Presence Of Digitonin Research Articles

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.

Role of a callose synthase zymogen in regulating wall deposition in pollen tubes of Nicotiana alata Link et Otto

The callose synthase (CalS) activity of membrane preparations from cultured Nicotiana alata Link & Otto pollen tubes is increased several-fold by treatment with trypsin in the presence of digitonin, possibly due to activation of an inactive (zymogen) form of the enzyme. Active and inactive forms of CalS are also present in stylar-grown tubes. Callose deposition was first detected immediately after germination of pollen grains in liquid medium, at the rim of the germination aperture. During tube growth the 3-linked glucan backbone of callose was deposited at an increasing rate, reaching a maximum of 65 mg h−1 in tubes grown from 1 g pollen. Callose synthase activity was first detected immediately after germination, and then also increased substantially during tube growth. Trypsin caused activation of CalS throughout a 30-h time course of tube growth, but the degree of activation was higher for younger pollen tubes. Over a 10-fold range of callose deposition rates, the assayed CalS activity was sufficient to account for the rate of callose deposition without trypsin activation, implying that the form of CalS active in isolated membranes is responsible for callose deposition in intact pollen tubes. Sucrose-density-gradient centrifugation separated a lighter, intracellular membrane fraction containing only inactive CalS from a heavier, plasma-membrane fraction containing both active and inactive CalS, with younger pollen tubes containing relatively more of the inactive intracellular enzyme. The increasing rate of callose deposition during pollen-tube growth may thus be caused by the transport of inactive forms of CalS from intracellular membranes to the plasma membrane, followed by the regulated activation of these inactive forms in this final location.

CD19 Is Linked to the Integrin-associated Tetraspans CD9, CD81, and CD82

The CD19-CD21-CD81 complex regulates signal transduction events critical for B lymphocyte development and humoral immunity. CD81, a molecule with 4 transmembrane domains, member of the tetraspan superfamily, is engaged, together with other tetraspans such as CD9, CD53, CD63, and CD82, in multimolecular complexes containing beta1 integrins and major histocompatibility complex antigens. Here we demonstrate that two other tetraspans, CD82 and the early B cell marker CD9, are coimmunoprecipitated with CD19 from Brij97 lysates of B cell lines. Moreover, CD9 was coprecipitated from lysates of purified CD10(+) early B cells. These associations were confirmed by the cocapping of CD19 with CD9 or CD82. The CD9/CD19 association was disrupted in the presence of digitonin, contrary to the CD81/CD19 association, indicating that CD9 and CD81 interact with CD19 in different ways. The CD9/CD81 association is also disrupted in the presence of digitonin, suggesting that CD9 associates with CD19 only through CD81. To characterize the regions involved in the CD81/CD19 association, two reciprocal CD9/CD81 chimeric molecules were tested for the association with CD19, but none of them could be coprecipitated with CD19 in digitonin, indicating that the domain of CD81 responsible for its association with CD19 is complex. Finally, engagement of CD9 could induce the tyrosine phosphorylation of different proteins, including CD19 itself, suggesting that the CD9/CD19 association is functionally relevant. Thus, a physical and functional link is formed between the CD19-CD21-CD81 complex and the integrin-tetraspan complexes, which is dynamically modulated in the process of B cell differentiation.

The Extracellular Domain of the Sodium Pump β Isoforms Determines Complex Stability with α1

Both subunits of the Na,K-ATPase are encoded by several genes giving rise to at least six isozymes. To examine whether β isoforms assemble with α1 in a selective manner, we have overexpressed wild-type and chimeric β subunits in L929 cells and examined assembly as a function of resistance towards detergent-mediated dissociation. In the presence of digitonin all β chimeras coimmunoprecipitate the endogenous α1 subunit. Only β proteins with the ectodomain of β1 coimmunoprecipitate α1 in the presence of Triton X-100. All β chimeras stimulate Na,K-ATPase activity in L929 cells. These data indicate that the β subunit ectodomains mediate interactions with α1 and influence the stability of this complex.

