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At least 19 recordsLinked to original sources
PubMed1982

Properties of rat erythrocyte membrane cytoskeletal structures produced by digitonin extraction: digitonin-insoluble beta-adrenergic receptor, adenylate cyclase, and cholera toxin substrate.

Rat erythrocyte plasma membranes have been extracted exhaustively with digitonin at low temperature, and the residual, detergent-extracted membrane cytoskeletal material is compared to that prepared with Triton X-100 with respect to protein, glycoprotein, phospholipid, and cholesterol content. Digitonin, a weaker detergent than Triton X-100, solubilizes only 26% of the phospholipids and none of the cholesterol. SDS-polyacrylamide gel electrophoresis reveals that differences between the proteins extracted by the two detergents are primarily quantitative. In terms of functional preservation, digitonin retains in the cytoskeleton 28% of the beta-adrenergic receptor binding activity (with the balance accounted for in the supernatant), greater than 90% of the adenylate cyclase and greater than 90% of the 45,000 mol wt polypeptide cholera toxin substrate. The cytoskeletal-associated beat-adrenergic receptor retains binding properties for antagonist and agonist which are identical to those of the native membrane receptor. The digitonin-extracted cytoskeleton containing the beta-adrenergic receptor may provide a useful vehicle for the reconstitution of a hormone-sensitive adenylate cyclase.

Adenylyl Cyclases↗
PubMed1980

Effect of digitonin on photophosphorylation and light-induced H+ uptake in isolated spinach chloroplasts.

The effects of the presence of digitonin in the reaction mixture on photophosphorylation, light-induced H+ uptake, and the size of isolated spinach chloroplasts were studied. Digitonin inactivated photosystem II and dissipated light-induced pH increase without affecting the photophosphorylation driven by photosystem I. Digitonin increased the concentration of NH4Cl required for uncoupling. This effect of digitonin was diminished by valinomycin which barely affects phosphorylation by itself. Although such properties have all been observed with digitonin subchloroplast particles, digitonin at the concentration required for acquiring such properties had little effect on the size of chloroplasts. These results suggest that the characteristic effects of digitonin subchloroplast particles are caused by thylakoid membranes penetrated by digitonin but not by the size of membrane vesicles. Both in chloroplasts with digitonin in the reaction mixture and in digitonin subchloroplast particles, N,N'-dicyclohexylcarbodiimide, purine nucleoside di- and tri-phosphates, and organic bases with low pKa values (pyridine and aniline) made the light-induced pH increase detectable, and this was lost upon the addition of proton ionophores. These results suggest that the lack of the light-induced pH increase by chloroplasts with digitonin in the reaction mixture and by digitonin subchloroplast particles is caused both by a membrane leaky to protons and by the loss of internal buffering capacity of thylakoids. Furthermore, the phosphorylation of chloroplasts with digitonin in the reaction mixture and of digitonin subchloroplast particles is considered to be driven by the proton motive force as is that of chloroplasts without digitonin.

Chloroplasts↗
PubMed1989

Inactivation and activation of various membranal enzymes of the cholesterol biosynthetic pathway by digitonin.

The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Stimulation of specific binding of [3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin.

Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.

Animals↗
PubMed1985

Plasma membrane and chromaffin granule characteristics in digitonin-treated chromaffin cells.

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.

Adenosine Triphosphate↗
PubMed1986

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators.

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.

Adenosine Triphosphate↗
PubMed1994

Effect of mitochondrial protein concentration on the efficiency of outer membrane removal by the cholesterol-selective detergent digitonin.

The effects of different mitochondrial protein concentrations on the efficiency of digitonin titration of the outer mitochondrial membrane were investigated in isolated rat liver mitochondria. Isolated mitochondria were subjected to treatment with digitonin concentrations ranging from 0 to 0.40 mg digitonin per mg protein. This digitonin concentration range was used in incubations containing 5 to 50 mg mitochondrial protein per ml. Significant differences in the efficiency of outer membrane removal by digitonin titration were noted at protein concentrations of less than 20 mg per ml. Estimation of the effective concentration of digitonin required to remove 50% of the outer membrane indicated that in general, as the protein concentration decreases, the amount of digitonin required to remove the outer membrane increases. Significant differences were also noted in the amount of digitonin needed for removal of 95% of the outer membrane between 5, 10 and 20 mg/ml with the effect of protein concentration disappearing above 20 mg/ml. No effect of protein concentration was found on the disruption of the inner membrane by digitonin as judged by leakage of matrix marker enzyme activity and by release of inner membrane marker enzyme activity. The conclusions of these studies indicate that at relatively low mitochondrial protein concentrations (< 20 mg/ml), the efficiency of digitonin in removing the outer membrane is substantially reduced.

