The regulation of amino acid transport from the vacuolar reservoir into the cytoplasm has been studied in hyphal cells of Penicillium cyclopium. To avoid artifacts caused by the isolation of vacuoles, efflux was examined “in situ,”i.e. in cells whose plasma membranes were permeabilized for micromolecules by a treatment with nystatin. The ATP-dependent proton gradient and amino acid transport activities at the vacuolar membrane remained intact under these conditions. Accumulation of amino acids in the vacuole proved to be the result of a dynamic equilibrium of active, ATP-dependent uptake and energy-independent efflux. The latter was strongly accelerated after the vacuolar amino acid content had surpassed a threshold level.Efflux of vacuolar amino acids was specifically controlled by extravacuolar adenylates: ATP, 5′-adenylyl imidodiphosphate (an ATPase-resistant ATP-analogue), ADP, or AMP caused a strong inhibition in the concentration range around 200 μmol/liter, whereas both lower and higher concentrations allowed significant efflux rates. Estimates of the cytosolic adenylates (which consisted mainly of ATP) were close to 2 mmol/liter in glucose-metabolizing cells, which concentration allowed maximum rates of both vacuolar uptake and efflux. During 24 h of carbon and nitrogen starvation, the adenylate level decreased toward the efflux-inhibiting region around 200 μmol/liter, whereas 3–4 d of carbon and nitrogen starvation caused a further decline of the adenylate content, leading again to efflux-permitting concentrations. Thus, the cytosolic adenylate pool appears to effectively control the availability of vacuolar amino acids for the cellular metabolism. The regulation of amino acid transport from the vacuolar reservoir into the cytoplasm has been studied in hyphal cells of Penicillium cyclopium. To avoid artifacts caused by the isolation of vacuoles, efflux was examined “in situ,”i.e. in cells whose plasma membranes were permeabilized for micromolecules by a treatment with nystatin. The ATP-dependent proton gradient and amino acid transport activities at the vacuolar membrane remained intact under these conditions. Accumulation of amino acids in the vacuole proved to be the result of a dynamic equilibrium of active, ATP-dependent uptake and energy-independent efflux. The latter was strongly accelerated after the vacuolar amino acid content had surpassed a threshold level. Efflux of vacuolar amino acids was specifically controlled by extravacuolar adenylates: ATP, 5′-adenylyl imidodiphosphate (an ATPase-resistant ATP-analogue), ADP, or AMP caused a strong inhibition in the concentration range around 200 μmol/liter, whereas both lower and higher concentrations allowed significant efflux rates. Estimates of the cytosolic adenylates (which consisted mainly of ATP) were close to 2 mmol/liter in glucose-metabolizing cells, which concentration allowed maximum rates of both vacuolar uptake and efflux. During 24 h of carbon and nitrogen starvation, the adenylate level decreased toward the efflux-inhibiting region around 200 μmol/liter, whereas 3–4 d of carbon and nitrogen starvation caused a further decline of the adenylate content, leading again to efflux-permitting concentrations. Thus, the cytosolic adenylate pool appears to effectively control the availability of vacuolar amino acids for the cellular metabolism. The metabolism of amino acids in fungal and plant cells is strongly influenced by transport activities of the vacuole. The majority of free amino acids is usually concentrated in this organelle, the relation between vacuolar and cytosolic pools being different for individual amino acid species (1Sakano K. Tazawa M. Plant Cell Physiol. 1984; 25: 1477-1486Crossref Scopus (39) Google Scholar, 2Kitamoto K. Yoshizawa K. Ohsumi Y. Anraku Y. J. Bacteriol. 1988; 170: 2683-2686Crossref PubMed Scopus (145) Google Scholar, 3Horak J. Biochim. Biophys. Acta. 1986; 864: 223-256Crossref PubMed Scopus (108) Google Scholar, 4Dietz K.-J. Jäger R. Kaiser G. Martinoia E. Plant Physiol. 