Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)

The ATPase activity of the chloroplast coupling factor 1 (CF₁) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca²⁺ -dependent activity. The Ca²⁺ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca²⁺ to Mg²⁺. Both the Ca²⁺ -dependent and Mg²⁺ -dependent ATPase activities of heat-treated Dunaliella CF₁ are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF₁. However, when assayed under identical conditions, the Ca²⁺ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg²⁺ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF₁ can exist in a variety of conformations, at least one of which catalyzes a Ca²⁺ -dependent ATPase and two or more of which catalyze an Mg²⁺ -dependent ATPase.     

Tight nucleotide binding sites and ATPase activities of theRhodospirillum rubrum RrF1-ATPase as compared to spinach chloroplast CF1-ATPase

SolubilizedRhodospirillum rubrum RrF₁-ATPase, depleted of loosely bound nucleotides, retains 2.6 mol of tightly bound ATP and ADP/mol of enzyme. Incubation of the depleted RrF₁ with Mg²⁺-ATP or Mg²⁺-AMP-PNP, followed by passage through two successive Sephadex centrifuge columns, results in retention of a maximal number of 4 mol of tightly bound nucleotides/mol of RrF₁. They include 1.5 mol of nonexchangeable ATP, whereas all tightly bound ADP is fully exchangeable. A similar retention of only four out of the six nucleotide binding sites present on CF₁ has been observed after its passage through one or two centrifuge columns. These results indicate that the photosynthetic, unlike the respiratory, F₁-ATPases have fasterk _off constants for two of the Mg-dependent nucleotide binding sites. This could be the reason for the tenfold lower Mg²⁺ than Ca²⁺-ATPase activity observed with native RrF₁, as with -depleted, activated CF₁. An almost complete conversion of both RrF₁ and CF₁ from Ca²⁺- to Mg²⁺-dependent ATPases is obtained upon addition of octylglucoside, at concentrations below its CMC, to the ATPase assay medium. Thus, octylglucoside seems to affect directly the RrF₁ and CF₁ divalent cation binding site(s), in addition to its proposed role in relieving their inhibition by free Mg²⁺ ions. The RrF₁-ATPase activity is 30-fold more sensitive than CF₁ to efrapeptin, and completely resistant to either inhibition or stimulation by the CF₁ effector, tentoxin. Octylglucoside decreases the inhibition by efrapeptin and tentoxin, but exposes on CF₁ a low-affinity, stimulatory site for tentoxin.Abbreviations: CF₁, EcF₁, MF₁, and TF₁, the soluble F₁-ATPase from chloroplasts, PE. coli, mitochondria,R. rubrum, and the thermophilic bacterium PS3, respectively: AMP-PNP, adenylyl-, -imidodiphosphate; CMC, critical micellar concentration; DTT, dithiothreitol, LDAO, lauryl dimethylamine oxide.Dedicated to Professor Achim Trebst in honor of this 65th birthday.     

Chloroplast genes encoding subunits of the H+-ATPase complex of Chlamydomonas reinhardtii are rearranged compared to higher plants: sequence of the atpE gene and location of the atpF and atpI genes

Summary The chloroplast gene for the epsilon subunit (atpE) of the CF₁/CF₀ ATPase in the green alga Chlamydomonas reinhardtii has been localized and sequenced. In contrast to higher plants, the atpE gene does not lie at the 3 end of the beta subunit (atpB) gene in the chloroplast genome of C. reinhardtii, but is located at a position 92 kb away in the other single copy region. The uninterrupted open reading frame for the atpE gene is 423 bp, and the epsilon subunit exhibits 43% derived amino acid homology to that from spinach. Codon usage for the atpE gene follows the restricted pattern seen in other C. reinhardtii chloroplast genes.The genes for the CF₀ subunits I (atpF) and IV (atpI) of the ATPase complex have also been mapped on the chloroplast genome of C. reinhardtii. The six chloroplast ATPase genes in C. reinhardtii are dispersed individually between the two single copy regions of the chloroplast genome, an organization strikingly different from the highly conserved arrangement in two operon-like units seen in chloroplast genomes of higher plants.Abbreviations bp base pairs - CF₁ chloroplast coupling factor 1 - CF₀ chloroplast coupling factor 0 - F₁ coupling factor 1 - F₀ coupling factor 0 - kb kilobase pairs     