Animals↗
PubMed1988

Digitonin treatment activates specific genes including the heat-shock genes in salivary glands of Drosophila melanogaster.

A large number of chromosomal sites were found to form puffs in Drosophila salivary glands after treatment with the mild detergent digitonin and incubation in a defined medium for 2 hr. The cytological locations of these puffs were determined, and the puff size was measured at 43 loci in both digitonin-treated salivary glands and intact glands. On the basis of comparisons of puffing between digitonin-treated and intact salivary glands, the puffs were classified into three categories: (1) digitonin-unaffected preexisting puffs (8 sites), (2) digitonin-activated preexisting puffs (6 sites), and (3) digitonin-induced new puffs ("digitonin puffs", 29 sites). The digitonin puffs included some of the developmentally regulated puffs and all the heat-shock puffs known in Drosophila melanogaster. The activation of the specific loci by digitonin treatment suggests that gene expression at these loci is suppressed in salivary glands by a mechanism(s) sensitive to digitonin.

Animals↗
PubMed1993

Effects of digitonin on the intracellular content of rat hepatocytes: implications for its use in the study of intralobular heterogeneity.

Anterograde or retrograde perfusion of rat liver with digitonin selectively permeabilizes the periportal or the perivenous zone of the hepatic lobule. Digitonin perfusion is used to analyze the effluents released by permeabilized hepatocytes or, combined with collagenase perfusion, to obtain cell suspensions enriched in either periportal or perivenous hepatocytes. Despite the wide use of digitonin to study lobular heterogeneity, its affects on rat hepatocytes are not well documented. We therefore analyzed the effects of digitonin perfusion on the intracellular content of rat hepatocytes by combining electron microscopy, histoenzymology, immunohistochemistry, and in situ hybridization. At the concentration currently used for the study of lobular heterogeneity, digitonin perfusion induced a marked cytosolic clarification of permeabilized hepatocytes, while most organelles except mitochondria were well preserved. In the digitonin-altered zones, there was no histochemical detection of non-membrane-bound enzymes (lactate dehydrogenase, glutamate dehydrogenase), whereas membrane-bound enzymes (succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, NADPH dehydrogenase, glucose-6-phosphatase) were still detected. Immunohistochemistry and in situ hybridization revealed significant amounts of several plasma proteins (albumin, alpha 2-macroglobulin, alpha 1-inhibitor 3, alpha 1-acid glycoprotein) and their respective mRNAs in digitonin-permeabilized hepatocytes. The demonstration that digitonin-permeabilized hepatocytes retain many intracellular constituents shows that biochemical analysis of cellular effluents released from digitonin-permeabilized hepatocytes must be interpreted with caution and that the apparent characteristics of cell suspensions obtained by the digitonin-collagenase technique might be significantly altered by contamination with permeabilized hepatocytes from the opposite zone.

Animals↗
PubMed1980

Influence of temperature on hemolysis of erythrocytes by digitonin.

The kinetics of hemolysis of pig erythrocytes by digitonin was continuously monitored by a potassium selective electrode. The following minimal mechanism of hemolysis was postulated, based upon kinetic measurements: 2 D + E (1)in equilibrium E x D2 (2)leads to (E x D2) (3)leads to (E x D2) where D denotes a digitonin molecule and E a specific digitonin binding-site on the membrane. The first step (1) represents a rapid reversible combining of digitonin with specific binding-sites on the membrane. The second step (2) is prelytic, related to the time required for bound digitonin molecules to alter the membrane structure so much that hemolysis may take place; this step has a transition temperature at 26 degrees C, probably related to the "melting" of specific membrane structures at that temperature. The third step (3) is hemolytic, and comprises the changes within the digitonin-altered membrane during hemolysis; it is stongly influenced by temperature. Lowering of temperature slows down the rate of hemolysis and increases the quantity of digitonin required to obtain a fixed extent of hemolysis. It appears that two molecules of digitonin combine with a single binding-site on the outer face of the membrane in a digitonin-membrane complex.

Animals↗
PubMed1997

Digitonin enhances the efficacy of carboplatin in liver tumour after intra-arterial administration.

