Differential lysis of plasma membranes and granules of human neutrophils by digitonin

The effect of the detergent digitonin on lysis of granule and plasma membranes of human neutrophils was studied. Either linear or sigmoid dose-response for release of the cytoplasmic marker lactate dehydrogenase and the granule markers lysozyme, beta-glucuronidase, lactoferrin, and myeloperoxidase was noted using digitonin concentrations ranging from 0.001 to 0.1 mM. However, release of the cytosol compartment was far more sensitive to the detergent than the granule compartment, with more release of lactate dehydrogenase than of lysozyme at 0.01-0.08 mM digitonin. Distinction between the two compartments was optimal at 0.025 mM digitonin. By examining in parallel the digitonin-induced release of exogenous fluorescent or luminescent indicators, a granule location was demonstrated for the pH indicator 9-aminoacridine, while the calcium probes aequorin and Quin 2 were released coincident with release of the cytosolic enzyme lactate dehydrogenase. These findings were employed to validate use of the indicators for monitoring of ion translocation in the intact cell. The differential effect of this detergent on subcellular membranes provides a broadly applicable technique for rapid assessment of the subcellular localization of tracer substances. Rapid monitoring may help to avoid problems of redistribution during cell fractionation.     

Human neutrophils permeabilized with digitonin respond with lysosomal enzyme release when exposed to micromolar levels of free calcium

We have recently reported that human neutrophils can be permeabilized with the cholesterol complexing agent saponin and that these cells can be induced to secrete the granule enzyme lysozyme in response to micromolar levels of free calcium. We now report that digitonin can be used in place of saponin and that it has several advantages. Permeabilization of human neutrophils was accomplished with 10 micrograms/ml digitonin in a high potassium medium. Normally impermeant solutes such as [14C]sucrose and inulin [14C]carboxylic acid gained access to one half of the intracellular water space marked with [3H]H2O. Between 30 and 100% of the cytoplasmic enzyme, lactate dehydrogenase, leaked from the intracellular space. The permeabilization process and calcium-triggered granule secretion were critically dependent upon temperature, time and digitonin concentration. Permeabilized neutrophils secreted beta-glucuronidase, lysozyme and vitamin B-12 binding-protein, constituents of both azurophil and specific granules, when exposed to micromolar levels of free calcium. Release of specific granule constituents appeared to be more sensitive to free calcium than release from azurophil granules. Although the amount of permeabilization varied considerably with each batch of cells, release of these granule markers was a consistent finding. Release of granule markers was accompanied by resealing of the cells to high-molecular-weight (Mr greater than 5000) solutes. Electron microscopic evidence also suggested that granule and plasma membranes were intact following digitonin treatment and that fusion of these membranes occurred in response to calcium. These results suggest that elevation of intracellular free-calcium levels is a sufficient condition for lysosomal enzyme release.     

Immunocytochemical Characterization and Subcellular Localization of Human Myristoyl-CoA: ProteinN-Myristoyltransferase in HeLa Cells

Antisera have been raised to three synthetic peptides based on the sequence of human myristoyl-CoA:proteinN-myristoyl transferase (NMT) and to the purified enzyme following its expression inEscherichia coli.These antisera have been affinity purified and shown to react both with theE. coliexpressed human NMT, and specifically with a protein of molecular weight of 63 kDa in immunoblots of the human cell line HeLa. The affinity purified antibodies have also been used to localize NMT in methanol/acetone permeabilized HeLa cells by immunofluorescent staining. The immunofluorescence showed a diffuse staining pattern throughout the cell, suggesting that the enzyme is predominantly cytosolic. This was confirmed by determining the distribution of NMT activity in different subcellular fractions of HeLa cells. Over 90% of NMT enzymatic activity was released from cell lysates during either hypotonic or isotonic homogenization. However, a small amount of enzymatic activity remained associated with cell membranes, despite extensive washing, and this was confirmed by immunoblot analysis of these membranes for NMT. In comparison, over 99.5% of lactate dehydrogenase activity was released under the same conditions, which suggests that the NMT was genuinely associated with the cell membranes. The membrane-bound enzyme behaved like a peripheral membrane protein. Permeabilization of HeLa cells with 50 μMdigitonin resulted in the release of 90–93% of lactate dehydrogenase compared to 73–85% of NMT, again suggesting that the majority of the enzyme is cytosolic, but that some may be associated with cell membranes or organelles.     