Activatory effect of calcium-binding protein regucalcin on ATP-dependent calcium transport in the basolateral membranes of rat kidney cortex.

The effect of regucalcin, a calcium-binding protein, on ATP-dependent Ca2+ transport in the basolateral membranes isolated from rat kidney cortex was investigated. The prepared membranes were in inside-out oriented and membrane vesicles. Ca(2+)-ATPase activity in the basolateral membranes was progressively elevated by increasing concentrations of regucalcin (10(-8) to 10(-6) M) in the reaction mixture. This increase was dependent on Ca2+ addition. The activatory effect of regucalcin on the enzyme is inhibited by the presence of digitonin (5 x 10(-3)%) which can solubilize the membranous lipids. Moreover, the regucalcin effect was clearly abolished by the presence of vanadate (0.1 mM) or N-ethylmaleimide (5.0 mM). However, the effect of calmodulin (6 x 10(-7) M) to increase Ca(2+)-ATPase activity was not significantly inhibited by vanadate or N-ethylmaleimide, indicating that the action mode of regucalcin differs from that of calmodulin. Also, the activatory effect of regucalcin on Ca(2+)-ATPase was appreciably inhibited by addition of dibutyryl cAMP (10(-5) and 10(-3) M), while inositol 1,4,5-trisphosphate (10(-7) and 10(-5) M) had no effect. Dibutyryl cAMP itself did not have an effect on the enzyme activity. Furthermore, the 45Ca2+ uptake by the basolateral membranes was clearly increased by the presence of regucalcin (10(-7) and 10(-6) M). This increase was completely blocked by the presence of vanadate (0.1 mM), N-ethylmaleimide (5.0 mM) or dibutyryl cAMP (10(-4) and 10(-3) M) in the reaction mixture. These results clearly demonstrate that regucalcin, which is expressed in rat kidney cortex, can increase Ca(2+)-ATPase activity and Ca2+ uptake in the basolateral membranes. Regucalcin may play a cell physiologic role as an activator in the ATP-dependent Ca2+ pumps in the basolateral membranes from rat kidney cortex.

The human natural killer cell receptor for major histocompatibility complex class I molecules. Surface modulation of p58 molecules and their linkage to CD3 ζ chain, FcϵRI γ chain and the p56lck kinase

The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183-EB6+ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4+ C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183+/EB6+ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the zeta family (i.e. CD3 zeta and Fc epsilon RI gamma chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58+ NK cells indicated a preferential association with CD3 zeta chains either in the form of covalently linked zeta-zeta homodimers or in the form of zeta-gamma heterodimers, while gamma-gamma homodimers were detectable in low amounts. However, p58+ clones displaying a unique association with gamma-gamma homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 zeta chains, no tyrosine phosphorylation was detected in the p58-associated CD3 zeta chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the gamma chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompanied by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 zeta chains.

Particulate binding sites for steroid hormones in subcellular fractions of the ovine corpus luteum: properties and hormone specificity