Platinum-containing drugs enter the cell slowly and have a poor tissue penetration. Increasing the permeability of the cell membrane might increase the intracellular drug concentration. Digitonin, a detergent that increases cell permeability by binding to cholesterol molecules in the cell membrane, can increase cisplatin accumulation and reduce tumour growth in vitro. The aim of this study was to determine whether digitonin could increase the efficacy of carboplatin (CBDCA) in vivo. In LH rats, a hepatoma was implanted in the liver. At 7 days after implantation, digitonin (or saline in the control group) was infused via the hepatic artery and, 10 min later, CBDCA was injected. Biopsies from the tumour and liver parenchyma were obtained after 1 h. The concentration of platinum measured in the liver tumours was higher in the digitonin group than in the control groups. In the liver parenchyma the concentrations were of the same magnitude. Measured with the 133Xe-clearance technique, digitonin did not alter the tumour blood flow. Digitonin enhanced the tumour-growth-retarding effect of CBDCA given intra-arterially at 5 mg/kg but not at 25 mg/kg. No increase in toxicity was observed for digitonin given together with CBDCA at 5 mg/kg. Systemic administration of CBDCA was not influenced by digitonin. These findings demonstrate that pretreatment with digitonin increases the tumour uptake of CBDCA and potentiates the cytotoxic effect of CBDCA.

Animals↗
PubMed1975

The effects of digitonin on photochemical activities of isolated chloroplasts.

The addition of digitonin to chloroplasts stimulated the rate of oxygen evolution followed by a gradual inhibition. The effect of digitonin was dependent on the digitonin to chlorophyll ratio and on temperature and time. The initial stimulation of oxygen evolution appeared to be a result of uncoupling as digitonin did not stimulate oxygen evolution by uncoupled chloroplasts. The stimulatory effect occurred more rapidly at high digitonin to chlorophyll ratios but the extent of stimulation was low and inhibition occurred soon after addition of the detergent. The inhibition of electron flow by digitonin was due to a site of action near photosystem II which resembled the inhibition reported for tris buffer and resulted in photobleaching. However, digitonin inhibition could not be recovered by washing with reducing agents and was only partially recovered by the addition of artificial electron donors to photosystem II. Electron flow mediated by photosystem I was unaffected by the addition of digitonin but was decreased when the chloroplasts were separated by subsequent centrifuging. This suggested that digitonin solubilizes photosystem I components which remain active in the soluble form.

Journal Article↗
PubMed1993

Advantages of digitonin extraction to reveal the intracellular structure of rat glomerular podocytes for high-resolution scanning electron microscopy.

Kidneys of anesthetized rats were perfused with digitonin to extract cytosolic proteins of glomerular podocytes so that the remaining intracellular structures could be examined by three-dimensional stereo high-resolution scanning electron microscopy (HRSEM). Cytoskeleton, consisting of microtubules and intermediate filaments, was preserved with each applied concentration of digitonin. High concentrations of digitonin (1.0 mg/ml) produced a corrugated appearance in plasma membranes likely due to the formation of digitonin-cholesterol complexes. At 1.0 mg/ml digitonin, the Golgi complex became vesicularized, and mitochondria were well extracted and their ultrastructure preserved. Lower concentrations of digitonin (0.1 and 0.2 mg/ml) were less disruptive to both the plasma membrane and the Golgi complex. Mitochondria, rough endoplasmic reticulum, coated vesicles, nuclear membrane, and chromatin were well preserved. Extraction with digitonin, at the optimal concentration and perfusion time, simultaneously maintains both the cytoskeleton and membranous organelles inside the cell and provides a method to elucidate the interactions between these two components. Furthermore, digitonin extraction should preserve antigenic sites, thereby allowing the localization of intracellular proteins by backscattered electron imaging of immunogold labels in the scanning electron microscope.

Animals↗
PubMed1989

High zone-selectivity of cell permeabilization following digitonin-pulse perfusion of rat liver. A re-interpretation of the microcirculatory zones.

The plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and Grunnet 1987). Brief pulses of digitonin applied with antegrade and retrograde perfusion of the liver caused selective elution of cytosolic enzymes and metabolites from the periportal and the perivenous zone of the same liver. In the present study a light microscopical examination of the liver fixed immediately after the digitonin pulse confirmed the very high zonal selectivity of the method inferred from the marker enzyme pattern of the eluates: Only cells around the port of entry of digitonin were affected and the borderline between affected and non-affected cells was always sharp. The typical periportal lesion was triangular in shape, enclosing the portal space, while the perivenous lesion was roughly circular, concentric with the hepatic vein. Assuming that the digitonin lesion reflects the microcirculatory flow pattern these findings seem to be at variance with the acinar model of Rappaport (Rappaport et al. 1954). The lesion in the lobuli near the surface of the liver as reflected by the discoloration pattern observed on the surface was the same as the lesion of deeper lobuli. The conducting vessels of the liver were only insignificantly affected by digitonin. At the cellular level only the sinusoidal luminal surface of the hepatocytes was affected. The cytoplasmic matrix of the cells including glycogen appeared thinned. All cell types of the liver parenchyma seemed to be equally affected by the digitonin treatment.