1990; 92: 123-129Crossref PubMed Scopus (56) Google Scholar, 5Carroll A.D. Stewart G.R. Phillips R. Plant Physiol. 1992; 100: 1808-1814Crossref PubMed Scopus (6) Google Scholar). Vacuolar amino acids are used as a reservoir to maintain the concentrations of cytosolic amino acids within distinct limits, thus compensating for changes of the external or biosynthetic supply and metabolic needs. As amino acid precursors are required for many essential anabolic and catabolic activities, the control over their vacuolar pool is assumed to have a high significance for metabolic regulation. Classical examples are the controlled exchange of vacuolar and cytoplasmic arginine and ornithine during the transition between anabolic and catabolic steady states inNeurospora crassa (6Davis R.H. Bowman B.J. Weiss R.L. J. Supramol. Struct. 1978; 9: 473-488Crossref PubMed Scopus (11) Google Scholar, 7Drainas C. Weiss R.L. J. Bacteriol. 1982; 150: 770-778Crossref PubMed Google Scholar, 8Drainas C. Weiss R.L. J. Bacteriol. 1982; 150: 779-784Crossref PubMed Google Scholar) or the response of the vacuolar amino acid pool to nutrient-induced changes of the rate of protein synthesis in leaves of barley or spinach (9Dietz K.-J. Martinoia E. Heber U. Biochim. Biophys. Acta. 1989; 984: 57-62Crossref Scopus (16) Google Scholar). In eukaryotic microorganisms various tonoplast transporters have been identified that catalyze the uptake of amino acids into the vacuole at the expense of proton-motive force generated by the tonoplast H+-ATPase and tonoplast H+-pyrophosphatase (10Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar, 11Cooper T.G. Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 399-461Google Scholar, 12Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11505-11508Abstract Full Text PDF PubMed Google Scholar, 13Paek Y.L. Weiss R.L. J. Biol. Chem. 1989; 264: 7285-7290Abstract Full Text PDF PubMed Google Scholar, 14Homeyer U. Litek H. Huchzermeyer B. Schultz G. Plant Physiol. 1989; 89: 1388-1393Crossref PubMed Google Scholar, 15Martinoia E. Bot. Acta. 1992; 105: 232-245Crossref Scopus (84) Google Scholar). In plant cells similar evidence is less abundant (16Boller Th Dürr M. Wiemken A. Eur. J. Biochem. 1975; 54: 81-91Crossref PubMed Scopus (57) Google Scholar, 17Boller Th Wiemken A. Annu. Rev. Plant Physiol. 1986; 37: 137-164Crossref Google Scholar), and proton-motive force-independent transport (stimulated by ATP and its ATPase-resistant analogue) has been reported as well (4Dietz K.-J. Jäger R. Kaiser G. Martinoia E. Plant Physiol. 1990; 92: 123-129Crossref PubMed Scopus (56) Google Scholar, 9Dietz K.-J. Martinoia E. Heber U. Biochim. Biophys. Acta. 1989; 984: 57-62Crossref Scopus (16) Google Scholar,18Martinoia E. Thume M. Rentsch D. Dietz K.-J. Plant Physiol. 1991; 97: 644-650Crossref PubMed Scopus (24) Google Scholar). The cellular control over the vacuolar amino acid pools suggests that tonoplast transport systems are involved in regulatory circuits that sense cytosolic conditions and respond by switching between accumulation into the organelle and efflux into the cytosol. Whereas some information about the regulation of vacuolar uptake can be deduced from kinetic and regulatory properties of amino acid transport systems at the tonoplast (3Horak J. Biochim. Biophys. Acta. 1986; 864: 223-256Crossref PubMed Scopus (108) Google Scholar, 4Dietz K.-J. Jäger R. Kaiser G. Martinoia E. Plant Physiol. 1990; 92: 123-129Crossref PubMed Scopus (56) Google Scholar, 10Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar, 15Martinoia E. Bot. Acta. 1992; 105: 232-245Crossref Scopus (84) Google Scholar, 17Boller Th Wiemken A. Annu. Rev. Plant Physiol. 1986; 37: 137-164Crossref Google Scholar, 18Martinoia E. Thume M. Rentsch D. Dietz K.-J. Plant Physiol. 1991; 97: 644-650Crossref PubMed Scopus (24) Google Scholar) and the energizing proton pumps (19Rea P.A. Sanders D. Physiol. Plant. 1987; 71: 131-141Crossref Scopus (279) Google Scholar, 20Anraku Y. Umemoto N. Hirata R. Wada Y. J. Bioenerg. Biomembr. 