evoked transient increase in cytoplasmic Ca2+ was reduced by isoflurane. METHODS: Experiments were performed at 37 degrees C on continuously perfused monolayers of human neuroblastoma SH-SY5Y cells using Fura-2 as the cytoplasmic Ca2+ indicator. Carbachol (1 mm) was applied to evoke a transient increase in cytoplasmic Ca2+. RESULTS: Isoflurane (1 mm) reduces the carbachol-evoked transient increase in cytoplasmic Ca2+, and this isoflurane action is eliminated when the cells are continuously stimulated with 200 mm KCl or pretreated with 10 mm caffeine or 200 microm ryanodine. CONCLUSIONS: Isoflurane reduction of the carbachol-evoked transient increase in cytoplasmic Ca2+ requires full caffeine-sensitive Ca2+ stores and Ca2+ release from the caffeine-sensitive stores through the ryanodine-sensitive Ca2+ release channels. The results indicate that isoflurane interferes with a muscarinic Ca2+ signaling through a mechanism downstream from the muscarinic receptors.

Caffeine↗
PubMed1984

Cyclosporine does not prevent cytoplasmic calcium changes associated with lymphocyte activation.

The recent development of fluorescent reagents that are sensitive to calcium ions has made it possible to quantitate cytoplasmic calcium levels of lymphocytes. The cytoplasmic calcium concentration more than doubles within the first few minutes following lymphocyte stimulation. Here I show that it is likely that this increase in calcium is an absolute requirement for mitogenic commitment. Brief exposure of T cells to concanavalin A (Con A) is sufficient to stimulate cell division, provided that extracellular calcium ions are available to the cells at the time of exposure to mitogen--no mitogenesis occurs if extracellular calcium is below 10(-5)M. Commitment to mitosis requires both Con A acting at the cell surface and a source of extracellular calcium, so it follows that the plasma membrane is implicated in the control of cytoplasmic calcium changes. The lipid-soluble nature of cyclosporine (Cys) results in the partitioning of this drug into the plasma membrane of lymphocytes; the possibility arises that Cys might inhibit lymphocyte activation by perturbing membrane structure so as to alter regulatory fluxes in cytoplasmic calcium. Experiments reported here show that early changes in cytoplasmic calcium levels of activated T cells occur normally in the presence of Cys.

Aminoquinolines↗
PubMed1995

Antineutrophil cytoplasm autoantibodies and vasculitis.

Antineutrophil cytoplasm autoantibodies are useful diagnostic serologic markers for a variety of well-known primary vasculitic syndromes, including Wegener's granulomatosis, microscopic polyangiitis, and idiopathic necrotizing and crescentic glomerulo-nephritis. More recently antineutrophil cytoplasm autoantibodies have been found in other vasculitic syndromes, such as Churg-Strauss syndrome, Henoch-Schönlein purpura, and some nonvasculitic diseases such as rheumatoid arthritis, inflammatory bowel disease, and autoimmune hepatobiliary diseases. There is now evidence to suggest that infection might be an important etiologic factor in the development of antineutrophil cytoplasm autoantibody-associated vasculitides. This link has been strengthened by in vitro data that suggest that antineutrophil cytoplasm autoantibodies are directly involved in the pathogenesis of antineutrophil cytoplasm autoantibody-associated vasculitides.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2003

Angiomyolipoma and PEComa are immunoreactive for MyoD1 in cell cytoplasmic staining pattern.

The family of tumors derived from mesenchymal perivascular epithelioid cells (so-called PEComas) includes angiomyolipoma, lymphangioleiomyomatosis, clear cell sugar tumor of the lung, clear cell myomelanocytic tumor of ligamentum teres/falciform ligament, and abdominopelvic sarcoma of perivascular epithelioid cells. These tumors were characterized by coexpression of melanocytic (HMB-45) and muscle markers. MyoD1 transcription factor has crucial role in commitment and differentiation of mesenchymal progenitor cells to myogenic lineage. Antibodies to MyoD1 protein (nuclear immunoreactivity) have been shown highly valuable adjuncts in the diagnosis of rhabdomyosarcomas. To evaluate expression of the transcription factor MyoD1 in PEComas, we performed immunohistochemistry. Monoclonal antibody 5.8A for MyoD1 was used on a series of cases of formalin-fixed, paraffin-embedded angiomyolipoma (n = 19), lymphangioleiomyomatosis (n = 3), clear cell sugar tumor of the lung (n = 1), and abdominopelvic sarcoma of perivascular epithelioid cells (n = 2). All cases showed strong granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 antibody. Cytoplasmic reactivity was noted in the spindle cells, fat cells, and epithelioid cells. Nuclei were negative in all tumors studied, and a clean background was obtained. Several normal and neoplastic human tissues have also been immunostained for MyoD1 without any positive cytoplasmic staining, with the exception of 2 alveolar soft part sarcomas. Cytoplasmic immunostaining with monoclonal antibody 5.8A for MyoD1 in PEComas may correspond to cross-reactivity with an undetermined cytoplasmic protein. Great caution should be exercised in interpreting the immunostaining results with anti-MyoD1 antibody 5.8A.

