scholarly journals Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Michael J. Cohen ◽  
Brianne Philippe ◽  
Peter N. Lipke
Keyword(s):  
Cell Wall ◽  
Propidium Iodide ◽  
Membrane Integrity ◽  
Extracellular Enzyme ◽  
Glycolytic Enzyme ◽  
Cell Permeability ◽  
Specific Marker ◽  
Intact Cells ◽  
Gapdh Activity ◽  
Mammalian Cell Cultures

ABSTRACT In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100 μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells. IMPORTANCE Eukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate nonconventional secretion more generally.

scholarly journals Enzymatic analysis of yeast cell wall-resident GAPDH and its secretion

2020 ◽  
Author(s):  
Michael J. Cohen ◽  
Brianne Philippe ◽  
Peter N. Lipke
Keyword(s):  
Cell Wall ◽  
Propidium Iodide ◽  
Membrane Integrity ◽  
Extracellular Enzyme ◽  
Glycolytic Enzyme ◽  
Cell Permeability ◽  
Specific Marker ◽  
Intact Cells ◽  
Gapdh Activity ◽  
Mammalian Cell Cultures

AbstractIn yeast, many proteins are found both in the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish non-conventional secretion from cellular leakage. We therefore monitored extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for non-conventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide crosslinks. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30-60-minute lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level one hour after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells.ImportanceEukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi, and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells, while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme, and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate non-conventional secretion more generally.

scholarly journals THE LYSIS OF GROUP A HEMOLYTIC STREPTOCOCCI BY EXTRACELLULAR ENZYMES OF STREPTOMYCES ALBUS

1952 ◽  
Vol 96 (6) ◽  
pp. 569-580 ◽  
Author(s):  
Maclyn McCarty
Keyword(s):  
Cell Wall ◽  
Extracellular Enzymes ◽  
Group A Streptococci ◽  
Carbohydrate Component ◽  
Streptococcal Cell Wall ◽  
Intact Cells ◽  
Enzymatic Lysis ◽  
Streptomyces Albus ◽  
Group A ◽  
Isolated Cell

Cell wall preparations of uniform chemical constitution have been obtained from several strains of group A streptococci. The isolated cell walls are dissolved by the same fractions of the Streptomyces albus enzymes that are effective in the lysis of intact cells, and it is likely that enzymatic lysis of group A streptococci is effected by an attack on the cell wall. The streptococcal cell wall, as prepared in this study, consists of approximately two-thirds carbohydrate and one-third protein. Small amounts of other components may be present. The carbohydrate component, which is composed primarily of N-acetyl-glucosamine and rhamnose, is the group-specific C carbohydrate. The evidence indicates that one of the streptomyces enzymes is directed toward the carbohydrate component of the cell wall.

EFFECT OF ALKYL BENZENE SULPHONATE ON YEAST METABOLISM

1963 ◽  
Vol 9 (1) ◽  
pp. 117-127
Author(s):  
E. R. Blakley
Keyword(s):  
Cell Wall ◽  
Candida Utilis ◽  
Complete Inhibition ◽  
Enzymatic Reactions ◽  
Alkyl Benzene ◽  
Intact Cells ◽  
Yeast Metabolism ◽  
Ph Values ◽  
Upper Limit ◽  
Yeast Protoplasts

The rate of fermentation of glucose by suspensions of Candida utilis at acid pH values is reduced by alkyl benzene sulphonate in the range 75 to 250 γ/ml. Concentrations of alkyl benzene sulphonate below 75 γ/ml decrease the rate of fermentation of glucose above pH 7 and respiration at all pH values. An upper limit of 70 to 90% inhibition of fermentation or respiration is obtained at concentrations of alkyl benzene sulphonate above 250 γ/ml, except at pH 4.2 where complete inhibition is obtained. The effect of alkyl benzene sulphonate on the fermentation of glucose by yeast protoplasts is similar to the effect observed for intact yeasts. Some enzymatic reactions of cell-free extracts are inhibited by concentrations of alkyl benzene sulphonate lower than that required to affect fermentation by intact cells. The enzyme components of the cell-free preparation appear to vary in their sensitivity to the surfactant. The results support the view that the surfactant in the micellar form disrupts the cell wall of the yeast, and unassociated molecules inactivate some enzymes vital for the metabolism of the cell.