5-O-β-d-galactopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin,an inhibitor of photophosphorylation in spinach chloroplasts

5-O--d-galactopyranosyl-7-methoxy-3,4-dihydroxy-4-phenylcoumarin isolated from Exostema caribaeum (Rubiaceae) has been found to act as an energy-transfer inhibitor in spinach chloroplasts. ATP synthesis and phosphorylating (coupled) electron flow were inhibited by 89 and 72%, respectively, at a concentration of 400 M. H⁺-uptake, basal and uncoupled electron transport were not affected by the coumarin. The light-activated Mg⁺²-ATPase activity from bound membrane thylakoid chloroplasts was slightly inhibited by the coumarin. Also, the heat-activated Ca⁺²-ATPase activity of the isolated coupling factor protein was insensitive to this compound. In chloroplasts partially stripped of coupling factor 1 by an EDTA treatment, the coumarin showed a restoration of the proton uptake process. These results suggest that the 4-phenylcoumarin under investigation inhibited phosphorylation in chloroplasts by specifically blocking the transport of protons through a membrane-bound component or a carrier channel (CF_O) located in a hydrophobic region at or near the functional binding site for the coupling factor 1.Abbreviations CF₁ chloroplast coupling factor 1 - CF_O coupling factor zero - DCCD dicyclohexylcarbodiimide - DTT dithiothreitol - EDTA ethylene-diaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - MES 2-(N-morpholino) ethanesulphonic acid - TCA trichloroacetic acid Taken in part from PhD thesis of M.R. Calera.     

The role of chloroplast membranes in the location of chloroplast DNA during the greening ofPhaseolus vulgaris etioplasts

Summary The location of DNA containing nucleoids has been studied in greening bean (Phaseolus vulgaris L.) etioplasts using electron microscopy of thin sections and the staining of whole leaf cells with the fluorochrome DAPI. At 0 hours illumination a diffuse sphere of cpDNA surrounds most of the prolamellar body. It appears to be made up of a number of smaller nucleoids and can be asymmetric in location. The DNA appears to be attached to the outside of the prolamellar body and to prothylakoids on its periphery. With illumination the nucleoid takes on a clear ring-like shape around the prolamellar body. The maximum development of the ring-like nucleoid at 5 hours illumination is associated with the outward expansion of the prolamellar body and the outward growth of the prothylakoids. At 5 hours the electron transparent areas lie in between the prothylakoids radiating out from the prolamellar body. Between 5 hours and 15 hours observations are consistent with the growing thylakoids separating the nucleoids as the prolamellar body disappears and the chloroplast becomes more elongate. At 15 hours the fully differentiated chloroplast has discrete nucleoids distributed throughout the chloroplast with evidence of thylakoid attachment. This is the SN (scattered nucleoid) distribution ofKuroiwa et al. (1981) and is also evident in 24 hours and 48 hours chloroplasts which have more thylakoids per granum. The changes in nucleoid location occur without significant changes in DNA levels per plastid, and there is no evidence of DNA or plastid replication.The observations indicate that cpDNA partitioning in dividing SN-type chloroplasts could be achieved by thylakoid growth and effectively accomplish DNA segregation, contrasting with envelope growth segregating nucleoids in PS-type (peripheral scattered nucleoids) chloroplasts. The influence of plastid development on nucleoid location is discussed.     

Effects of freezing on the release and inactivation of chloroplast coupling factor 1

The three high-molecular-weight subunits of chloroplast coupling factor (CF₁) are the primary proteins released from pyrophosphate-washed thylakoids exposed to freezing. Identical subunit profiles are found in the supernatant proteins of thylakoids exposed to different intensities of freezing stress by the inclusion of sugars with varying degrees of cryoprotective efficiency. Isolated CF₁ is inactivated by freezing in the presence of NaCl, glucose, and sucrose but raffinose can protect against loss of enzymatic activity during freezing. The low specific activity of the supernatant proteins released from the thylakoid and the inability to recover the Ca²⁺-dependent ATPase activity lost from the membrane suggest that inactivation accompanies release of CF₁ during freezing.     