We have described previously the presence of binding sites in particulate fractions of the porcine corpus luteum (CL) which were specific for progesterone. We now demonstrate the presence of similar progesterone-specific binding sites in particulate fractions of the ovine CL. Preincubation of ovine luteal membranes with radiolabelled steroids demonstrated binding of progesterone and pregnenolone to a low-density particulate fraction (1.07–1.09 g/cm 3). Preincubation with digitonin perturbed the buoyant density of this fraction (to1.10–1.14 g/cm 3) without causing release of steroid. Androgens and oestrogens did not bind appreciably to control luteal membranes, but were bound when preincubated with digitonin. In contrast, steroid conjugates (oestrone sulfate, pregnanediol glucuronide), cortisol, fatty acids (arachidonic acid, prostaglandin F 2α) and cholesterol ester failed to bind to ovine luteal membranes, with or without digitonin pretreatment. The effects of digitonin on steroid binding led us to examine its effects on steroid binding to ovine luteal membrane fractions in vitro. Specific progesterone binding was absent in the absence of digitonin, even at very high membrane concentrations. However, binding of 3H-labelled progesterone was stimulated 5–15-fold in a dose-dependent fashion by increasing digitonin concentrations, reaching a plateau at about 100 μM. In the presence of digitonin, [ 3H]progesterone binding increased linearly with luteal membrane concentration. Other detergents, saponins and cardiotonic steroids tested did not stimulate progesterone binding to ovine luteal membranes. [ 3H]Progesterone binding was dependent on the pH, duration and temperature of incubation. Unlabelled progesterone decreased binding of [ 3H]progesterone (half-maximal displacement of specific binding (IC 50) at about 60 nM) whereas androgens were less potent (IC 50, 500–3300 nM), whilst a wide range of other steroids and inhibitors of steroidogenic enzymes were neffective, except at very high concentrations. Similarly, a number of progesterone receptor agonist and antagonist analogues failed to compete for progesterone binding to luteal membranes, suggesting that these binding sites were unrelated to progesterone receptors. Modifications to the 3,4, 5 and 11 positions of progesterone, removal of the steroid side-chain or aromatization of the A-ring decreased binding potency dramatically, whereas changes to the 17 or 20 positions had relatively minor effects. Our results indicate the presence of a low density particulate fraction in ovine corpora lutea which contains specific binding sites for endogenous and exogenous progesterone.

Activating effect of regucalcin on (Ca(2+)-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group.

The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca(2+)-Mg2+)-ATPase in the plasma membranes of rat liver was investigated. (Ca(2+)-Mg2+)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5-5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25-1.0 microM) significantly increased (Ca(2+)-Mg2+)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca(2+)-Mg2+)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 microM) increasing (Ca(2+)-Mg2+)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca(2+)-Mg2+)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme.

Specificity studies of particulate binding sites for steroid hormones in subcellular fractions of the porcine corpus luteum

We have studied the binding of a number of radiolabelled steroids and lipophilic substances to porcine corpus luteum (CL) particulate fractions. Following preincubation of CL homogenates with radiolabelled progesterone or pregnenolone prior to fractionation on continuous sucrose density gradients, a broad peak of binding was observed associated with a particulate fraction of buoyant density 1.05-1.10 g/cm3. Progesterone content also peaked at a similar buoyant density (1.06-1.12 g/cm3). Pretreatment of luteal homogenates with digitonin perturbed the buoyant density of the progesterone-binding particulate fraction to 1.10-1.14 g/cm3 and sharpened the binding peak. Progesterone content was also perturbed to a similar extent by digitonin pretreatment, without release of the steroid. Oestrogens were also sequestered by this fraction, but steroid precursors (cholesterol, cholesterol ester), corticosteroids (cortisol, corticosterone), sterol conjugates (oestrone sulphate, pregnanediol glucuronide) and other lipophilic substances (arachidonic acid, phospholipid, prostaglandins E1, E2 and F2 alpha) were not bound. Androgens were bound weakly by fractions from control gradients but, in the presence of digitonin, significant binding could be demonstrated. Radiolabelled steroids were shown to interact directly with luteal membrane fractions, rather than interacting first with cytosolic steroid receptors which then bound to membranes. Furthermore, [3H]progesterone was not bound by porcine granulosa cell particulate fractions. These observations suggest that this fraction may be involved in sequestration or packaging of progesterone for secretion by the luteal cell.