Phytotoxic Effects of Treated Wastewater Used for Agricultural Irrigation On Root Hydraulic Conductivity and Plant Growth

2021 ◽  
Author(s):  
Sare Asli ◽  
Nedal Massalha ◽  
Muhamad Hugerat
Keyword(s):  
Cell Wall ◽  
Hydraulic Conductivity ◽  
Plant Growth ◽  
Root Elongation ◽  
Dry Weight ◽  
Cell Permeability ◽  
Treated Wastewater ◽  
Pressurized Water ◽  
Pore Sizes ◽  
Physical Effects

Abstract AimsTo determine the effects of treated wastewater (TWW) and dialyzed TWW (DTWW) through dialysis tube with a cut-off at 6000-8000 Da, on the water transport characteristics of maize seedlings (Zea mays L). MethodsLaboratory experiments were conducted to determine the effect of TWW on the hydraulic conductivity of excised roots. Moreover, the effect on transpiration, plant growth, root cell permeability and on the plant fresh and dry weight was determined. ResultsPressurized water flow through the excised primary roots was reduced by 25%-52%, within 90 min of exposure to TWW or DTWW. In hydroponics, DTWW affected root elongation severely by 58 %, while cell-wall pore sizes of same roots were little reduced (by 6%). Additionally, the exposure to TWW or DTWW caused inhibition of both leaf growth rate by (26%-70%) and transpiration by (14%-64%). While in soil growth, the plant fresh and dry weight was also significantly affected but not with secondary DTWW. Conclusions These impacts appeared simultaneously to involve phytotoxic and physical clogging impacts. First, the inhibition in hydraulic conductivity through live roots (phytotoxic and physical effects) after exposure to secondary DTWW was by 22%, while through killed roots accepted after hot alcohol disruption of cell membranes (physical effects only); was only by 14%. Second, although DTWW affected root elongation severely by 58%, cell-wall pore sizes of same roots were little reduced by 6%. We conclude that large molecules, such as polypeptides, remained after the dialysis process, may have produced hormone-like activity that affected root water permeability.

scholarly journals Ionic Relations of Cells of Ohara Australis I. Ion Exchange in the Cell Wall

1959 ◽  
Vol 12 (4) ◽  
pp. 395 ◽  
Author(s):  
J Dainty ◽  
AB Hope
Keyword(s):  
Cell Wall ◽  
Ion Exchange ◽  
Free Space ◽  
Cell Walls ◽  
Plant Cells ◽  
External Solution ◽  
Exchangeable Cations ◽  
Intact Cells ◽  
Donnan Free Space ◽  
Isolated Cell

Measurements of ion exchange were made between isolated cell walls of Ohara australis and an external solution. Comparison between intact cells and cell walls showed that nearly all the easily exchangeable cations are located in the cell wall. The wall is hown to consist of "water free space" (W.F.S.) and "Donnan free space" (D.F.S.); the concentration of in diffusible anions in the D.F.S. is about O� 6 equivjl. This finding is contrary to past suggestions that the D.F.S. is in the cytoplasm of plant cells.

scholarly journals The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Arunas Stirke ◽  
Raimonda Celiesiute-Germaniene ◽  
Aurelijus Zimkus ◽  
Nerija Zurauskiene ◽  
Povilas Simonis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Yeast Cell ◽  
Recovery Process ◽  
Plasma Membrane Permeability ◽  
Intact Cells ◽  
Sytox Green ◽  
Membrane Resealing ◽  
Nucleic Acid Stain ◽  
Main Barrier ◽  
Non Equilibrium