1989; 21: 589-603Crossref PubMed Scopus (43) Google Scholar, 21Sze H. Ward J.M. Lay S. J. Bioenerg. Biomembr. 1992; 24: 371-381Crossref PubMed Scopus (180) Google Scholar), the mechanism(s) governing the efflux of accumulated amino acids into the cytosol are largely unknown. Such knowledge would promote the understanding of the regulatory properties of the vacuolar transporters and of their impact on enzymic processes in the cytoplasm, whose conversion rates might be controlled by the efflux of precursors from their vacuolar pools (e.g. see Ref. 22Roos W. Brossi A. The Alkaloids. 39. Academic Press, New York1990: 63-97Google Scholar). In N. crassa, Weiss and co-workers (8Drainas C. Weiss R.L. J. Bacteriol. 1982; 150: 779-784Crossref PubMed Google Scholar, 23Legerton T.L. Weiss R.L. J. Biol. Chem. 1984; 259: 8875-8879Abstract Full Text PDF PubMed Google Scholar) described a stimulating effect of glutamine limitation on the energy-requiring efflux of vacuolar arginine. In vacuoles isolated from barley mesophyll protoplasts Dietz et al. (9Dietz K.-J. Martinoia E. Heber U. Biochim. Biophys. Acta. 1989; 984: 57-62Crossref Scopus (16) Google Scholar) found the efflux of amino acids to be stimulated by ATP and its ATPase-resistant analogue AMP-PNP 1The abbreviations used are: AMP-PNP, 5′-adenylyl imidodiphosphate; 4-MUP, 4-methylumbelliferyl phosphate; G6PDH, glucose-6-phosphate dehydrogenase; CCCP, carbonylcyanidem-chlorophenylhydrazone; fwt, fresh weight; MES, 4-morpholineethanesulfonic acid; CTAB, cetyltrimethylammonium bromide. 1The abbreviations used are: AMP-PNP, 5′-adenylyl imidodiphosphate; 4-MUP, 4-methylumbelliferyl phosphate; G6PDH, glucose-6-phosphate dehydrogenase; CCCP, carbonylcyanidem-chlorophenylhydrazone; fwt, fresh weight; MES, 4-morpholineethanesulfonic acid; CTAB, cetyltrimethylammonium bromide. and to be inhibited by neutral cytosolic amino acids. In the present report we describe two novel control mechanisms of vacuolar efflux that became apparent in fungal cells whose plasma membrane was selectively permeabilized for micromolecules: (a) stimulating as well as inhibitory effects exerted by distinct concentrations of extravacuolar adenylates and (b) a sigmoidal concentration dependence of amino acid efflux. Submerged batch cultures of Penicillium cyclopium, strain SM 72a, were cultivated from conidiospores as described elsewhere (24Pönitz J. Roos W. J. Bacteriol. 1994; 176: 5429-5438Crossref PubMed Google Scholar). The culture liquid contained (mmol/liter): sucrose, 117; glucose, 55; ammonium tartrate, 35; MgSO4, 4.0; KH2PO4, 2.75; FeSO4, 0.18; ZnSO4, 0.17; trace elements (μmol/liter): Mn2+, 14; Co2+, 1.7; Cu2+, 0.32; MoO42−, 0.41; Triton X-100, 0.003%. 100-ml cultures were grown in 300-ml Erlenmeyer flasks on rotary shakers (250 rpm) at 24 °C. After 2 d of growth, a 100-ml submerged culture was diluted with 500 ml of maleate buffer, pH 4.5, 50 mmol/liter, and filtered through a nylon mesh (pore diameter ∼500 μm) to remove hyphal pellets. The fine, hairy grown mycelium was washed twice with 250 ml of the same buffer by suction filtration. The cells were then resuspended (4 mg fwt/ml) in half-concentrated culture medium without ammonium tartrate and shaken for another 12 h in order to allow the derepression of nitrogen-regulated transport activities (25Roos W. Biochim. Biophys. Acta. 1989; 978: 119-133Crossref PubMed Scopus (19) Google Scholar). After 2 d of growth, submerged hyphae (approximately 100 mg fwt) were filtered and washed by suction filtration with 20 ml of sodium citrate buffer, 5 mmol/liter, pH 4.5, containing 1 mol/liter sorbitol, resuspended in 10 ml of the same buffer, and gently shaken for 5 min at 24 °C. Then 5 μl of a nystatin solution (10 mg/ml in Me2SO) were added, and the cells were incubated with shaking for another 6 min (or times indicated otherwise). The effect of nystatin was stopped by adding 20 ml of HEPES buffer, pH 7.0, 50 mmol/liter containing 150 mmol/liter KCl, 50 mmol/liter NaCl, 600 mmol/liter sorbitol, 5 