Malate regulation of Mg2+-ATPase of chloroplast coupling factor 1

The regulatory effects of malate on chloroplast Mg²⁺-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF₁ Mg²⁺-ATPase activity. The subunit of CF₁ may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg²⁺-ATPase was also controlled by the Mg²⁺/ATP ratio in the reaction medium. The enhancing effect of malate on Mg²⁺-ATPase activity depended on the presence of high concentrations of Mg²⁺ in the reaction mixture. Kinetic study showed that malate raised the V_max of catalysis without affecting the K_m for Mg²⁺ ATP. The experiments imply that the stimulation of Mg²⁺-ATPase by malate is probably correlated with the P_i binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF₁ chloroplast coupling factor 1 - CF₁ (-) and CF₁ (-) CF₁ deficient in the and subunit - MF₁ mitochondria coupling factor 1 - NEM N-ethylmaleimide - PMS phenazine methosulfate - OG n-octyl--d-glucopyranoside     

Interaction of Sulfite with the Noncatalytic and Catalytic Sites of Chloroplast Coupling Factor CF1

The interaction between sulfite, an efficient Mg²⁺-dependent F₁-ATPase activator, and chloroplast CF₁-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF₁. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF₁ catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg²⁺-dependent ATPase activity of CF₁ is caused by sulfite binding to its catalytic sites.     

Influence of Ca2+ on the thylakoid lumen violaxanthin de-epoxidase activity through Ca2+ gating of H+ flux at the CFo H+ channel

Part of the chloroplast photoprotection response to excess light absorption involves formation of zeaxanthin (and antheraxanthin) via the violaxanthin deepoxidase enzyme, the activity of which requires lumen acidity near or below pH 6.0. Clearly, the violaxanthin de-epoxidase activity is strongly regulated because at equivalent energization levels (including the parameters of H⁺ accumulation and ATP formation rates), there can be either low or high violaxanthin de-epoxidase enzyme activity. This work shows that the factor or factors responsible for regulating the violaxanthin deepoxidase correlate directly with those which regulate the expression of membrane-localized or delocalized proton gradient (Δ~μ_H+) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca²⁺ binding to the lumen side of the thylakoid membrane; in particular, Ca²⁺ binding to the 8 kDA subunit III of the CF_o H⁺ channel. The activity of violaxanthin deepoxidase in pea (Pisum sativa) and spinach (Spinacea oleracea) thylakoids is shown here to be strongly correlated with conditions known from previous work to displace Ca²⁺ from the CF_o H⁺ channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

Characterisation of a plasma membrane-associated phospholipase A2 activity increased in response to cold acclimation

We here demonstrate the presence of a plasma membrane-associated phospholipase A₂ (EC 3.1.1.4; PLA₂) activity in spinach (Spinacia oleracea) leaves. The pH profile of the spinach plasma membrane PLA₂ activity revealed two peaks, one at pH 4.4 and one at pH 5.5. The activity at pH 5.5 had an absolute requirement of Ca²⁺, with full enzyme activity at 10 μmol/L Ca²⁺. The Ca²⁺-dependent PLA₂ activity was both heat sensitive and stimulated by diacylglycerol, whereas ATP completely inhibited the activity. Thus, the spinach plasma membrane contains a Ca²⁺-dependent PLA₂ activity, which has not previously been characterised in plants. Cold acclimation of spinach resulted in a 2.2-fold higher plasma membrane PLA₂ activity whereas the plasma membrane phospholipase D activity remained unaffected. Taken together, our data suggest a role of PLA₂ in cold acclimation in plants.     

