Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

The prestalk/prespore differentiation and polarized cell movement inDictyostelium discoideum slugs. A possible involvement of the intracellular Ca2+-concentration

Summary Intracellular free calcium ion concentrations ([Ca²⁺]_i) in the anterior prestalk and posterior prespore cells of theDictyostelium discoideum slug were determined, using the highly selective Ca²⁺ indicators, quin-2/AM and fura-2/AM. Temporal changes in [Ca²⁺]_i in response to chemotactic stimulation with cAMP were also monitored at the single-cell level and compared between the two types of cells. The results obtained showed that resting [Ca²⁺]_i in the prestalk cells is considerably higher than that in the prespore cells. Moreover, transient increase in [Ca²⁺]_i upon stimulation with a low concentration of cAMP (20 nM) was noticed only in the prestalk cells, but not in the prespore cells. These facts are discussed in relation to the polarized movement and cellular differentiation in the migrating slug.Abbreviations cAMP 3,5-cyclic adenosine monophosphate - DIF differentiation-inducing factors - IP₃ inositol 1,4,5-triphosphate     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Ca2+ triggers toxicyst discharge inDidinium nasutum

Summary A carnivorous ciliate,Didinium nasutum, captures a prey,Paramecium spp., by discharging extrusomes (i.e., toxicysts) from the proboscis. To directly examine the role of Ca²⁺ for the discharge, we injected Ca²⁺ intoD. nasutum. Injection of Ca²⁺ evoked discharge of toxicysts, if the site of the injection was the periphery region of the proboscis. After the discharge,D. nasutum, opened the proboscis and swallowed the discharged toxicysts. These observations demonstrate that a rise in cytoplasmic Ca²⁺ level is an actual cause of toxicyst discharge inD. nasutum.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate     

Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-buffered media (-log [Ca²⁺] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca²⁺]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca²⁺] in M. scalaris that was nearly independent of the external [Ca²⁺] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca²⁺ from intracellular stores. Increased cytoplasmic [Ca²⁺] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca²⁺-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.Abbreviations EGTA ethylene glycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - indo-1 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,Ntetraacetic acid - pCa log [Ca²⁺] - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - x_G geometric mean Dedicated to Professor Wolfgang Haupt on the occasion of his 70th birthdayThis paper is part of the Ph.D. thesis of U. Russ at the Justus-Liebig-Universitat Giessen (FRG). Part of this work has been presented at a meeting on Calcium and intracellular signalling in plants in Plymouth, UK, Dec. 1990We are indebted to Dr. G. Seibold and Dipl. Phys. H. Weintraut for their advice on the technique of microspectrofluorometry and for allowing access to the microspectrophotometric facilities in the Strahlenzentrum der Justus-Liebig-Universität, Giessen, FRG. We thank Mrs. A. Quanz for reliable culture of the algae and evaluation of the videotapes. Bay-K8644 was a generous gift of Bayer AG, Wuppertal, FRG. U. russ was supported by a scholarship according to the Hessisches Graduierten Förderungsgesetz. This work was supported by the Deutsche Forschungsgemeinschaft.     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

The effect of organic calcium channel modulators on the germination ofVaucheria longicaulis aplanospores

Summary InVaucheria longicaulis var.macounii aplanospore germination and filament growth are severely inhibited by the Ca²⁺-channel antagonists (–)202–791, diltiazem, nifedipine and verapamil, whereas the agonists (+)202–791, Bay K-8644 and CGP-28392 stimulate those processes. Both antagonist and agonist actions suggest that voltage-controlled Ca²⁺ influx plays a major role in the regulation of the initial events of germination and filament growth. Increases in⁴⁵Ca²⁺ influx are observed after pretreatment of the aplanospores with low temperature shocks of brief duration or FCCP. Both agents are known to depolarize the surface membrane.⁴⁵Ca²⁺ influx is reduced in material treated with FC, an agent known to hyperpolarize cell membranes. The results indicate that Ca²⁺ influx takes place through voltage-sensitive Ca-channels.Abbreviations Bay K-8644 methyl 1,4-dihydro-2,6-dimethy1-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate - CGP CGP-28392 - CTC chlorotetracycline - dil diltiazem - DMSO dimethyl sulphoxide - DTE dithioerythritol - EGTA ethyleneglycol-bis-(-aminoethylether) N,N¹-tetraacetic acid - FC fusicoccin - FCCP p-fluoromethoxy-(carbonylcyanide)phenylhydrazone - nif nifedipine - PCMB p-chloromercuribenzoate - ver verapamil     

Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii

Specific binding of the calcium antagonist [³H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K⁺-stimulated, Mg²⁺-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [³H]verapamil were found at 22°C, one with higher and a second with lower affinity to [³H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [³H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [³H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [³H]verapamil binding in both fractions.Abbreviations B_max maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC₅₀ concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid     

The role of calcium ions in phytochrome-mediated germination of spores of Onoclea sensibilis L.