Subcellular distribution of hexokinase isoenzymes in pancreatic islet cells exposed to digitonin after incubation at a low or high concentration of D-glucose

It was recently proposed that stimulation of pancreatic islet by D-glucose results in the translocation of glucokinase from the perinuclear area to the cell periphery, where the enzyme might conceivably interact with either the glucose transporter GLUT-2 or some other proteins and, by doing so, become better able to express its full catalytic activity. To explore the possible interaction between glucokinase and the cell boundary, dispersed rat pancreatic islet cells were preincubated for 60 min at a low (2.8 mM) or high (16.7 mM) concentration of D-glucose, then exposed for 1 min to digitonin (0.5 mg/ml) and eventually centrifuged through a layer of oil for separation of the cell pellet from the supernatant fraction containing the material released by digitonin. Under these conditions, the bulk of lactate dehydrogenase and glutamate dehydrogenase activities were recovered in the supernatant fraction and cell pellet, respectively. The measurement of hexokinase isoenzyme activities in th e two subcellular fractions, as conducted at low or high hexose concentrations and in either the absence or presence of exogenous hexose phosphates (3.0 mM glucose 6-phosphate and 1.0 mM fructose 1-phosphate) indicated a preferential location of the low-Km hexokinase in the cell pellet and of the high-Km glucokinase in the cytosolic fraction. Such a distribution pattern failed to be significantly affected by the concentration of D-glucose used during the initial incubation of the dispersed islet cells. These findings argue against the view that the glucose-induced translocation of glucokinase would result in any sizeable binding of the enzyme to a plasma membrane-associated protein. (Mol Cell Biochem 175: 131–136, 1997)     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5′-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Characterization of a vasoactive intestinal peptide-sensitive adenylate cyclase in rat intestinal epithelial cell membranes

A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 · 10⁻¹⁰−1 · 10⁻⁷ M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 ± 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive inteetinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 μg protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5–10 mM Mg²⁺ and was inhibited by 1 · 10⁻⁵ M Ca²⁺. At sufficiently high concentrations, both ATP (3 mM) and Mg²⁺ (40 mM) inhibited the enzyme.Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E₁ and E₂ at 1 · 10⁻⁵ M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Isolation of pig platelet membranes and granules distribution and validity of marker enzymes

A method for the subcellular fractionation of pig platelet homogenates by sucrose density gradient centrifugation is described. The procedure is simple, highly reproducible and yields two major particulate fractions and a soluble phase. One particulate fraction consists almost entirely of membrane fragments and is relatively free from granule contamination. The other particulate zone contains the platelet granules and mitochondria. The distribution on the gradients of the enzymes lactate dehydrogenase, succinate dehydrogenase, 5′-nucleotidase, leucyl β-naphthylamidase and cholinesterase has been studied and organelle localisation further substantiated by electron microscopy. The degree of solubilisation of certain marker enzymes during homogenisation has been investigated and the parallel release of these enzymes with the soluble phase marker enzyme lactate dehydrogenase, suggests they have a true biphasic location between the soluble and particulate components of the cell. No significant difference was found in the molar ratios of cholesterol to phospholipid in the subcellular fractions but the content of each lipid was twice as high in the membrane fraction as in the granule fraction.     

ENZYMATIC PROPERTIES OF THE INNER AND OUTER MEMBRANES OF RAT LIVER MITOCHONDRIA

Treatment of rat liver mitochondria with digitonin followed by differential centrifugation was used to resolve the intramitochondrial localization of both soluble and particulate enzymes. Rat liver mitochondria were separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes localized between the membranes plus some solublized outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase were found primarily in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, β-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase were found in the inner membrane-matrix fraction. Nucleoside diphosphokinase was found in both the outer membrane and soluble fractions; this suggests a dual localization. Adenylate kinase was found entirely in the soluble fraction and was released at a lower digitonin concentration than was the outer membrane; this suggests that this enzyme is localized between the two membranes. The inner membrane-matrix fraction was separated into inner membrane and matrix by treatment with the nonionic detergent Lubrol, and this separation was used as a basis for calculating the relative protein content of the mitochondrial components. The inner membrane-matrix fraction retained a high degree of morphological and biochemical integrity and exhibited a high respiratory rate and respiratory control when assayed in a sucrose-mannitol medium containing EDTA.     

