In situ assay of intracellular enzymes of yeast (Kluyveromyces fragilis) by digitonin permeabilization of cell membrane

The yeast, Kluyveromyces fragilis was permeabilized to a number of low-molecular-weight substrates using digitonin. The activities of intracellular yeast enzymes, viz., alcohol dehydrogenase (ADH), beta-galactosidase, glucose-6-phosphate dehydrogenase, aspartase, and hexokinase were found to be much higher in the permeabilized cells than the untreated cells. The optimum conditions for permeabilization with reference to ADH were 0.1% digitonin at 37 degrees C for 15 min. The ADH activity in permeabilized cells was several-fold higher than that in cell free extracts prepared by either physical or chemical methods.     

Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells

Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01–0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation.     

Improvement in cell‐bound phytase activity of Pichia anomala by permeabilization and applicability of permeabilized cells in soymilk dephytinization

Aims: Whole cell permeabilization of Pichia anomala to ameliorate the cell‐bound phytase activity and usability of permeabilized cells in dephytinization of soymilk. Methods and Results: The cells of P. anomala were subjected to permeabilization using the surfactant Triton X‐100 to overcome the permeability barrier and prepare whole cell biocatalysts with high phytase activity. The statistical approach, response surface methodology (RSM) was used to optimize the operating conditions for permeabilization. The treatment of cells with 5% Triton X‐100 for 30 min resulted in c. 15% enhancement in cell‐bound phytase activity. The shrinkage of protoplast was observed, although cell viability and phytase stability were not significantly altered. The free as well as immobilized permeabilized cells hydrolysed soymilk phytate, and the latter could be reused over four consecutive cycles. Conclusions: Whole cell permeabilization of P. anomala using Triton X‐100 led to enhancement in cell‐bound phytase activity. The viability and integrity of yeast cells were not significantly affected because of permeabilization. The permeabilized P. anomala cells effectively dephytinized soymilk, and the permeabilized cells immobilized in alginate could be reused because of sustained phytase activity. Significance and Impact of the Study: This is the first report on the use of permeabilized yeast cells for mitigating phytate content of soymilk. Alginate entrapment of permeabilized P. anomala allows reuse of cells for soymilk dephytinization, thus suggesting a potential application in food industry.     

Production of trehalose by permeabilized Micrococcus QS412 cells

Permeabilized Micrococcus QS412 cells were used to produce trehalose from starch through catalysis of maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase in the cells. The permeabilized cells could omit the enzyme purification and simplify the immobilization of intracellular enzymes. The reagent, reagent dosage and time of cell permeabilization treatment were determined. The maximum trehalose biosynthesis activity was obtained after the cells were treated with 5% (w/v) of toluene at 30 °C for 40 min. Reaction conditions of trehalose synthesis of permeabilized cells were optimized. The yield of trehalose was up to 188 mg/g wet permeabilized cells in pH 8.0, 100 mmol/l phosphate buffer at 30 °C after 12 h reaction. Batch reactions showed that the permeabilized cells could be reused for 16 cycles in the biosynthesis reaction. The total trehalose yield was up to 2.5 g/g wet permeabilized cells. Development of permeabilized cells provide a new cheaply alternative technology for trehalose production.     

Electropermeabilization of dense cell suspensions

This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 × 10⁶ cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 × 10⁶ to 400 × 10⁶ cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 × 10⁶ and 400 × 10⁶ cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.     

Improved localization of intracellular sites of phosphatases using cerium and cell permeabilization

A simple permeabilization method has been developed that allows for intracellular localization of acid phosphatase in neutrophils and several types of tissue culture cells with cerium. This permeabilization procedure also facilitates intracellular alkaline phosphatase localization in neutrophils without the loss of cell surface reaction in this cell type. Only the cell surface reaction was detected in the absence of permeabilization. Glutaraldehyde-fixed cells were permeabilized with detergent during the cytochemical reaction. Triton X-100 at 0.0001-0.0002% gave the best results for the enzymes and cell types tested.     