Unlike in other monocotyledonous plants such as wheat and rice, the region containing the tRNA (UCC), tRNA (UCU) and CF1 ATPase α-subunit genes has not undergone recombination in the leek chloroplast DNA

The nucleotide sequence of a 1.1 kbp BamHI fragment of the leek chloroplast DNA (Allium porrum., fam. Liliaceae) has been determined. The fragment contains the 3' part of the tRNA^Gly (UCC) gene and the tRNA^Arg (UCU) gene on the same strand, and the 3' end of the atpA gene encoding the CF₁ ATPase α-subunit which is located on the opposite strand. The gene arrangement and nucleotide sequence of this fragment are similar to those of the corresponding region in the tobacco chloroplast DNA but differ significantly from what has been observed in other monocotyledonous plants such as wheat and rice, in which the region containing these genes has undergone intensive rearrangement.     

Covalent Binding of 1,N 6-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP synthase

Catalytic and noncatalytic sites of the chloroplast coupling factor (CF₁) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N⁶-ethenoadenosine diphosphate. The modified CF₁ was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N⁶-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N⁶-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N⁶-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N⁶-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed.     

Examination of ribosome-like particles in isolated prolamellar bodies

Potential methods for the preparation of fractions enriched in prolamellar bodies (PLBs) were examined in detail. Sucrose density gradient centrifugation methods gave fractions consisting almost exclusively of PLBs whilst those methods employing differential centrifugation were quite successful but contained greater quantities of lamellar membranes. Greater difficulty was experienced in obtaining detached PLBs which retained their ribosome-like lattice particles. No modification to density gradient procedures was found which retained these particles but the omission of ethylene diaminetetraacetic acid (EDTA) from all media including that of lysis gave a hint that this was possible with differential centrifugal methods. This was developed to produce a successful method for the preparation of PLBs which retain the ribosome-like particles of the lattice. Such fractions from Avena sativa L. and Hordeum vulgare L. were treated with ribonuclease which completely removed these particles from the lattice structures implying that they may be ribosomal in nature. EDTA apparently has a critical effect on PLB structure at concentration lower than those that effect the chloroplast coupling factor particles but it is not known if it is a direct effort of PLB membranes, on the lattice particles or both.Abbreviations PLB prolamellar body - EDTA Ethylene diaminetetra-acetic acid - MOPS morpholinopropane sulphonic acid - CF₁ chloroplast coupling factor particles - SDS sodium dodecyl sulphate     

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

pH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803

It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557–567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483–493) that pH dependent reversible Ca²⁺ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a proton gate proposed to exist in the F₀ H⁺ channel of the F₀F₁ ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H⁺/ATP ratio than the wild type and therefore shows ATP synthesis at low values of _H ⁺. The presence of 1 mM CaCl₂ during the preparation and storage of these vesicles blocked acid–base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the pH of the acid–base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl₂, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca²⁺ was present. This is consistent with Ca²⁺ being displaced by H⁺ ions from the F₀ on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca²⁺ to F₀. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca²⁺ binding effect on acid–base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by ⁴⁵Ca²⁺- ligand blot assays on organic solvent soluble preparations containing the 8 kDa F₀ subunit c from the S-3-C mutant ATP synthase, which showed ⁴⁵Ca²⁺ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca²⁺ and EGTA treated vesicles from both strains were almost equal showing that Ca²⁺ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca²⁺ binding to the F₀ H⁺ channel can block H⁺ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca²⁺, opening the CF₀ H⁺ channel between the thylakoid lumen and H⁺ conductive channel.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Ion-translocating ATPases in tendrils ofBryonia dioica Jacq.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg²⁺ATP-driven transport of⁴⁵Ca²⁺. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca²⁺-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca²⁺-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K⁺, Mg²⁺-ATPase and the Ca²⁺-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM calmodulin - CCCP carbonylcyanidem-chlorophenylhydrazone - IC₅₀ concentration giving 50% inhibition - PM plasma membrane - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - FC fusicoccin - U₃+U₃ the two PM-rich upper phases obtained after phase partitioning of microsomal membranes The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support.