Abstract An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure  to  PEF indicate a strong coupling between these parts of the cell.

scholarly journals Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide

Zygote ◽  
2011 ◽  
Vol 20 (4) ◽  
pp. 333-337 ◽  
Author(s):  
Kenzo Uchikura ◽  
Masashi Nagano ◽  
Mitsugu Hishinuma
Keyword(s):  
Propidium Iodide ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Non Invasive ◽  
Maturation Rate ◽  
Cell Layers ◽  
The Relationship

SummaryWe examined the relationship between integrity of cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturational competence of feline oocytes. Feline cumulus–oocyte complexes (COCs) were collected from either small (400–800 μm) or large (≥800 μm) follicles. Immediately after collection, cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI), which penetrates only non-viable cells. Cumulus cells without PI staining were judged as having good membrane integrity. After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89.8%) was higher (p < 0.05) than that from small follicles (60.8%). There was no difference in the maturation rate of oocytes from follicles with the same size regardless of cumulus morphology. In contrast, oocytes that had cumulus cells with good membrane integrity showed a higher maturation rate (93.8%) than oocytes with poor cumulus integrity (76.9%) in large follicles (p < 0.05). We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide staining can be used to predict the maturational competence of oocytes.

scholarly journals Daptomycin Exerts Bactericidal Activity without Lysis of Staphylococcus aureus

2008 ◽  
Vol 52 (6) ◽  
pp. 2223-2225 ◽  
Author(s):  
Nicole Cotroneo ◽  
Robert Harris ◽  
Nancy Perlmutter ◽  
Terry Beveridge ◽  
Jared A. Silverman
Keyword(s):  
Electron Microscopy ◽  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Division ◽  
Bactericidal Activity ◽  
Membrane Integrity ◽  
Cell Lysis

ABSTRACT The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.

Dynamic Recording of Cell Death in the In Vitro Dorsal Vagal Nucleus of Rats in Response to Metabolic Arrest

2003 ◽  
Vol 89 (1) ◽  
pp. 551-561 ◽  
Author(s):  
Michael Müller ◽  
Klaus Ballanyi
Keyword(s):  
Cell Death ◽  
Propidium Iodide ◽  
Neuronal Death ◽  
Time Course ◽  
Time Window ◽  
Membrane Integrity ◽  
Brain Slices ◽  
Limited Information ◽  
Histological Techniques ◽  
Metabolic Arrest

Anoxic/ischemic neuronal death is usually assessed in cell cultures or in vivo within a time window of 24 h to several days using the nucleic acid stain propidium iodide or histological techniques. Accordingly, there is limited information on the time course of such neuronal death. We loaded acute rat brain stem slices with propidium iodide for dynamic fluorometric recording of metabolic arrest-related cell death in the dorsal vagal nucleus. This model was chosen because dorsal vagal neurons show a graded response to metabolic inhibition: anoxia and aglycemia cause a sustained hyperpolarization, whereas ischemia induces a glutamate-mediated, irreversible depolarization. We found that the number of propidium iodide–labeled cells increased from 27% to 43% of total cell count within 1–7 h after preparation of slices. Compared with these untreated control slices, cyanide-induced anoxia (30 min) or aglycemia (1 h) did not cause further cell death, whereas 3-h aglycemia destroyed an additional 13% of cells. Ischemia (1 h) due to cyanide plus iodoacetate immediately labeled an additional 20% of cells, and an additional 48% of cells were destroyed within the following 3 h of postischemia. Continuous recording of propidium iodide fluorescence showed that loss of membrane integrity started within 25 min after onset of the ischemic depolarization and the concomitant intracellular Ca2+ rise. The results show that propidium iodide can be used to monitor cell death in acute brain slices. Our findings suggest that pronounced cell death occurs within a period of 1–4 h after onset of metabolic arrest and is apparently due to necrotic/oncotic mechanisms.

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