The synthesis of palmitoylcarnitine by etio-chloroplasts of greening barley leaves

CoASH, Mg²⁺, ATP and (-)-carnitine were found to be essential for the production of palmitoylcarnitine from palmitate by purified barley etio-chloroplasts. It was concluded that long-chain acyl CoA synthetase (palmitoyl CoA synthetase, EC 6.2.1.3) and carnitine long-chain acyl-transferase (carnitine palmitoyltransferase, EC 2.3.1.21) activity were present in the etio-chloroplasts. It is suggested that the long-chain acylcarnitine formed may move more easily through membrane barriers than the long-chain acyl CoA compound. Also or alternatively this enzyme may spare CoA by transferring long-chain acyl groups from long-chain acyl CoA to carnitine.     

K+ current inhibition by amphiphilic fatty acid metabolites in rat ventricular myocytes

Fatty acid metabolites accumulate in the heart underpathophysiological conditions that affect -oxidation and can elicit marked electrophysiological changes that are arrhythmogenic. The purpose of the present study was to determine the impact of amphiphilic fatty acid metabolites on K⁺currents that control cardiac refractoriness and excitability. Transient outward(I_to) andinward rectifier(I_K1)K⁺ currents were recorded by thewhole cell voltage-clamp technique in rat ventricular myocytes, and theeffects of two major fatty acid metabolites were examined:palmitoylcarnitine and palmitoyl-coenzyme A (palmitoyl-CoA).Palmitoylcarnitine (0.5-10 µM) caused a concentration-dependent decrease in I_todensity in myocytes internally dialyzed with the amphiphile; 10 µMreduced mean I_todensity at +60 mV by 62% compared with control(P < 0.05). In contrast, externalpalmitoylcarnitine at the same concentrations had no effect, nor didinternal dialysis significantly alterI_K1. Dialysiswith palmitoyl-CoA (1-10 µM) produced a smaller decrease inI_to densitycompared with that produced by palmitoylcarnitine; 10 µM reduced meanI_to density at+60 mV by 37% compared with control(P < 0.05). Both metabolites delayedrecovery of I_tofrom inactivation but did not affect voltage-dependent properties.Moreover, the effects of palmitoylcarnitine were relatively specific,as neither palmitate (10 µM) nor carnitine (10 µM) alone significantly influencedI_to when added tothe pipette solution. These data therefore suggest that amphiphilicfatty acid metabolites downregulateI_to channels by amechanism confined to the cytoplasmic side of the membrane. Thisdecrease in cardiac K⁺ channelactivity may delay repolarization under pathophysiological conditionsin which amphiphile accumulation is postulated to occur, such asdiabetes mellitus or myocardial infarction.

     

Carnitine long-chain acyltransferase and oxidation of palmitate,palmitoyl coenzyme A and palmitoylcarnitine by pea mitochondria preparations

Palmitoylcarnitine was oxidised by pea mitochondria.l-carnitine was an essential addition for the oxidation of palmitate or palmitoylCoA. When palmitate was sole substrate, ATP and Mg²⁺ were also essential additives for maximum oxidation. Additions of CoA inhibited the oxidation of palmitate. It was shown that CoA was acting as a competitive inhibitor of the carnitine-stimulated O₂ uptake. It is suggested that palmitoylacarnitine and carnitine passed through the mitochondrial barrier with ease but palmitoylCoA and CoA did not. The presence of carnitine long-chain acyl (palmitoyl)transferase (EC 2.3.1.21) in pea-cotyledon mitochondria was shown. This enzyme may play a role in the transport of long-chain acyl groups through membrane barriers.Abbreviation Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

L-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2

Maternofetal transport of L-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that L-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K_m = 11.09 ± 1.32 µM; V_max = 41.75 ± 0.94 pmol·mg protein^–1·min^–1), and was unchanged over the pH range from 5.5 to 8.5. L-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of L-carnitine, including D-carnitine, acetyl-D,L-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-L-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na⁺-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for L-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans. membrane transport; valproate; maternofetal; xenobiotics; acylcarnitine     