mmol/liter CaCl2, and 5 mmol/liter MgCl2. Finally, the cells were washed by suction filtration and resuspended in the last buffer containing 1 mmol/liter ATP. The success of permeabilization and the intactness of the vacuolar accumulation capacity was checked prior to each transport experiment by following the vacuolar accumulation of neutral red from 2 mmol/liter solutions in the presence and absence of 1 mmol/liter ATP (see Fig. 2). This was done both microscopically and by following the decrease ofA 530 of the supernatant. The test is based on the observation that ATP does not support dye accumulation in the presence of bafilomycin and that intact cells stain much slower and do not react to ATP, which is analogous to the uptake of Phe (see TableI).Figure 2Visualization of an ATP-dependent pH gradient across the vacuolar membrane in situ.Transmission microphotographs, obtained with the Nikon Optiphot microscope. Nystatin-treated and control cells were incubated with neutral red (2 mmol/liter) for 10 min in HEPES buffer, pH 7.0, 50 mmol/liter, containing 150 mmol/liter KCl, 50 mmol/liter NaCl, 600 mmol/liter sorbitol, 5 mmol/liter CaCl2, and 5 mmol/liter MgCl2. a, control (no nystatin treatment);b, after 6 min of nystatin treatment, staining in the presence of 1 mmol/liter MgATP; c as b, MgATP omitted; d, as in b, after 12 min of nystatin treatment; e, after 2-min treatment with 0.02% CTAB, staining in the presence of 1 mmol/liter MgATP. Accumulated dye is seen as dark spots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IComparison of Phe transport by intact and nystatin-treated hyphal cellsUptake of [14C]Phe, C o = 50 μmol/liter1-aData are means of four experiments, S.D. = 7–11%.Intact cellsNystatin treatedpH optimum3.57.0Accumulation ratio (C in/C out) in the presence of 1 mmol/liter ATP15001250Initial uptake rates (%)1-b100% of initial uptake rate represent 7.2 nmol of [14C]Phe/min/mg of protein, determined with intact cells in the absence of ATP and osmotic stabilizers.in the presence of 1 mmol/liter ATP + sorbitol 1 mol/liter94118 No sorbitol1073 bafilomycin, 2 μmol/liter966 NO3−, 100 mmol/liter10032 Vanadate, 1 mmol/liter4893 Azide, 0.1 mmol/liter15100In the absence of ATP10051-a Data are means of four experiments, S.D. = 7–11%.1-b 100% of initial uptake rate represent 7.2 nmol of [14C]Phe/min/mg of protein, determined with intact cells in the absence of ATP and osmotic stabilizers. Open table in a new tab Hyphal cells were resuspended (4 mg fwt/ml) in 60 mmol/liter sodium maleate buffer, pH 3.5 (intact cells), or in 60 mmol/liter sodium MES buffer, pH 7.0, with 0.4 mol/liter sorbitol, 0.15 mol/liter KCl, and 1 mmol/liter MgATP (permeabilized cells). These suspensions were gently aerated through a glass sinter funnel. After 1 min the unlabeled amino acid (final concentration 50 μmol/liter), together with the tracer amino acid (14C- or3H-labeled, final concentration <1 μmol/liter), and effectors in the appropriate buffer were added. Further details for uptake and efflux protocols are given in Figs. 3 and 4. The content of labeled amino acids given in the figures refers to the amount of free amino acids extractable from the cells with 80% ethanol. It was determined in 5-ml samples, which were rapidly injected into 10 ml of the same buffer at 0 °C, sucked onto cellulose nitrate filters (1.2 μm cutoff), and washed with 15 ml of this buffer (intact cells) or with 0.7 mol/liter sorbitol (permeabilized cells) at 0 °C. The cell pellet was then immersed in 2 ml of ethanol 80% (v/v), shaken for 2 h, and centrifuged at 5000 × g for 10 min, and radioactivity in 1 ml of the supernatant was counted by liquid scintillation. A Hewlett Packard LS counter was used in a dual channel mode that allowed the simultaneous detection of 14C and3H in discrete energy windows by automatically compensating for overlapping radiation via internal calibration curves. The separate detection of the isotopes was confirmed using test mixtures. Initial rates of uptake and efflux were computed from