Rat-kidney lysosomes: isolation and properties

1. The activities of lysosomal enzymes in the cortexes and medullas and the principal subcellular fractions of rat kidney were measured. 2. A method is described for the isolation of rat-kidney lysosomes and a detailed analysis of the enzymic composition of the lysosomes is reported. Enzyme analysis of the other principal subcellular fractions is included for comparison. 3. Studies of the distribution of α-glucosidase showed that the lysosomal fraction contained only 10% of the total enzyme activity. The microsomal fraction contained most of the particulate α-glucosidase. Lysozyme was concentrated mainly in the lysosomal fraction with only small amounts present in the microsomal fraction. Lysosomal α-glucosidase had optimum pH5 whereas the microsomal form had optimum pH6. Both lysosomal and microsomal lysozyme had optimum pH6·2. 4. The stability of lysosomal suspensions was studied. Incubation at 37° and pH7 resulted in first an increased availability of enzymes without parallel release of enzyme. This was followed by a second stage during which the availability of enzymes was closely related to the release of enzymes. These changes were closely paralleled by changes in light-scattering properties of lysosomes. 5. The latent nature of the α-glucosidase and lysozyme of intact kidney lysosomes was demonstrated by their graded and parallel release with other typical lysosomal enzymes. 6. Isolated lysosomes were unstable at pH values lower than 5, most stable at pH6–7 and less stable at pH 8–9. Lysosomes were not disrupted when the osmolarity of the suspending medium was decreased from 0·6m to 0·25m. 7. The discussion compares the properties and composition of kidney lysosomes, liver lysosomes and the granules of macrophages. 8. The possible origin of the lysozyme in kidney lysosomes by reabsorption of the lysozyme in blood is discussed.     

Rat pancreatic acini permeabilised with streptolysin O secrete amylase at Ca2+ concentrations in the micromolar range, when provided with ATP and GTP gamma S.

The aim of this study was to define the conditions required for exocytosis in pancreatic acini permeabilised with the bacterial toxin streptolysin O. Treatment of a suspension of acini with streptolysin O caused the release of both the cytoplasmic enzyme lactate dehydrogenase and the zymogen granule enzyme amylase. The release of amylase occurred more quickly than that of lactate dehydrogenase and was smaller in magnitude. In addition, a component of amylase release occurred only in the presence of Ca2+ (at concentrations in the micromolar range), ATP and GTP gamma S. We conclude that this component represents an exocytotic event, but that the release of lactate dehydrogenase occurs through toxin-generated lesions. The concentrations of Ca2+, ATP and GTP gamma S causing half-maximal exocytosis were 0.7 microM, 0.2 mM and 10 microM, respectively. This system should permit a study of the mechanisms underlying regulated exocytosis in this cell type.     

The osmotically sensitive potassium and sodium compartments of synaptosomes

1. Synaptosomes are pinched-off nerve terminals whose components can be liberated by osmotic `shock'. A synaptosome preparation run through a Sephadex column that was eluted with an iso-osmotic solution retained its small ions, whereas when the column was eluted hypo-osmotically the small ions were lost. In this way the osmotically sensitive Na<sup>+</sup> and K<sup>+</sup> of synaptosomes were measured. Measurements of the lactate dehydrogenase occluded within the synaptosome were also made. The release of osmotically sensitive Na<sup>+</sup> and K<sup>+</sup> and occluded lactate dehydrogenase had similar characteristics with respect to the degree of osmotic `shock' necessary and the action of lytic agents. 2. The distribution of osmotically sensitive Na⁺, K⁺ and occluded lactate dehydrogenase in the subfractions of a crude mitochondrial preparation was examined. The synaptosome fraction was the richest source of these constituents. 3. On standing at 5° in iso-osmotic solution Na⁺ and K⁺ were lost from synaptosomes, whereas the amount of occluded lactate dehydrogenase remained stable, suggesting that the synaptosome membrane retained its integrity but that Na⁺ and K⁺ diffused through it out of the osmotically sensitive compartment. 4. The uptake of Na⁺ and K⁺ into the osmotically sensitive compartment was examined. At 5° the rates of uptake of Na⁺ and K⁺ were found to be equal to the rates of loss of these ions when correction to a uniform concentration gradient had been made. K⁺ travelled across the membrane slightly faster than Na⁺, the rate of K⁺ movement being about 1·0μμequiv.cm.⁻²sec.⁻¹ under a concentration gradient of 0·1m. Active transport is not thought to contribute to the ion movements under the conditions used. 5. The amount of K⁺ taken up into the osmotically sensitive compartment as a function of the external concentration was examined. Since the uncharged molecule d-[¹⁴C]galactose distributes across the synaptosome membrane similarly to K⁺ there is not thought to be a synaptosomal trans-membrane potential. The volume of the osmotically sensitive compartment was measured by this method and found to agree with estimates of the synaptosomal volume made from morphological studies. In media of low ionic strength synaptosomes exhibit a Donnan effect. 6. It is concluded from these studies that the osmotically sensitive compartment represents the inner volume of the synaptosome, which is completely separated from the outside environment by a diffusion barrier having many of the general properties of a biological membrane.     