Angiomyolipoma↗
PubMed1990

Temporal and spatial correlation of fertilization current, calcium waves and cytoplasmic contraction in eggs of Ciona intestinalis.

Eggs of the ascidian Ciona intestinalis were loaded with the calcium indicator fura-2 via whole-cell clamp electrodes and changes in cytoplasmic calcium and cell currents were monitored during fertilization either in separate eggs or simultaneously in the same egg. The first indication of egg activation was the fertilization current; which reached peak values around 1 nA after 30 s. A wave of elevated calcium was detectable between 5 s and 30 s (mean = 21 s) after the start of the fertilization current. This wave spread across the egg increasing cytoplasmic calcium levels to at least 10 microM. When the fertilization current and calcium wave were complete and cytoplasmic calcium levels were decreasing to prefertilization levels, a cortical contraction wave spread across the egg surface. In eggs showing normal fertilization current, the calcium wave and the contraction wave were in the same direction. A region of elevated calcium persisted at the animal pole. Changing cytoplasmic calcium levels locally by local application of ionophore A23187 caused a contraction wave originating at the site of ionophore application. Increasing cytoplasmic calcium uniformly by facilitating calcium entry through voltage-regulated channels did not result in a contraction wave.

Animals↗
PubMed1978

Pre-lytic release of foot-and-mouth disease virus in cytoplasmic blebs.

The pre-lytic release mechanism of foot-and-mouth disease virus was investigated by immunofluorescence, acridine orange staining, and electron microscopy in infected bovine and porcine kidney coverslip cultures. Cells with cytoplasmic fluorescence and which were positive for single stranded RNA with acridine orange staining were observed at 2 h after infection. Scanning electron microscopy showed cytoplasmic blebs in all cultures examined 2 h after infection. Rounded cells with virus inclusions began to appear 3 h after infection. Rounded cells and cytoplasmic blebs were shown to have single stranded RNA by acridine orange staining. Immunofluorescence and transmission electron microscopy with immunoferritin tagging demonstrated foot-and-mouth disease virus in cytoplasmic blebs. This study presents evidence for a pre-lytic release of foot-and-mouth disease virus through virus-containing cytoplasmic blebs emerging from infected cells.

Aphthovirus↗
PubMed2005

Anti-Saccharomyces cerevisiae and perinuclear anti-neutrophil cytoplasmic antibodies in coeliac disease before and after gluten-free diet.

BACKGROUND: Anti-Saccharomyces cerevisiae and perinuclear anti-neutrophil cytoplasmic autoantibodies are markers of Crohn's disease and ulcerative colitis respectively. AIM: To determine the prevalence of anti-S. cerevisiae and perinuclear anti-neutrophil cytoplasmic autoantibodies in a large series of coeliac disease patients before and after gluten free diet, and to correlate anti-S. cerevisiae-positivity with intestinal mucosal damage. METHODS: One hundred and five consecutive coeliac disease patients and 141 controls (22 ulcerative colitis, 24 Crohn's disease, 30 primary sclerosing cholangitis, 15 postenteritis syndrome, 50 blood donors) were tested for anti-S. cerevisiae by enzyme-linked immunosorbent assay and for perinuclear anti-neutrophil cytoplasmic autoantibodies by indirect immunofluorescence. RESULTS: In coeliac disease anti-S. cerevisiae (immunoglobulin G and/or immunoglobulin A) were slightly less frequent (59%) than in Crohn's disease (75%, P = 0.16) and significantly more frequent than in ulcerative colitis (27%), primary sclerosing cholangitis (30%), postenteritis syndrome (26%) and blood donors (4%) (P = 0.009, P = 0.0002, P = 0.025, P < 0.0001). No correlation was found between anti-S. cerevisiae and degree of mucosal damage. Perinuclear anti-neutrophil cytoplasmic autoantibodies were detected only in one coeliac. After gluten free diet the disappearance of anti-S. cerevisiae-immunoglobulin A (93%) was more frequent than that of immunoglobulin G (17%, P = 0.0001); perinuclear anti-neutrophil cytoplasmic autoantibodies disappeared in the only coeliac positive at diagnosis. CONCLUSION: More than half of untreated coeliacs are anti-S. cerevisiae-positive irrespective of the severity of mucosal damage. Differently from immunoglobulin A, anti-S. cerevisiae-immunoglobulin G persisted in more than 80% after gluten free diet. The high prevalence of anti-S. cerevisiae in coeliac disease suggests that they may be the effect of a non-specific immune response in course of chronic small bowel disease.