the experimental curves fitted to a hyperbolic equation (uptake curves) or the equation of exponential decay, y =a(e − Kx) + b, (efflux curves). At least 88% of the radioactivity extracted from the cells after 80 min of incubation with [14C]Phe was chromatographically identical with phenylalanin as confirmed by reversed phase high performance liquid chromatography amino acid analysis after derivatization with Fmoc-chloride (9-fluorenylmethyl chloroformate) (26Einarsson S. Josefsson B. Anal. Chem. 1987; 59: 1191-1195Crossref PubMed Scopus (302) Google Scholar). After two extraction steps with ethanol 80% (v/v), the total protein solubilized with 10% SDS (see below) contained <10% of the radioactivity found in the ethanol-soluble fraction. Hyphal suspensions were filtered by suction, the cell pellet (approximately 10 mg fwt) was rapidly immersed in boiling glycine buffer (100 mmol/liter, pH 11.0) and extracted for 10 min at 100 °C. After cooling to 20 °C, the extract was centrifuged at 6000 × g for 15 min, and the adenylates in the supernatant were assayed luminometrically. For ATP, 10-μl aliquots were injected into 300 μl of a luciferin/luciferase mixture reconstituted from lyophilized preparations (Boehringer Mannheim) in glycine buffer 100 mmol/liter, pH 7.4, and the luminescence counts integrated over 10 s in a luminometer (Clinilumat, Fa. Berthold). The method was calibrated with ATP solutions in the extraction buffer and validated via internal standards or addition of ATPase. S.D. = 8%, n = 5. ADP was assayed as the amount of ATP formed from phosphoenolpyruvate via pyruvate kinase as outlined by Hampp (27Hampp R. Bergmeyer H.U. Methods of Enzymatic Analysis. VII. VCH Verlagsgesellschaft, Weinheim1989: 370-379Google Scholar). AMP was assayed as the amount of ATP used by the adenylate kinase reaction. The reaction mixture contained adenylate kinase (0.9 unit/10 μl) and 10 mmol/liter MgS04 in 25 mmol/liter HEPES-K+, pH 7.5. ATP present in the native sample was used as the phosphate donor as its concentration always enabled a sufficient excess over AMP (see Fig.5 B). ADP and AMP could be determined in the presence of a ≤20-fold (ADP) or ≤7-fold (AMP) excess of ATP as estimated from calibration experiments at ATP = 10 μmol/liter. The intracellular water volume was determined as the difference between the total water of a wet hyphal pellet (dry loss) and the extracellular water. The latter was estimated as the partial volume of the wet cell pellet that was accessible to the non-membrane-penetrating solute bovine serum albumin. Glucose-6-phosphate dehydrogenase (G6PDH) activities were assayed in hyphal suspensions after exposure to nystatin for various times (see permeabilization procedure above). 2.5 mg of cells (fwt) in 1 ml were mixed with the same volume of triethanolamine buffer, 100 mmol/liter, pH 7.6, containing 0.7 mol/liter sorbitol. After centrifugation at 16,000 × g for 10 min, both the cell pellet (after resuspension in the original volume of the last mentioned buffers) and the supernatant were used to determine conversion rates of glucose 6-phosphate: 800 μl of cell suspension or supernatant were mixed with 150 μl of MgCl2 solution, 100 mmol/liter, and 60 μl of NADP+ solution, 15 mmol/liter. After reading the initial extinction the conversion was started by adding 60 μl of glucose 6-phosphate solution, 30 mmol/liter, and reciprocal shaking at 24 °C. After 30 and 60 s, the reaction was stopped by rapid cooling on ice, and the mixture was centrifuged at 10,000 ×g for 10 min at 0 °C. The OD of the supernatants was read at 340 nm after warming to room temperature. Total cellular protein was extracted from cell pellets by shaking with 10% SDS for 4 h. After centrifugation at 6000 ×g for 10 min, the supernatant was assayed by the Lowryet al. (28Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) procedure. Hyphal cells of P. cyclopium are able to concentrate amino acids to a considerable extent. For example, nitrogen-starved cells accumulate externally added 