Insulin receptor β-subunit serine phosphorylation in permeabilized cultured fetal rat hepatocytes

Regulation of cellular protein phosphorylation by insulin was investigated after short exposure at 37°C prior to applying the permeabilization/phosphorylation step in the presence of digitonin and [γ- 32P]ATP for 30 min at 4°C. The results revealed major 32P incorporation into a limited number of membrane polypeptides exhibiting a molecular mass of 95, 58 and 51 kDa. Phosphorylation of 95 kDa protein was selectively inhibited with Ca 2+-free EGTA-containing permeabilization/phosphorylation buffer and became predominant in the presence of Ca 2+. Considering in particular its immunoprecipitation by a monoclonal antibody directed against insulin receptor, the 32P-labeled 95 kDa protein represented the β-subunit of the insulin receptor. Its phosphorylation was transiently stimulated after exposure to insulin (35% after 2 min), and concerned mostly serine residues under both basal and stimulated conditions. Vanadate had a similar effect and both agents favored glycogenesis, whereas heparin which inhibited 95 kDa protein phosphoseryl phosphorylation had an opposite effect on glycogenesis. These results suggest a biological role for the membrane-associated phosphoseryl-protein kinase(s) and phosphatase(s) acting on the insulin receptor β-subunit in cultured fetal hepatocytes.

Spectral analysis of cytochromes in rat heart myocytes: Transient and steady-state photodiode array spectrophotometry measurements

Myocytes prepared from rat heart have been studied by optical spectroscopy using a photodiode array spectrophotometer adapted to a stopped flow apparatus (PASF). The isolated cells were viable for 3–4 h (i.e., over the total time of the experiments), as tested employing morphological parameters of cell damage, reactivity toward trypan blue, and the ability to use succinate in the absence and presence of digitonin. Respiration was activated by addition of sodium ascorbate and tetramethyl- para-phenylendiamine (TMPD) as exogenous reductants, in order to single out the contributions of cytochrome c and cytochrome c oxidase among the complexes of the mitochondrial respiratory chain. TMPD was shown to be freely permeable across cytoplasmic and mitochondrial membranes, with a measured K D = 0.9 mM. The use of singular value decomposition analysis coupled to PASF acquisition proved very powerful in resolving statically and kinetically, in the millisecond time region, the spectral contributions of the cytochromes. Spectral analysis was improved by adding carbon monoxide at concentrations which did not affect cytochrome c oxidase activity, but kept myoglobin fully saturated (and thus uninfluential to absorbance changes).

Evidence for the simulaneous translocation of muscarinic acetylcholine receptor and G protein by carbachol

Interactions between guanine nucleotide regulatory proteins (G proteins) and muscarinic acetylcholine receptors (mAChRs) were studied in vivo following carbachol treatment. Rat brain homogenates were separated by high speed ultracentrigation into heavy and light membrane and 300,000 g supernate franctions. The G proteins were partially purified by Sephadex-G200 and heptylamine-Sepharose and the mAChRs by (3,2′-aminobenzhydryloxy)-tropane-(ABT)-affinity chromatographies. Radioligand binding assays showed that acute carbachol induced a biphasic translocation of the mAChRs and G proteins into the light membrane fraction with an initial release at 5–10 min and a second phase at 60 min. Portions of the released mAChRs and the G proteins, were found in the 300,000 g supernates and light membranes and were eluted in the same peak fractions from a Sephadex G-200 column. This dually labelled peak dissociated in the presence of digitonin, suggesting close association between the mAChR and G protein. ABT-affinity chromatography yielded dually labelled mAChR-G protein fractions which eluated as a single radioactive peak on a second ABT column. the partially purified G proteins from these fractions were photoaffinity labelled with 8-azidoguanosine-5′-triphosphate, [γ- 32P]. SDS-PAGE autoradiography revealed the presence of G 0ξ and G iα which may be released simultaneously with the mAChRs from the plasma membrane. In addition, a 110,000 molecular weight polypeptide was dually labelled by [ 3H]-PrBCM and [γ- 32P]-8-azido-GTP suggesting the presence of a “mAChR-G protein complex.” These findings provide direct evidence for the release of mAChRs and G proteins and a mAChR-G protein complex by agonist occupation of the mAChRs.

Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100 000 × g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.

The Cellular Dynamics of Hepatic Receptors for α2-Macroglobulin.Protease Complex and for Insulin Are Different1

Making freshly isolated rat hepatocytes permeable by 0.4 g/liter digitonin doubled the number of binding sites for alpha 2-macroglobulin.trypsin complex without changing the affinity. Thus, digitonin unmasked a receptor pool, probably of intracellular origin. The total cellular binding capacity was measured in the presence of digitonin, the surface-exposed in its absence. Upon preincubation of the cells at 37 degrees C, the total cellular binding capacity for alpha 2-macroglobulin.trypsin decreased over a 2-h period to 0.26 of the initial value. By contrast, the surface-exposed binding capacity initially increased in response to a preincubation at 37 degrees C, reached after 20 min a peak value 1.74 times that at 0 time, followed by a decrease. Neither the increase in nor the loss of surface-exposed binding capacity was influenced by inhibitors of lysosomal functions, protein synthesis and glycosylation. Colchicine abolished the increase in surface-exposed binding capacity but not the disappearance. By contrast, phenylarsine oxide (inhibitor of endocytosis), N-ethylmaleimide, and phenylmethanesulphonyl fluoride inhibited the receptor loss, suggesting that the loss occurred by proteolysis. The insulin receptor concentration, studied in parallel, remained practically constant in the investigated period in the presence and absence of digitonin. Thus, the hepatic receptor for alpha 2-macroglobulin.protease complexes is regulated independently of other specialized plasma membrane proteins.

Some differences in the properties of carnitine palmitoyltransferase activities of the mitochondrial outer and inner membranes

Recent evidence has shown that the outer, overt, malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPTo) activity resides in the mitochondrial outer membrane [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382]. A comparison of CPTo activity of rat liver mitochondria with the inner, initially latent, carnitine palmitoyltransferase (CPTi) of the mitochondrial inner membrane has revealed that the presence of digitonin and several other detergents inactivates CPTo activity. The CPTi activity, in contrast, was markedly stimulated by various detergents and phospholipid liposomes. These findings explain why in previous studies, which used digitonin or other detergents to expose, separate and purify the CPT activities, the inferences were drawn that (a) the ratio of latent to overt CPT was quite high, (b) both the CPT activities could be ascribed to one active protein recovered, and (c) the observed lack of malonyl-CoA inhibition indicated possible loss/separation of a putative malonyl-CoA-inhibition-conferring protein. Although both CPTo and CPTi were found to catalyse the forward and the backward reactions, CPTo showed greater capacity for the forward reaction and CPTi for the backward reaction. The easily solubilizable CPT, released on sonication of mitoplasts or of intact mitochondria under hypo-osmotic conditions, resembled CPTi in its properties. When octyl glucoside was used under appropriate conditions, 40-50% of the CPTo of outer membranes became solubilized, but it showed limited stability and decreased malonyl-CoA sensitivity. Malonyl-CoA-inhibitability of CPTo was decreased also on exposure of outer membranes to phospholipase C. When outer membranes that had been exposed to octyl glucoside or to phospholipase C were subjected to a reconstitution procedure using asolectin liposomes, the malonyl-CoA-inhibitability of CPTo was restored. A role of phospholipids in the malonyl-CoA sensitivity of CPTo is thus indicated.

Measurement of cytoplasmic calcium in aleurone protoplasts using indo-1 and fura-2