Adolescent↗
PubMed1986

Electron-immunocytochemical localization of neuron-specific enolase in cytoplasm and on membranes of primary and metastatic cerebral tumours and on glial filaments of glioma cells.

A series of primary and metastatic human brain tumours was evaluated immunocytochemically for the electron microscopic localization of neuron-specific enolase (NSE). All contained cells which, regardless of the cell type, demonstrated an irregular distribution of NSE in their cytoplasm and on membranes. This was in contrast to the staining pattern in normal central nervous system (CNS) cells which, as previously reported (Vinores et al. 1984b), show only diffuse cytoplasmic staining usually not associated with membranes. In the tumours, the interior of nuclei and the cristae and matrices of mitochondria were consistently negative, as in normal CNS cells. Except in one low-grade fibrillary astrocytoma, the cytoplasmic filaments in neoplastic astrocytes were often, but not invariably, stained for NSE. The fine structural localization of NSE in neoplastic cells suggests that the conversion of 2-D-glycerophosphate to phosphoenolpyruvate by enolase may occur on the membrane and, in the case of astrocytic tumours, on the cytoplasmic filaments as well as in the cytoplasm. When cells which contain only the non-neuronal form of enolase (NNE) transform to neoplastic cells, they may acquire the ability to produce NSE. This presumably enables them to accommodate the increased metabolic demands of neoplasia by allowing them to elude the regulatory controls that are specific for NNE.

Adolescent↗
PubMed2006

Cytoplasmic localization of p63 is associated with poor patient survival in lung adenocarcinoma.

AIMS: To determine the significance of p63 protein expression in the development and progression of lung adenocarcinoma. METHODS AND RESULTS: The expression of p63 was immunohistochemically investigated in 92 cases of lung adenocarcinoma with a maximum diameter of 30 mm or less. p63 expression was observed not only in the nuclei (46/92 cases, 50%), but also in the cytoplasm of neoplastic cells (47/92, 51%). Nuclear localization of p63 was correlated with nuclear accumulation of p53 (P=0.0120), whereas the presence of nuclear p63 had no apparent effect on patient survival. Cytoplasmic localization of p63 was found to be correlated with shorter survival periods by univariate and multivariate analyses (P=0.0486 and P=0.0488, respectively) and the relation was independent of clinicopathological factors. Cytoplasmic localization of p63 was further confirmed by immunoblots of the cytoplasmic fraction of HLC-1, a lung adenocarcinoma cell line which predominately expressed DeltaNp63alpha transcript relative to TAp63 transcript by quantitative reverse transcriptase-polymerase chain reaction. CONCLUSIONS: Cytoplasmic expression of p63 is an adverse prognostic factor in patients with adenocarcinoma of the lung.

Adenocarcinoma↗
PubMed1992

Phylogeny of cytoplasmic incompatibility micro-organisms in the parasitoid wasp genus Nasonia (Hymenoptera: Pteromalidae) based on 16S ribosomal DNA sequences.

Cytoplasmic incompatibility results in embryo mortality in diploids, or all male offspring in haplodiploids, when individuals carrying different cytoplasmic factors are crossed. Cytoplasmic factors have been identified as intracellular micro-organisms. Microbe-induced cytoplasmic incompatibility is found in many insect taxa and may play a role in reproductive isolation between populations. Such micro-organisms cause bidirectional incompatibility between species of the parasitoid wasp genus Nasonia. The phylogenetic relationship of cytoplasmic incompatibility microorganisms (CIM) of different Nasonia species was analysed using their 16S ribosomal DNA (rDNA) sequence. Two 16S rDNA operons were detected in the CIM of each Nasonia species. Sequence analysis indicates that the Nasonia CIM are closely related and belong to the alpha group of the Proteobacteria.

Animals↗
PubMed1988

Identification of cytoplasmic nodule-associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum.

The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion-exchange chromatography into four different fractions. Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak. During nodule development an increase in malate dehydrogenase activity per gram of material was observed. This increase occurred concomitantly with the increase in nitrogenase activity. The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied. The Km values for malate (2.6 mM), NAD+ (27 microM), oxaloacetate (18 microM) and NADH (13 microM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 microM and 70 microM respectively). Binding of malate by the enzyme-NADH complex from root nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH. The dominant root malate dehydrogenase does not form the abortive complex. From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacteroid and the infected plant cell. The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration. Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.