14C-labeled amino acids from 5 μmol/liter solutions by the following concentration factors (C in/C out, calculated on the basis of total intracellular water): Arg, 2600-fold; Glu, 2000-fold; Leu, 1700-fold; Phe, 1300-fold; even from 5 mmol/liter solutions concentration factors of 38 (Arg), 34 (Glu), 32 (Leu), and 24 (Phe) are reached. As to be expected, the majority of absorbed amino acids accumulated in the vacuolar compartment; after selective lysis of the plasma membrane (see next section) the vacuoles retained 89% of total [14C]Arg, 65% of total [14C]Glu, 93% of total [14C]Leu, and 81% of total [14C]Phe, each preaccumulated from a 5 μmol/liter solution. Vacuoles liberated from intact hyphal cells via protoplasts contained approximately 65% of the total ninhydrin-positive compounds but showed very low accumulation capacities for added amino acids compared with the protoplasts of their origin (e.g. less than 20% in the case of [14C]Phe or [14C]Arg). This situation was only marginally influenced by the isolation procedure,i.e. lysis with DEAE-dextran (29Dürr M. Boller T. Wiemken A. Arch. Microbiol. 1975; 105: 319-327Crossref PubMed Scopus (65) Google Scholar), EGTA-induced lysis (according to Kringstad et al. (30Kringstad R. Kenyon W.H. Black C.C. Plant. Physiol. 1980; 66: 379-382Crossref PubMed Google Scholar)), lysis by shearing forces via ultracentrifugation (31Pugin A. Montrichard F. Le-Quoc K. Le-Quoc D. Plant Sci. 1986; 47: 165-172Crossref Scopus (15) Google Scholar), all followed by flotation in sorbitol/Ficoll density gradients. Addition to isolated vacuoles of a 20,000 × g supernatant (obtained after disintegration of protoplasts in a glass homogenizer at 0 °C, centrifugation and 3 h flow dialysis against 10 mmol/liter MES, pH 6.5) increased their accumulation capacity for [14C]Phe and [14C]Arg by 30–50% as well as their lifetime detectable by microscopic observation. 2W. Roos, unpublished results. Thus, contact with cytosolic macromolecules appears to be critical for maintenance of tonoplast transport capacities that can therefore not reliably be assayed with isolated vacuoles. Similar objections have been raised against the use of isolated vacuoles in an artificial environment for the elucidation of regulatory functions of vacuolar ion channels (32Weiser Th Nonselective Cation Channels: Pharmacology, Physiology and Biophysics. Birkhäuser, Basel1993: 305-310Crossref Scopus (0) Google Scholar).In situ systems can serve as a verifiable compromise between the reduced complexity of the isolated organelle and the functional stability and variability of the intact cell. Therefore we decided to probe the dynamics of vacuolar accumulation and efflux with an in situ system to be established by selective permeabilization of the plasma membrane for micromolecules under conditions that keep the vacuole functionally intact within its macromolecular intracellular environment. Selective lysis of the fungal plasmalemma without damaging the tonoplast has been reported hitherto only for yeast cells that had been treated with either DEAE-dextran (33Huber-Wälchli V. Wiemken A. Arch. Microbiol. 1979; 120: 141-149Crossref Scopus (46) Google Scholar) or CuCl2 (34Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar). While experiences with the CuCl2 procedure argue for a damaging side effect on the tonoplast H+-ATPase (uptake of arginine or Ca2+ was not only reduced compared with intact cells but also insensitive to uncouplers, cf. Ohsumi et al.(34Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar)), treatment with DEAE-dextran permeabilized hyphal cells of our strain of P. cyclopium only partially. In this fungus, treatment with the polyene antibiotic nystatin in a buffer of low ionic strength proved to permeabilize the plasma membrane with sufficient efficiency and selectivity. Polyenes, such as nystatin or amphotericin B, are known for their pore-forming effect, which is based on their binding to membrane sterols of fungal cells (35Schlegel R. Grigoriev P.A. Thrum H. Stud. Biophys. 