Oxygraphic Evaluation of Mitochondrial Function in Digitonin-Permeabilized Mononuclear Cells and Cultured Skin Fibroblasts of Patients with Chronic Progressive External Ophthalmoplegia

For quantitative elucidation of maximal mitochondrial oxidation capacities in human mononuclear cells, cultured human skin fibroblasts and human thrombocytes the optimal amount of digitonin for plasma membrane permeabilization was determined to be 5, 10, and 0.1 μg/10⁶ cells, respectively. Using these concentrations the rate of respiration of permeabilized cells with the mitochondrial substrates succinate (+ rotenone) or glutamate + malate can be stimulated between two- and fourfold by ADP and inhibited by carboxyatractyloside. The maximal respiratory activities of well-characterized preparations of permeabilized mononuclear cells of five patients with chronic progressive external ophthalmoplegia were compared to healthy controls and a 30 to 50% decrease of the ADP-stimulated respiration rates with glutamate + malate and succinate + rotenone was detected. This is an indication for the presence of the mitochondrial defect in respiratory active blood cells. Additionally, for two of these patients the mitochondrial defects were proven to be detectable by the determination of maximal oxygen consumption rates of digitonin-permeabilized cultured skin fibroblasts. Therefore, the determination of maximal oxidation capacities of a well-defined cell population using strictly standardized conditions of digitonin permeabilization is judged as a useful and sensitive method for the elucidation of mitochondrial function in extramuscular tissue.     

Permeabilization of yeast cells: Application to study on the regulation of AMP deaminase activity in situ

A permeabilization method which allows the assay of several intracellular enzymes within the boundaries of the yeast cell wall is described. Toluene treatment was found to make yeast cells completely permeable to exogenous substrates, and intracellular enzymes did not leak out of the treated cells. This method was also compared with the permeabilization techniques reported previously. Electron microscopic examination of toluene-treated cells indicated that they were essentially intact. The kinetic properties of AMP deaminase, examined in the permeabilized cells, including allosteric regulation by polyamine and Zn²⁺, suggest some differences in protein interactions for AMP deaminase in situ and in vitro.     

Effect of moderate electric field frequency and growth stage on the cell membrane permeability of Lactobacillus acidophilus

Changes in growth kinetics and metabolic activity of microorganisms under the presence of a moderate electric field (MEF) have been hypothesized as being due to temporary permeabilization of cell membranes. We investigated herein the effects of frequency and growth stage on cell membrane permeabilization of Lactobacillus acidophilus OSU 133 during MEF fermentation. Cells were stained with two fluorescent nucleic acid stains: the green, nonselective, cell membrane permeable SYTO 9, and the red, cell membrane impermeable propidium iodide (PI). Fluorescence exhibition post‐treatment was assessed using fluorescence microscopy. Total plate counting was done to determine whether or not the permeabilized population represented live cells. Fermentation treatments investigated were conventional (control) and MEF (2 V/cm, 45, 60, 1,000, 10,000 Hz) at 30°C. Studies were conducted at 45 Hz for lag, exponential, and stationary phases of growth. Low frequency MEF treated cells exhibited significantly greater numbers of red cell counts than conventional treatments; further, no significant differences existed in viable counts between MEF and conventional treatments, suggesting that the red counts represent permeabilized live cells. MEF treatments at the early stage of bacterial growth at 45 Hz exhibited the maximum permeabilization followed by treatments at 60 Hz. MEF treated samples at frequencies higher than 60 Hz did not exhibit red fluorescence. Cells at lag phase showed the greatest susceptibility to permeabilization followed by those at exponential phase. No evidence of electroporation was observed during the stationary phase. To our knowledge, these observations provide the first evidence that cell membrane permeabilization occurs under the presence of electric fields as low as those under MEF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Use of a selectively permeabilized isolated rat hepatocyte preparation to study changes in the properties of overt carnitine palmitoyltransferase activity in situ.

1. A permeabilized isolated rat liver cell preparation was developed to achieve selective permeabilization of the cell membrane to metabolites and to allow the assay of mitochondrial overt carnitine palmitoyltransferase (CPT I) activity in situ. By performing the digitonin-induced permeabilization in the presence of fluoride and bivalent-metal-cation sequestrants, it was possible to demonstrate that the activity of other enzymes, which are regulated by reversible phosphorylation, was preserved during the procedure and subsequent washing of cells before assay. 2. CPT activity at a sub-optimal palmitoyl-CoA concentration was almost totally (approximately 90%) inhibited by malonyl-CoA, indicating that mitochondrial CPT I was largely measured in this preparation. 3. The palmitoyl-CoA-saturation and malonyl-CoA-inhibition curves for CPT activity in permeabilized cells were very similar to those obtained previously for the enzyme in isolated liver mitochondria. Moreover, starvation and diabetes had the same effects on enzyme activity, affinity for palmitoyl-CoA and malonyl-CoA sensitivity of CPT I in isolated cells as found in isolated mitochondria. These physiologically induced changes persisted through the cell preparation and incubation period. 4. Neither incubation of cells with glucagon or insulin nor incubation with pyruvate and lactate before permeabilization resulted in alterations of these parameters of CPT I in isolated cells. 5. The results are discussed in relation to the temporal relationships of changes in the activity and properties of CPT I in vivo in relation to the effects of insulin and glucagon on fatty acid metabolism in vivo.     