Biological Transport↗
PubMed1988

Yeast adenylate kinase is active simultaneously in mitochondria and cytoplasm and is required for non-fermentative growth.

Displacement of the single copy structural gene for yeast adenylate kinase (long version) by a disrupted nonfunctional allele is tolerated in haploid cells. Since adenylate kinase activity is a pre-requisite for cell viability, the survival of haploid disruption mutants is indicative of the presence of an adenylate kinase isozyme in yeast, capable of forming ADP from AMP and, thus, of complementing the disrupted allele. The phenotype of these disruption mutants is pet, showing that complementation occurs only under fermentative conditions. Even on glucose, growth of the disruption mutants is slow. Adenylate kinase activity is found both in mitochondria and cytoplasm of wild type yeast. The disruption completely destroys the activity in mitochondria, whereas in the cytoplasmic fraction about 10% is retained. An antibody raised against yeast mitochondrial adenylate kinase recognizes cross-reacting material both in mitochondria and cytoplasm of the wild type, but fails to do so in each of the respective mutant fractions. The data indicate that yeast adenylate kinase (long version, AKY2) simultaneously occurs and is active in mitochondria and cytoplasm of the wild type. Nevertheless, it lacks a cleavable pre-sequence for import into mitochondria. A second, minor isozyme, encoded by a separate gene, is present exclusively in the cytoplasm.

Adenosine Diphosphate↗
PubMed1991

Kinetic studies of the variations of cytoplasmic pH, nucleotide triphosphates (31P-NMR) and lactate during normoxic and anoxic transitions in maize root tips.

We have followed the dynamic evolution of intracellular pH and of the intracellular concentration of nucleotides (NDP, NTP), Pi and lactate in maize root tips during the course of normoxia and anoxia transition. The intracellular pH, determined from the 31P-NMR chemical shift of the cytoplasmic P1 peak, dropped from 7.5 to 6.9 during the first few minutes after anaerobiosis. It increased again, then settled to a steady-state value of 7.1-7.2, 25 min after the beginning of the anoxic treatment. Following oxygenation, the chemical shift of the cytoplasmic Pi peak drifted gradually to its initial value. The cytoplasmic pH followed an oscillatory time course which was almost identical to the time course of NTP. Intracellular lactate accumulated steadily during the first 30 min after anaerobiosis, then its intracellular concentration remained almost constant. Following oxygenation, the intracellular concentration of lactate decreased slowly. The cytoplasmic pH followed a time course which was not identical to the time course of lactate. Following hypoxia, the pH dropped to low values long before the intracellular lactate concentration reached a steady-state equilibrium. Conversely, subsequent to oxygenation, the pH returned to normal values long before lactate. These results do not agree with the statement that cytoplasmic acidification in hypoxic maize root tips is necessarily associated with lactic acid synthesis.

Cytoplasm↗
PubMed1997

Extraction of Hoechst 33342 from the cytoplasmic leaflet of the plasma membrane by P-glycoprotein.

P-glycoprotein is an ATP-dependent plasma membrane multidrug transporter of broad specificity. A common chemical property of its substrates is that all are lipophilic. Using Hoechst 33342 as the substrate, we have previously shown that P-glycoprotein extracts the substrate directly from the lipid bilayer [Shapiro, A. B., Corder, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 115-121]. In this paper, we determined the leaflet of the plasma membrane from which P-glycoprotein extracts Hoechst 33342. The initial rate of Hoechst 33342 transport upon ATP addition to P-glycoprotein-rich inside-out plasma membrane vesicles decreased slightly with the amount of time previously elapsed for slow diffusion of Hoechst 33342 to the extracellular leaflet. This result is consistent with transport from the cytoplasmic leaflet. Fluorescence resonance energy transfer from donor Hoechst 33342 to acceptor 2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-sn-glycero- 3-phosphocholine (Nbd-C6-HPC) in the cytoplasmic leaflet was used to monitor the amount of Hoechst 33342 in the cytoplasmic leaflet versus time. The initial rate of decrease of the energy-transfer-related Nbd-C6-HPC fluorescence after ATP addition exceeded that of the Hoechst 33342 fluorescence and continued to decrease after decrease of the Hoechst 33342 fluorescence had ceased. These effects were consistent with transport of Hoechst 33342 from the cytoplasmic leaflet to the aqueous interior of the vesicles, followed by rebinding to the extracellular leaflet. This demonstrates that P-glycoprotein transports drugs from the cytoplasmic leaflet of the plasma membrane directly to the aqueous extracellular medium. This finding has implications for efforts to localize the drug-binding site(s) within P-glycoprotein.

4-Chloro-7-nitrobenzofurazan↗