1982; 92: 135-140Google Scholar, 36Gale E.F. Cundliffe E. Reynolds P.E. Richmond M.H. Waring M.J. The Molecular Basis of Antibiotic Action. Wiley, New York1981Google Scholar, 37Kinsky S.C. J. Bacteriol. 1961; 82: 889-897Crossref PubMed Google Scholar). Selectivity toward the plasma membrane can be expected because the sterol (mainly ergosterol) content of this membrane is more than tenfold higher than that of other cellular membranes. This is not only true for P. cyclopium (38Hernández A. Cooke D.T. Clarkson D.T. Biochim. Biophys. Acta. 1994; 1195: 103-109Crossref PubMed Scopus (11) Google Scholar), but for other fungal species as well. The breakdown of the plasma membrane permeability barrier was indicated by the increasing availability of extracellular substrates for intracellular (“latent”) enzymes. As seen in Fig. 1, a cytosolically localized indicator enzyme, glucose-6-phosphate dehydrogenase (G6PDH) reports an increasing availability of its external substrates NADP+ and glucose 6-phosphate during a 6-min incubation with nystatin. Similar findings were obtained with the conversion of added 4-methylumbelliferyl phosphate (4-MUP) by cellular phosphatases (data not shown). The leakage of bulk protein, measured as A 280 in the supernatant, remained very low for up to 8 min of nystatin treatment (less than 2% compared with detergent treated cells) and correlated with the appearance of marker enzyme activities in the outer medium. Thus it appears very likely that a 6-min treatment with 5 μg/ml nystatin permeabilizes the plasma membrane for micromolecules as NADP+, glucose 6-phosphate, and 4-MUP 3The indicator reactions reported the permeabilization process with different sensitivity: the dephosphorylation of 4-MUP, a smaller and less hydrophilic substrate than NADP+, required shorter times (4 min) of nystatin treatment to accelerate compared with the G6PDH reaction. Both reactions, after their initial rates had reached a plateau (after 4 or 6 min of nystatin treatment, respectively) showed the sameK m in permeabilized cells compared with the liberated enzymes. Hence, the permeabilization procedure released the indicator reactions from any limitation caused by the membrane transfer of their substrates. without causing a substantial loss of protein. On the other hand, this treatment did not significantly impair the functional integrity of the vacuoles as indicated by the following data. (a) The vacuoles were able to maintain an ATP-dependent proton gradient. Microscopic observations on the vacuolar trapping of neutral red indicated that this dye accumulated much faster in permeabilized than in intact cells (Fig. 2,a and b). The diagnostic value of neutral red accumulation to probe the intactness and energization of the vacuole is supported by showing that the dye accumulates neither in the absence of ATP nor after prolonged nystatin treatment or addition of the detergent CTAB (Fig. 2, c–e). Furthermore, the nucleic acid-based viability probe Fungo-light® (Molecular Probes) accumulated in vacuoles of permeabilized cells supplied with ATP much like in those of intact cells (data not shown). ATP-dependent accumulation of neutral red was used as a test criterion in routine checks of successful and selective permeabilization. (b) The uptake of 14C-labeled amino acids meets various criteria for vacuolar transport energized by a H+-ATPase (Table I). The most evident properties diverging from intact cells are a pH optimum near 7.0, strong dependence on ATP supply and osmotic stabilization, sensitivity toward bafilomycin and nitrate (inhibitors of tonoplast H+-ATPases), but not toward vanadate and azide (inhibitors of plasma membrane- and mitochondrial H+-ATPases, respectively). Quantitative data suggest that the transport capacities of the permeabilized cells are close to that of vacuoles in intact cells; the accumulation ratio (C in/C out at an external concentration of 50 μmol/liter) reached 80% for Phe or Arg and 75% for Leu of that observed with intact cells. The