Phytochrome is confirmed to be the photoreceptor pigment in the germination response of Onoclea sensibilis L. by demonstrating red-far-red (R-FR) photoreversibility. External Ca²⁺ is required for this response with a threshold at a submicromolar concentration. Ethylene glycol-bis(-amino-ethyl ether)-N,N,N,N-tetraacetic acid, La³⁺ and Co²⁺ reversibly inhibit germination. Lanthanum only inhibits germination when applied before or during irradiation, indicating that the external Ca²⁺ requirement is transient, although in the absence of Ca²⁺ the R-stimulated system remains maximally poised to accept the ion for over 4 h after irradiation. The ability to respond to Ca²⁺ 4.1 h after R-irradiation is not reversed by FR-irradiation, indicating that Ca²⁺ transport has been uncoupled from phytochrome. Barium and Sr²⁺, but not Mg²⁺ can substitute for Ca²⁺. Artificially increasing the concentration of intracellular free Ca²⁺ with the ionophore A 23187 stimulates germination in the dark. The Ca²⁺-calmodulin antagonists, trifluoperizine and chlorpromazine, reversibly inhibit germination. Calcium is required in phytochrome-mediated fern spore germination; it may be acting as a second messenger.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FR far-red light - R fed light     

Partial purification and properties of guanosine triphosphate cyclohydrolase from Drosophila melanogaster

The first enzyme (named GTP cyclohydrolase) in the pathway for the biosynthesis of pteridines has been partially purified from extracts of late pupae and young adults of Drosophila melanogaster. This enzyme catalyzes the hydrolytic removal from GTP of carbon 8 as formate and the synthesis of 2-amino-4-hydroxy-6-(d-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine triphosphate (dihydroneopterin triphosphate). Some of the properties of the enzyme are as follows: it functions optimally at pH 7.8 and at 42 C; activity is unaffected by KCl and NaCl, but divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺) are inhibitory; the K _m for GTP is 22 m; and the molecular weight is estimated at 345,000 from gel filtration experiments. Of a number of nucleotides tested, only GDP and dGTP were used to any extent as substrate in place of GTP, and these respective compounds were used only 1.8% and 1.5% as well as GTP.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Influence of calcium on blue-light-induced chloroplast movement in Lemna trisulca L.

The presence of calcium is essential for chloroplast movement induced by blue light in Lemna trisulca L. The regulatory role of calcium was confirmed by the inhibition of chloroplast movement by cytochalasin B and trifluoperazine. The calcium concentration in tissues was modified by ethylene glycol-bis(2-aminoethylether)-N,N,N, N-tetraacetic acid (EGTA), the calcium ionophore A23187 and La³⁺. Only a long period of incubation (12h) in EGTA or La³⁺ caused distrubances in chloroplast movement. This indicates that calcium influx is not essential for chloroplast movement. Those conditions that dramatically changed the internal calcium concentration, either applications of calcium ionophore A23187 and EGTA, or ionophore and La³⁺, markedly decreased the amplitude of response to blue-light pulses. This demonstrates that disturbances of chloroplast movement are observable only when internal stores of calcium are affected by Ca²⁺-antagonists. We suggest that the calcium involved in blue-light-induced chloroplast movement is derived from intracellular stores. The addition of Mg²⁺ to EGTA buffer counteracted its effect, indicating that Mg²⁺, as well as Ca²⁺, might possibly be involved in chloroplast movement.Abbreviations EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes 4(2-hydroxyethyl-1-piperazine) ethanesulfonic acid - A23187 calcium ionophore We express our gratitude to Professor W. Korohoda for valuable critical comments on this paper and stimulating discussion. We also thank Mr. P. Malec for help in preparing the experiment with trifluoperazine and Mr. A. Waloszek for taking the photographs. We are indebted to Mr. Tim Kline (International House, Krakow, Poland) for improving the English style. This research was supported by grant No. 1042/P2/92/03 from the State Committe for Scientific Research.     