In situ assays of fungal enzymes in cells permeabilized by osmotic shock

Permeabilization of yeast and other fungal cells by osmotic shock enabled the in situ assays of intracellular plasma membrane-bound enzymes, such as beta-1,3-glucan synthase, chitin synthase, and Na(+)/K(+) ATPase as well as the soluble, cytoplasmic enzymes, such as lactate dehydrogenase and alpha-glucosidase. The permeabilization was accomplished by rapid changes in osmolarity of the washing buffer at 0 degrees C whereby 0.5-3.5 M glycerol, sorbitol, and/or mannitol and/or 1 M KCl could be used as the osmolytes. No appreciable leakage of intracellular proteins occurred during the permeabilization procedure. The described procedure caused practically complete cell permeabilization while avoiding treatments with organic solvents, detergents, and other xenobiotics currently used for the permeabilization of microbial cells.     

Stabilization of D-amino acid oxidase and catalase in permeabilized Rhodotorula gracilis cells and its application for the preparation of alpha-ketoacids*

The cellular D-amino acid oxidase (DAAO) and catalase activities of Rhodotorula gracilis were greatly increased upon the treatment of the cells with cetyltrimethylammonium bromide (CTAB). However, these enzymes, slowly leaks out from the permeabilized cells. The released DAAO was rapidly inactivated in the absence of ethylenediaminotetraacetic acid (EDTA), beta-mercaptoethanol, and glycerol. DAAO within the permeabilized cells did not require these stabilizing agents. Treating the CTAB-permeabilized cells with 0.2% glutaraldehyde (GA) at 4 degrees C for 10 min prevented the leakage of both DAAO and catalase. Alternately, stabilized whole cell DAAO and catalase was prepared by treating the whole yeast cells with 1% GA at 4 degrees C for 60 min, followed by permeabilization with CTAB, a method which was equally efficient but easy to scale up. CTAB-permeabilized cells converted D-phenylalanine to 97% phenylpyruvate and 3% phenylacetate, and these cells were reused up to 3 cycles in a batchwise reaction. On the other hand, GA-treated CTAB-permeabilized cells produced more than 99% phenylpyruvate and the cells could be reused up to 20 cycles.     

On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeabilization of microorganisms by Triton X-100.

A simple permeabilization procedure has been developed which allows the reliable determination of enzyme activitiesin situ in a variety of different microorganisms. Permeabilization is obtained by freezing cell suspensions in the presence of a low concentration of the anionic detergent Triton X-100. After thawing, the cells can be used directly in the enzyme assays. The procedure has been optimized using the yeastSaccharomyces cerevisiae. Yeast cells are completely permeabilized by Triton X-100 concentrations of 0.05% (v/v), and permeabilization is independent of cell age and cell concentration. The treatment makes the cells freely diffusible for macromolecules up to molecular weights around 70,000. Cytoplasmic and mitochondrial amino acid biosynthetic enzymes as well as aminoacyl-tRNA synthetases could be readily measured in treated cells. The method has been successfully applied to the determination of enzyme activities in other fungi as well as in gram-positive and gramnegative bacteria.     

Measurement of in situ halophilic glyceraldehyde-3-phosphate dehydrogenase activity from the permeabilized cells of archaebacterium Haloarcula vallismortis

The cells of Haloarcula vallismortis, an extreme halophilic archaebacterium, were permeabilized by various chemical, physical, and biological treatments. Biological permeabilization by lysozyme and papain showed effective results as observed by studying the in situ activity of halophilic glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) as the model enzyme. Detergents N-cetyl-N, N, N-trimethyl ammonium bromide (CTAB) and digitonin also showed significant results. Other strains of halobacteria could also be permeabilized by lysozyme. The cell morphology did not show any significant change after permeabilization as observed by phase contrast microscopy. The enzyme characteristics of hGAPDH were studied in situ using permeabilized H. vallismortis cells. The properties, like optimum pH, Km for GAP and NAD(+), inhibition by heavy metals, sulphydryl reagents, and other compounds, showed remarkable similarity with those studied in vitro.     