high degree of inhibition by bafilomycin and the absence of inhibition by azide implies that other organelles of the permeabilized cells (e.g. mitochondria) did not significantly contribute to the uptake of the labeled amino acids. Summarizing, it seems justified to assume that the established procedure permeabilizes the plasma membrane for micromolecules without significantly impairing the ability of the vacuole to maintain a proton gradient and to accumulate external amino acids. Thus, reliable studies of the vacuolar amino acid transport can be performed in situ, i.e. with the vacuolar transport agency kept in place within its macromolecular environment. Initial rates of efflux of labeled amino acids were determined after the cells had accumulated these compounds over different periods of time and hence had reached different vacuolar concentrations. Most of the data were obtained by a double tracer technique (“indirect method”) that allowed the simultaneous monitoring of unidirectional uptake and net accumulation. Efflux was then calculated as the dif
Aggregating cells of Dictyostelium discoideum are able to release cyclic AMP periodically. The oscillations of cAMP generation are associated with changes in adenylate cyclase activity. Cyclic AMP receptors on the cell surface are functionally coupled to the oscillating system as evidenced by phase shifts that are induced by small pulses of extracellular cAMP. An important element of the oscillating system is the signal processing from surface receptors to the adenylate cyclase. This pathway exhibits adaptation resulting in the suppression of responses to constant, elevated concentrations of cAMP. The signal input for adenylate cyclase activation is, therefore, a change in the extracellular cAMP concentration with time. Oscillations in the absence of detectable changes of intra- or extracellular cAMP concentrations suggest the possibility that there is a metabolic network in D. discoideum cells that undergoes oscillations without coupling to adenylate cyclase. Cyclic GMP concentrations oscillate with a slight phase difference in advance of that of cAMP, suggesting that the two nucleotide cyclases might not be activated by the same mechanism. Elevation of extracellular calcium exerts an inhibitory effect on the accumulation of cAMP and on the second of the two cGMP peaks.
Neuron-specific enolase (NSE) represents the gamma gamma- and alpha gamma- isoforms of the dimeric glycolytic enzyme enolase. NSE is predominantly found in neurons and neuroendocrine cells and has proven to be a marker for tumors derived from these cells. It is widely accepted in the monitoring of patients with small cell lung cancer and is also of value as an aid in diagnosis. Recently it has become of interest in the monitoring of brain damage. Monoclonal antibodies against gamma-enolase were raised in mice and selected for optimal performance on the Cobas Core enzyme immunoassay system. The antibody combination of choice was MAb 18E5 for capturing and MAb 84B10 for detection which is accomplished by using a horseradish peroxidase conjugate and the substrate 3,3',5,5'-tetramethylbenzidine. The resulting assay is a one-step enzyme immunoassay of the sandwich type. It is performed on the fully automated Cobas Core immunoassay analyzer with a total assay time of 45 min. The sample volume is 10 microliters. Calibration is done by a 1-point recalibration using a lot-specific master calibration curve provided with the kit. The dynamic range is 0-200 ng/ml. The analytical detection limit (standard 0 + 2SD) of the Cobas Core NSE EIA II was 0.1 ng/ml. Intra- and interassay coefficients of variation were < 5% and < 6%, respectively. A Hook Effect was not observed up to a concentration of 20'000 ng/ml. Test results correlated closely with the well established polyclonal Cobas Core NSE EIA (r = 0.99). In summary, the Cobas Core NSE EIA II is a rapid, reliable and convenient test for measuring NSE in human serum.
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