Methylxanthines reversibly inhibit tracheary-element differentiation in suspension cultures of Zinnia elegans L.

Tracheary-element (TE) differentiation in suspension-cultured mesophyll cells of Zinnia elegans L. was completely inhibited by caffeine and theophylline only when these methylxanthines were applied at least 8 h prior to the appearance of secondary cell-wall thickenings. In contrast, the calcium-channel blocker nifedipine completely inhibited TE differentiation when applied only 2–3 h prior to the onset of secondary cell-wall deposition. This indicates the involvement of a methylxanthine-inhibitable event in TE differentiation that is distinguishable from an event dependent on influx of extracellular calcium. The correlation between the time of appearance of chlorotetracycline fluorescence (an indicator of sequestered Ca²⁺) and loss of methylxanthine effectiveness indicates that inhibition by methylxanthines may result from release of Ca²⁺ from intracellular stores. Methylxanthines with high potencies against adenosine 3 5-cyclic monophosphate (cAMP) phosphodiesterase and adenosine receptors were less effective inhibitors of TE differentiation, indicating that inhibition of differentiation by methylxanthines is independent of cAMP metabolism. The role of cAMP in transduction of the cytokinin signal, which was proposed previously on the basis of stimulation of TE differentiation by theophylline, was investigated using the non-hydrolyzable analog 8-bromo-cAMP. Although 8-bromo-cAMP stimulated differentiation in the absence of inductive concentrations of cytokinin, the non-cyclic analog 8-bromo-AMP was even more effective, indicating that 8-bromo-cAMP behaves as a cytokinin analog, rather than a second messenger, in stimulating TE differentiation.Abbreviations 8-bromo-AMP 8-bromoadenosine 5-monophosphate - 8-bromo-cAMP 8-bromoadenosine 3:5-cyclic monophosphate - BAP N⁶-benzylaminopurine - cAMP adenosine 3:5-cyclic monophosphate - TE tracheary element - TMB-8 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride This research was supported by a grant from the National Science Foundation (DCB-87-10243) to C.H.H.     

Tetracyclines,verapamil and nifedipine induce callose deposition at specific cell sites in Riella helicophylla

The Ca²⁺ indicator 7-chlorotetracycline has been shown to bind to a pore complex on both outer surfaces of all non-meristematic cells in the unistratose thallus of Riella (chlorotetracycline-binding surface region=CSR; Grotha, 1983, Planta 158, 473–481). Prolonged treatment of the thallus with 7-chlorotetracycline, 5-hydroxytetracycline, verapamil and desmethoxyverapamil induces the deposition of callose at the same region. The influence of various treatments on verapamil-induced CSR-callose was measured in situ by microfluorometry of aniline-blue-stained material. Callose deposition is maximal at 10^-4M verapamil or 5·10^-5M desmethoxyverapamil with 2·10^-4M Ca²⁺ or Mg²⁺ in the medium. The reaction is completely inhibited at pH 5.5 and is optimal between pH 6.5 and 7.5. The production of CSR-callose is absolutely light-dependent with callose being first visible after 30 min of light. La³⁺, ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid and amiprophosmethyl, antagonists of Ca²⁺ functions, and 2-deoxy-D-glucose suppress the verapamil induction of CSR-callose. Furthermore the ionophores A 23187, valinomycin and monensin effectively block the reaction. The deposition of CSR-callose is diminished at increasing external osmolarity and is abolished at osmotic values that stimulate plasmolysis-callose. Wounding causes the formation of wound-callose but inhibits the induction of CSR-callose in cells of the wound edge. Nifedipine increases or prolongs callose synthesis in cell plates. The Ca²⁺-channel blocker diltiazem is completely ineffective. It is suggested as a working hypothesis that verapamil-induced CSR-callose synthesis is caused by a local change in membrane permeability, possibly as a consequence of the opening of Ca²⁺ channels being involved in Golgi-vesicle mediated exocytosis (A. Kramer and H. Lehmann, 1986, Ber. Dtsch. Bot. Ges. 99, 111–121).Abbreviations APM amiprophosmethyl - APW artificial pond water - CSR chlorotetracycline-binding surface region - CTC 7-chlorotetracycline - DDG 2-deoxy-D-glucose - EGTA ethylone glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - OTC 5-hydroxytetracycline - Pipes 1,4-piperazinediethane sulfonic acid Dedicated to Professor Luise Stange on the occasion of her 60^th birthday     