Previous attempts to measure cytoplasmic Ca 2+ in plant cells using the new generation of fluorescent probes, indo-1 and fura-2, have been unsuccessful. We investigated the use of indo-1 and fura-2 to measure cytoplasmic Ca 2+ in barley aleurone protoplasts and found that indo-1 could be successfully used when it was loaded into protoplasts in the Ca 2+-sensitive form. The acetoxymethyl esters of both dyes accumulated in aleurone protoplasts, but fura-2 was sequestered in the vacuole and indo-1 was not adequately hydrolyzed. We developed a non-disruptive method for loading the Ca 2+-sensitive form of indo-1 into aleurone protoplasts in mildly acidic solutions. Using this approach, protoplasts accumulate indo-1 in a pH-dependent manner. The accumulated dye is Ca 2+-sensitive, it is not sequestered in vacuoles or the endomembrane system, and it is not rapidly secreted. Fluorescence from indo-1 in individual cells was quenched by Mn 2+ in the presence of digitonin. We estimate the cytoplasmic Ca 2+ concentration in aleurone protoplasts to be approximately 250 nM. The Ca 2+ ionophore, ionomycin does not induce changes in the fluorescence of protoplasts loaded with indo-1, but fluorescence changes could be induced by changes in extracellular Ca 2+ in the presence of digitonin. We conclude that the strategy of loading indo-1 at acidic pH provides a useful means of measuring cytoplasmic Ca 2+ in the barley aleurone that may also be applicable to other types of plant cells.

Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle.

Purified dihydropyridine-sensitive calcium channels from rabbit transverse-tubule membranes consist of three noncovalently associated classes of subunits: alpha (167 kDa), beta (54 kDa), and gamma (30 kDa). Cleavage of disulfide bonds reveals two distinct alpha polypeptides and an additional component, delta. The alpha 1 subunit, a 175-kDa polypeptide that is not N-glycosylated, contains the dihydropyridine binding site, cAMP-dependent protein kinase phosphorylation site(s), and substantial hydrophobic domain(s). alpha 2, a 143-kDa glycoprotein, has none of the properties characteristic of alpha 1 but binds lectins and contains about 25% N-linked carbohydrate. alpha 2 is disulfide-linked to delta, a 24- to 27-kDa glycopeptide. beta (54 kDa) contains a cAMP-dependent phosphorylation site but is not N-glycosylated and does not have a hydrophobic domain. gamma (30 kDa) has a carbohydrate content of about 30% and extensive hydrophobic domain(s). Precipitation with affinity-purified anti-alpha 1 antibodies or alpha 2-specific lentil lectin-agarose demonstrated that alpha 1 alpha 2 beta gamma delta behaves as a complex in the presence of digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, whereas the alpha 2 delta complex dissociates from alpha 1 beta gamma in the presence of Triton X-100. A model for subunit interaction and membrane insertion is proposed on the basis of these observations.

The development of monospecific antibodies against human thrombo-plastin apoprotein (apoprotein III) and their application in the immunocytochemical detection of the antigen in blood cells

Human thromboplastin apoprotein (apoprotein III) purified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was purified a further 2–4 fold by PAGE in the presence of digitonin. Subsequent line immunoelectrophoresis of the protein revealed several lines, only one of which contained inhibitory antibodies. New inhibitory antibodies which were raised by using this particular line to immunize rabbits produced only a single line in immunoelectrophoresis of apoprotein III, with precipitated inhibitory antibodies being present only in the line. When these antibodies were used in electroblot immunobinding studies of crude thromboplastin after SDS-PAGE staining was found mainly in a single band of MW about 50 000, but also to some extent in immunologically related higher MW material. Prior deglycosylation of the thromboplastin using trinitrobenzenesulfonic acid resulted in a shift of the bulk of the main band representing an apparent MW reduction of 16%, and a corresponding shift in the position of protein with the capacity to bind inhibitory antibodies. Besides being a good criterion of specificity of the antibodies this also suggests that non-carbohydrate parts of apoprotein III may be involved in the interaction with Factor VII. Immunoperoxidase staining of unstimulated or endotoxin stimulated blood cells using the antibodies revealed the presence of significant amounts of apoprotein III only in stimulated monocytes, apparently available on the surface of the cells since it was detectable also by preembedding staining of fixed cells in suspension. The result is strong evidence that apoprotein III is synthesized de novo in monocytes upon endotoxin stimulation.

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.

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.