Translocation of Mg2+-dependent phosphatidate phosphohydrolase between cytosol and endoplasmic reticulum in a permanent cell line from human lung

Incubation of A549 cells with digitonin for 4 min resulted in the release of over 90% of the lactate dehydrogenase activity into the medium. Approximately 80% of the Mg2+-dependent but only 7% of the Mg2+-independent phosphatidate phosphohydrolase activity was released in the presence of digitonin. Pretreatment of the cells with oleate reduced the efflux of the Mg2+-dependent phosphatidate phosphohydrolase activity to approximately 5% of total. Oleate did not affect the release of lactate dehydrogenase or the release of the Mg2+-independent phosphohydrolase activity. Incubation of A549 cells with [3H]oleate for 60 min led to incorporation of the label into phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, diacylglycerol, monoacylglycerol, and triacylglycerol, in ascending order. When the level of exogenous oleate was increased to over 2.0 mM, there was a marked increase in the incorporation into monoacylglycerol and diacylglycerol. Only small amounts of radioactivity were associated with phosphatidic acid. Time course studies revealed that the amount of radioactive phosphatidate remained low throughout the incubation period. These investigations were interpreted to indicate that free fatty acids can promote the translocation of the Mg2+-dependent phosphatidate phosphohydrolase activity from cytosol to membrane fractions. This translocation could, at least theoretically, function to facilitate the metabolism of increased amounts of phosphatidate.     

Tauroursodeoxycholate counteracts hepatocellular lysis induced by tensioactive bile salts by preventing plasma membrane-micelle transition

Ursodeoxycholic acid is widely used as a therapeutic agent for the treatment of cholestatic liver diseases. In these hepatopathies, the bile secretory failure produces accumulation of endogenous, tensioactive bile salts, leading to plasma membrane damage and, eventually, hepatocellular lysis. In the present study, we analyzed the capacity of the ursodeoxycholic acid endogenous metabolite, tauroursodeoxycholate (TUDC), to stabilize the hepatocellular plasma membrane against its transition to the micellar phase induced by the tensioactive bile salt taurochenodeoxycholate (TCDC), the main endogenous bile salt accumulated in cholestasis. The disruption of the plasma membrane was evaluated (i) in isolated hepatocytes, through the release of the enzyme lactate dehydrogenase to the incubation medium and (ii) in isolated plasma membranes, through the self-quenching assay of the membranotropic probe octadecylrhodamine B; this assay allows for detergent-induced transition from membrane bilayer to micelle to be monitored. Our results showed that isolated hepatocytes treated with TUDC are more resistant to TCDC-induced cell lysis. When this effect was evaluated in isolated plasma membranes, the TCDC concentration necessary to reach half of the transition from bilayer to micelle was increased by 22% (p < 0.05). This difference remained even when TUDC was removed from the incubation medium before adding TCDC, thus indicating that TUDC exerted its effect directly on the plasma membrane. When the same experiments were carried out using the non-ionic detergent TX-100 or the cholesterol-complexing detergent digitonin, no protective effect was observed. In conclusion, TUDC prevents selectively the bilayer to micelle transition of the hepatocellular plasma membrane induced by hydrophobic bile salts that typically build up and accumulate in cholestatic processes. Our results suggest that formation of a complex between negatively charged TUDC and cholesterol in the membrane favours repulsion of negatively charged detergent bile salts, thus providing a basis for the understanding of the TUDC protective effects.     

Release of unsaturated vitamin B12 binding capacity from human granulocytes by the calcium ionophore A23187

The ionophore A23187, in the presence of calcium ions, was capable of eliciting the release of granule-associated unsaturated vitamin B₁₂ binding capacity from human granulocytes. The ionophore-induced extrusion of unsaturated vitamin B₁₂ binding capacity was concentration, time and temperature-dependent. The release was blocked by 2-deoxyglucose and was unaccompanied by cytotoxicity (trypan-blue uptake and lactate dehydrogenase release). The unsaturated vitamin B₁₂ binding capacity release was accompanied by the release of lysozyme, a specific granule marker enzyme.     