Redistribution of potassium,chloride and calcium during the gravitropically induced movement of Mimosa pudica pulvinus

When the leaves of Mimosa pudica are changed from their normal position in the gravitational field, they perform reversible compensatory movements by means of pulvini. These movements are not the result of growth processes but involve reversible turgor variations. These variation are concomitant with ion migrations within pulvini: during the gravitropic movement, K⁺ and Cl^- shift towards the adaxial half of the motor organ whereas Ca²⁺ shifts towards the abaxial half. Compounds known to affect K⁺ transport, tetraethylammonium chloride and valinomycin, do not hinder the gravitropic movement but inhibit strongly the seismonastic reaction. The same general result is obtained with compounds affecting anion transport, disulfonic stilbenes and 9-anthracene carboxylic acid. Calcium chelators inhibit the gravitropic movement more efficiently than the seismonastic reaction and the calcium ionophore A 23 187 increases both movements. The data obtained with these various compounds indicate that ions do not have the same functional importance in the regulation of the two different pulvinar movements.Abbreviations abx abaxial half of the pulvinus - adx adaxial half of the pulvinus - 9-AC 9-anthracene carboxylic acid - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid - TEA tetraethylammonium chloride     

Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

Wound-healing motility in the green alga Ernodesmis: calcium ions and metabolic energy are required

Wounding a giant cell of the marine alga Ernodesmis verticillata (Kützing) Børgesen (Chlorophyta) induces two concomitant motility phenomena: longitudinal contraction of the protoplasm away from the wound site, and centripetal contraction of the cut end around the central vacuole. Healing is complete within 30 min of wounding. Mechanical extrusion of the protoplasm into the medium with a teasing needle is followed by contraction of the protoplasm into numerous spherical protoplasts within 60 min. Utilizing a simple defined medium, it is shown that motility is almost completely inhibited by the absence of exogenous free Ca²⁺, with 5.0 mM ethylene glycol bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid present. This inhibition is reversible by rinsing the cells with Ca²⁺-containing medium. Similarly, extruded cytoplasm fails to exhibit motility in Ca²⁺-free medium. The threshold concentration of exogenous free Ca²⁺ is approx. 10^-7 M for wound-induced contraction. The ions Ba²⁺, Cd²⁺ and Sr²⁺ will substitute for Ca²⁺, but the rate of contraction is one-half that with Ca²⁺ present. Although darkness has no inhibitory effect, lower temperature (15°C), cyanide, or micromolar amounts of phosphorylation uncouplers reversibly slow protoplasmic motility in wounded cells and extruded cytoplasm. Carbonylcyanide m-chlorophenylhydrazone and carbonylcyanide p-trifluoromethoxyphenylhydrazone are especially potent inhibitors. These results indicate that cellular wound healing utilizes metabolic energy and requires exogenous free Ca²⁺, implying that motility in Ernodesmis is a true contractile process. Since 1.0 mM La³⁺ completely and reversibly prevents contraction, it is postulated that Ca²⁺ fluxes may actually trigger motility.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DMSO dimethylsulfoxide - DNP 2,4-dinitrophenol - EGTA ethylene glycol bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone     

Regulation of myosin sliding alongChara actin bundles by native skeletal muscle tropomyosin

Summary Native tropomyosin from rabbit skeletal muscle introduced by intracellular perfusion intoChara cells inhibited the cytoplasmic streaming irrespective of the Ca²⁺ concentration. To find the action site of native tropomyosin inChara, the cytoplasmic streaming was reconstituted by introducing isolated endoplasm into actin donorChara cells from which native endoplasm had been removed. The reconstituted streaming was inhibited by pretreatment of the actin donor cells with native tropomyosin but not by that of the endoplasm, suggesting that the native tropomyosin inhibited the cytoplasmic streaming by binding toChara actin bundles. Staining of the actin bundles with FITC-labeled native tropomyosin also showed that the native tropomyosin could bind to the actin bundles. Streaming reconstituted fromChara actin bundles and skeletal muscle myosin was insensitive to Ca²⁺, but became sensitive on application of the native tropomyosin.Abbrevations APW artificial pond water - ATP adenosine 5-triphosphoric acid - BSA bovine serum albumin - EDTA ethylene diamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - FITC-NTM fluorescein isothiocyanate-labeled native tropomyosin - NTM native tropomyosin     

The role of Ca2+ in elicitation of phytoalexin synthesis in cell culture of onion (Allium cepa L.)