Animals↗
PubMed1980

Sensitivity of the (Na+ + k+)-atpase to state-dependent inhibitors. Effects of digitonin and Triton X-100.

Treatment of a purified (NA+ + 5+)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na+ + k+)-atpase reaction inhibited more than the K+-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na+ + k+)-atpase reaction but not the K+-phosphatase reaction; however, treatment with digitonin made the K+-phosphatase reaction almost as sensitive to oligomycin as the (Na+ + k+)-atpase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K+-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K+-phosphatase reaction more than the (Na+ + k+)-atpase reaction). Both digitonin and Triton markedly increased the K0.5 for K+ as activator of the K+-phosphatase reaction, with little effect on the K0.5 for K+ as activator of the (Na+ + k+)-ATpase reaction. In contrast, increasing the K0.5 for K+ in the K+-phosphatase reaction by treatment of the enxyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K+-phosphatase reaction by ATP and increased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E1 conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E2 state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K0.5 for K+ in the K+-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na+ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na+ plus nucleotide.

Animals↗
PubMed1987

Morphology and secretory activity of digitonin- and alpha-toxin-permeabilized chromaffin cells.

An ultrastructural examination of cultured bovine chromaffin cells permeabilized with Staphylococcus aureus alpha-toxin or digitonin revealed differences in the preservation of cell morphology. The toxin-treated cells closely resembled control cultured cells whereas digitonin-treated cells showed gradations in cytoplasmic densities suggesting extraction, some swelling of the endoplasmic reticulum and, occasionally, discontinuities in the plasma membrane and free granules in the extracellular medium. In both cell models, there was a swelling of the mitochondria. Horseradish peroxidase labelling of permeabilized cells marked the cytoplasm of digitonin-treated cells but only the surface of toxin-treated cells, demonstrating that larger lesions were caused by digitonin. In stimulated cells, the decrease in volumetric density of chromaffin granules correlated well with catecholamine release. The sites of secretory activity could be demonstrated in toxin-treated cells using horseradish peroxidase as a surface marker. Although both cell systems secrete catecholamines in response to calcium stimulation, their calcium requirements and the kinetics of release were different. In alpha-toxin-treated cells, 100 microM free calcium induced maximal catecholamine release. In digitonin-treated cells, 20 microM evoked maximal release but secretion was blocked at 100 microM. Catecholamine release terminated in digitonin-treated cells within 10 min but continued in alpha-toxin-treated cells for at least 60 min. In addition, the maximal release observed in toxin-treated cells (50%) was always greater than that observed in digitonin-permeabilized cells (20%). The results suggest that both exocytosis and granule translocation are operational in alpha-toxin-treated cells, but that the translocation step or the docking of granules at the plasma membrane may be impaired in digitonin-treated cells.

Adrenal Medulla↗
PubMed1986

Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and phenylethanolamine N-methyltransferase, EC 2.1.1.28) are involved in catecholamine biosynthesis and are considered soluble proteins. However, they may actually be localized on the surface of the chromaffin granule. We have used the detergent digitonin to permeabilize the plasma membrane of cultured adrenal chromaffin cells to investigate the subcellular localization of TyrOHase and PMTase. A digitonin titration of the release of proteins and catecholamines revealed the existence of at least three subcellular compartments that are distinguished by their digitonin sensitivity: (i) soluble proteins, which were released upon treatment of the cells with low digitonin concentrations (5 microM), (ii) a "digitonin-sensitive" cytoplasmic protein pool, which required higher concentrations of digitonin for release (10 microM) and included TyrOHase and PMTase, and (iii) the chromaffin granule, which was insensitive to digitonin. Analysis of the rates of release of all of these proteins revealed that the rate of TyrOHase and PMTase release was slower at 10 microM than at 40 microM digitonin, while the rates of release of the other proteins were similar at both concentrations and varied in proportion to their respective sizes. Treatment with cytoskeletal disrupting agents had no effect on TyrOHase or PMTase efflux. These data suggest that TyrOHase and PMTase are in a detergent-labile association in the cell. This is consistent with the concept that TyrOHase and PMTase may be localized on the surface of the chromaffin granule.

Animals↗