Introduction of high molecular weight (IgG) proteins into receptor coupled, permeabilized smooth muscle

The permeability to high molecular weight (IgG, 150 kD) proteins of the plasma membrane of receptor-coupled smooth muscles permeabilized with β-escin was determined using confocal microscopy of immunofluorescent tracers and measurement of lactate dehydrogenase (LDH, 135–140 kD) leakage. Permeabilized strips of rabbit portal vein and guinea pig ileum were incubated in a relaxing solution containing mouse anti-smooth muscle α-actin antibody and immunostained with F(ab′)₂ labeled with tetramethyl rhodamine isothiocyanate. Confocal light microscopy of Triton X-100 and β-escin permeabilized cells showed homogeneous staining of the cytoplasm, whereas in α-toxin treated and intact preparations only damaged cells at the edges of the strips were stained. Both the Ca²⁺-sensitizing effect of phenylephrine, in rabbit portal vein, and Ca²⁺ release by carbachol in guinea pig ileum, were retained after permeabilization and the treatment with the primary antibody. During the 30 min permeabilization, 38%, and within the next 75 min an additional approximately 30%, of the total LDH leaked out from the β-escin-treated group, but not from the α-toxin-treated group (3.2%). The responsiveness to agonist and maximum contractility was improved if the preparations were incubated during the introduction of proteins at 4°C, rather than 24°C. Ca²⁺-independent myosin light chain kinase (61 kD) contracted the permeabilized portal vein in the absence of free Ca²⁺ (pCa < 8). In conclusion, permeabilization with β-escin allows the transmembrane passage of 150 kD proteins under our experimental conditions that also retain receptor-coupled signal transduction.     

Micromolar concentrations of free calcium provoke secretion of lysozyme from human neutrophils permeabilized with saponin

Permeabilization of human neutrophils has been accomplished by using saponin, a cholesterol complexing agent, permitting experimental manipulation of the intracellular milieu. Access of ordinarily impermeable solutes, such as [14C]-inulin or [14C]-sucrose, to the water space of the cells was considered the main criterion for permeabilization. Other criteria were substantial (50 to 80%) release of cytoplasmic lactate dehydrogenase and permeability to trypan blue. Successful permeabilization did not cause substantial release of the granule enzymes lysozyme or beta-glucuronidase. Washing the neutrophils, to remove soluble saponin and released cytoplasmic contents, and resuspension did not alter their permeabilized character. By supplementing the medium with CaCl2, thereby obtaining free Ca2+ concentrations of 1.5 X 10(-7) M to 10(-4) M, it was possible to stimulate lysozyme secretion from washed or unwashed permeabilized neutrophils. A total of 20 to 30% of the total cellular lysozyme was released during an incubation of 5 min at 37 degrees C. Secretion was inversely related to cell concentration. No beta-glucuronidase was secreted under these conditions and no response was obtained by using unpermeabilized cells. Thus, permeabilized neutrophils respond to increases in free Ca2+ alone, without resorting to conventional secretagogues. This system also permits the manipulation of intracellular constituents important for stimulus-response coupling.     

Production of S‐lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols

The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts. Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S‐lactoylglutathione in the presence of glutathione, was used as the model system. The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells. Especially, the initial S‐lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4°C were high and were 364 and 582 times larger than those of untreated cells, respectively. These permeabilized yeast cells retained high activities during repeated batch reactions. Even in third batch reaction, they showed approximately 70–80% of the activity in the first batch. The plasma membrane of S. cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability. The initial S‐lactoylglutathione production rates of these permeabilized cells were 1.5–2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells. These results demonstrate that the recombinant S. cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 54–60, 1999.     

Studies on lactate dehydrogenase of Lactobacillus plantarum spp. involved in lactic acid biosynthesis using permeabilized cells

Lactate dehydrogenase (LDH, EC 1.1.1.27) catalyses the reduction of pyruvate to lactate in facultative anaerobes. Whole cells of Lactobacillus plantarum NCIM 2084 showed low levels of LDH activity but permeabilization of cells by treatment with organic solvents toluene, chloroform and diethyl ether increased the measurable LDH activities, ether treated cells showing the highest increase. The maximum intracellular activity was obtained upon treating the cells with ether (1%) at 28°C for 1 min. The LDH activity in permeabilized cells was nearly three-fold higher than that in the cell-free extract prepared by sonication. The kinetic properties of LDH in the permeabilized cells were comparable to that of cell-free extract, indicating that catalytically it functions similar to the isolated enzyme.