Summary Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione, were accumulated in cultured onion cells. Removal of extracellular Ca²⁺ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5-trimethoxybenzoate caused similar effects, whereas the addition of the Ca²⁺ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca²⁺ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca²⁺ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells.Abbreviations A23187 4-bromo-calcium ionophore - cAMP adenosine 3,5-cyclic monophosphate - [Ca²⁺]_cyt cytoplasmic Ca²⁺ concentration - EGTA ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid - EtOH ethanol - Et₂O diethyl ether - fr.wt fresh weight - HR hypersensitive response - PIPES piperazine N,N-bis-(2-ethanesulfonic acid) - TMB-8 [8-N,N-(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate - Tsl tsibulin     

Regulation of expression by divalent cations of a light-inducible gene in Arthrobacter photogonimos

Expression of the light-inducible (lipA) gene in Arthrobacter photogonimos is repressed by Ca²⁺ at a concentration greater than 0.1 M. Expression of lipA was induced by relatively high concentrations of Zn²⁺ Ni²⁺ or Co²⁺ in cell suspensions, an effect that was blocked by an increase in the concentration of Ca²⁺ in the medium. Zn²⁺ and other metals apparently overcame repression by Ca²⁺ by competing for a cellular binding site. Expression of lipA was also induced when the amount of free Ca²⁺ was lowered with ethylene-bis (oxyethylenenitrilo)tetraacetic acid (EGTA). Our results show that the lipA gene does not require Zn²⁺ or other divalent cation for expression and that it is regulated negatively by Ca²⁺.Accumulation of the mature product of this gene (light-inducible protein, LIP) was minimal in the presence of EGTA. Accumulation increased 10-to 20-fold when divalent cations such as Ca²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ were added to cell suspensions treated with chelator. These divalent cations, which allowed the protein to achieve a protease-resistant form on the cell surface, could be substituted by protease inhibitors such as antipain, leupeptin or 1,10-phenanthroline. Our data can be explained by a biparous mechanism in which divalent cations regulate both expression of the lipA gene and accumulation of the gene product.Abbreviations LIP light-inducible protein - BAPTA 1,2-bis(o-aminophenoxy)ethanc-N,N,N,N-tetraacetic acid     

Evidence for two types of electron transfer processes through Photosystem II

Inhibition of electron flow from H₂O to methylviologen by 3-(34 dichlorophenyl)-1,1 dimethyl urea (DCMU), yields a biphasic curve — an initial high sensitivity phase and a subsequent low sensitivity phase. The two phases of electron flow have a different pH dependence and differ in the light intensity required for saturation.Preincubation of chloroplasts with ferricyanide causes an inhibition of the high sensitivity phase, but has no effect on the low sensitivity phase. The extent of inhibition increases as the redox potential during preincubation becomes more positive. Tris-treatment, contrary to preincubation with ferricyanide, affects, to a much greater extent, the low sensitivity phase.Trypsin digestion of chloroplasts is known to block electron flow between Q _A and Q _B, allowing electron flow to ferricyanide, in a DCMU insensitive reaction. We have found that in trypsinated chloroplasts, electron flow becomes progressively inhibited by DCMU with increase in pH, and that DCMU acts as a competitive inhibitor with respect to [H⁺]. The sensitivity to DCMU rises when a more negative redox potential is maintained during trypsin treatment. Under these conditions, only the high sensitivity, but not the low sensitivity phase is inhibited by DCMU.The above results indicate the existence of two types of electron transport chains. One type, in which electron flow is more sensitive to DCMU contains, presumably Fe in a Q _A Fe complex and is affected by its oxidation state, i.e., when Fe is reduced, it allows electron flow to Q _B in a DCMU sensitive step; and a second type, in which electron transport is less sensitive to DCMU, where Fe is either absent or, if present in its oxidized state, is inaccessible to reducing agents.Abbreviations DCMU 3-(34 dichlorophenyl)-1, 1 Dimethyl urea - MV methyl viologen - PS II Photosystem II - Tris tris (hydroxymethyl)aminomethane