The involvement of ionic interactions during asbestos-induced enzyme release from polymorphonuclear leukocytes

Chrysotile asbestos permeabilizes the plasma membrane of rabbit polymorphonuclear leukocytes (PMNs) which is evident from the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) from the cell. When Ca2+ is present in the medium exocytosis is observed, evident from the release of the granule associated enzyme lysozyme which is not liberated in the absence of Ca2+. Asbestos-induced enzyme release is inhibited by polyanions or by removal of positive charges on asbestos, and resembles enzyme release induced by synthetic polycations. Pretreatment of PMNs with neuraminidase does not affect the ability of asbestos to induce enzyme release from these cells. Asbestos induces release of glucose from glucose-loaded liposomes, and this effect can be inhibited by the polyanion poly-D-glutamic acid. The results are compatible with the view that positive charges play a decisive role in the interaction between PMNs and asbestos, and that the primary target of asbestos could be the lipid bilayer of the membrane. The interaction results in a permeabilized plasma membrane. When Ca2+ is present in the medium it moves into the cell and causes exocytosis of the granule enzyme lysozyme. Inhibition of cytotoxicity by polyanion may cause a diminished Ca2+-influx and hence inhibition of lysozyme release.     

The aromatization of cyclohexanecarboxyl-CoA to hippuric acid by guinea pig liver mitochondria: submitochondrial localization

Summary The conversion of cyclohexanecarboxyl-CoA to hippuric acid in submit ochondrial fractions from guinea pig liver was studied using a gas chromatographic-mass spectrometric method employing selected ion monitoring. Comparison of the activities of the cyclohexanecarboxyl-CoA to hippuric acid converting system (CCoAHC-system) and marker enzymes in the various submit ochondrial fractions showed that the CCoAHC-system is localized in the mitochondrial matrix. Partial separation of the inner and outer membranes has been accomplished by treating mitochondria with digitonin in isotonic medium and fractionating the treated mitochondria by differential centrifugation. A digitonin-protein ratio of 2.6 mg of digitonin/10 mg of protein must be used in order to release significant amounts of amine oxidase activity (outer membrane marker) from low speed mitochondrial pellets. This pellet still contained most of the glutamate dehydrogenase activity and was insignificantly contaminated with adenylate kinase. Moderate concentrations of phenazine methosulfate (PMS) greatly stimulated the activity of the CCoAHC-system, even in intact mitochondria (optimal concentration of PMS: 1 mM) whilst higher concentrations (> 1 mM) decreased the activity. The formation of hippuric acid in these mitochondrial preparations was linear with time for at least 40 min and linear with respect to protein concentration up to approximately 2.0 mg mitochondrial protein·m¹.     

The sequestration of Ca2+ by mitochondria in rat heart cells

Rat heart ventricular cells, purified by Percoll density gradient centrifugation, were incubated in the presence of 1.3 mM CaCl2. After 20 min incubation, samples of the cells were lysed in medium containing 0.3 mM digitonin, ruthenium red and EGTA, and a mitochondrial fraction was isolated at intervals thereafter. Extrapolation of the mitochondrial 45Ca2+ contents to zero time enabled the endogenous 45Ca2+ to be estimated at the time of cell lysis. The lysis conditions yielded essentially complete release of lactate dehydrogenase from the cells, but caused negligible damage to the mitochondria as judged by their retention of glutamate dehydrogenase, and their ability to accumulate and retain Ca2+ in the absence of ruthenium red and EGTA. The data indicate that about 13% of total cell Ca2+ only may be mitochondrial in vivo.     

Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture

Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.     

Localization of enzymes by means of proteases

A method is described which makes it possible to determine whether a given enzyme is located on the outer surface of a mitochondrial membrane, or wether it is localized within a mithondrial compartment. The method combines the use of proteases with digitonin. Depending on the concentration of digitonin, enzymes behind the microsomal membranes and the outer mitochondrial membrane may be successively exposed to the action of proteases. Thus, enzymes located within microsomes contaminating the mitochondrial fraction may be easily distinguished from true intramitochondrial enzymes.Applying this technique to the mitochondrial fraction of rat liver, it is shown that lactate dehydrogenase (l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27) is located on the outer surface of the mitochondria and within microsomal contaminants.