Oxidation of acetate,acetyl CoA and acetylcarnitine by pea mitochondria

Acetylcarnitine was rapidly oxidised by pea mitochondria. (-)-carnitine was an essential addition for the oxidation of acetate or acetyl CoA. When acetate was sole substrate, ATP and Mg²⁺ were also essential additives for maximum oxidation. CoASH additions inhibited the oxidation of acetate, acetyl CoA and acetylcarnitine. It was shown that CoASH was acting as a competitive inhibitor of the carnitine stimulated O₂ uptake. It is suggested that acetylcarnitine and carnitine passed through the mitochondrial membrane barrier with ease but acetyl CoA and CoA did not. Carnitine may also buffer the extra- and intra-mitochondrial pools of CoA. The presence of carnitine acetyltransferase (EC 2.3.1.7) on the pea mitochondria is inferred.     

Active-site probes of carnitine acyltransferases. Inhibition of carnitine acetyltransferase by hemiacetylcarnitinium, a reaction intermediate analogue

Hemiacetylcarnitinium (2S,6R:2R,65)-6-carboxymethyl-2-hydroxy-2,4,4- trimethylmorpholinium) chloride is a relatively potent competitive inhibitor (Ki = 0.89 mM) of pigeon breast carnitine acetyltransferase (CAT) and of the crude rat liver CAT (Ki = 4.72 mM) but is neither an inhibitor nor an effective substrate for purified rat liver carnitine palmitoyltransferase (CPT). It does not inhibit state 3 oxygen consumption in isolated hepatic mitochondria using palmitoyl-CoA or palmitoylcarnitine as substrates. This compound is a reaction intermediate analogue of the proposed tetrahedral intermediate for acetyl transfer between acetylcarnitine and CoASH. Because the hemiketal carbon is chiral, a suggestion is made that one of the enantiomers has the same relative configuration as the proposed tetrahedral intermediate.     

Long-chain acyl CoA synthetase,carnitine and β-oxidation in the pea-seed mitochondrion

Mitochondria from pea (Pisum sativum L.) seeds were separated into two fractions, mitoplasts (intact inner membrane) and the outer-membrane fraction. The mitoplasts only oxidised palmitate in the presence of carnitine and added outermembrane fraction. Mitoplasts were able to oxidise palmitoylCoA in the presence of carnitine and added outer-membrane fraction had no effect on this oxidation. It was concluded that a long-chain acylCoA synthetase (EC 6.2.1.3) was located on the outer membrane and that the activity of this enzyme in assays was more than sufficient to account for any observed rate of O₂ uptake during palmitate oxidation by pea mitochondria. The location of carnitine long-chain acyltransferase (carnitine palmitoyl transferase EC 2.3.1.21) would appear to be the mitoplast i.e. the inner mitochondrial membrane, and confirms the previous work at Newcastle.Abbreviation Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate

1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50mumumoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by beta-oxidation takes place without detectable accumulation of acyl-CoA intermediates.     

Competitive inhibition of the response to Na+ by Ca2+ in water fibers of the frog glossopharyngeal nerve

Single water fibers of the frog glossopharyngeal nerve respondto low concentrations of CaCl₂ (1–2 mM) and to relativelyhigh concentrations of NaCl(>80 mM). However, stimulationby a mixture with a low concentration of CaCl₂ and relativelyhigh concentration of NaCl gives rise to only a small response,suggesting that the effects of Ca²⁺ and Na⁺ are mutually antagonistic.It has been reported that Na⁺ inhibits the response to Ca²⁺by competing with Ca²⁺ for a calcium receptor site (X_Ca; Kitadaand Shimada, 1980). In the present study, it was found tha Ca²⁺inhibited the response to Na⁺. Therefore, the sodium receptorsite (X_Na) responsible for the response to Na is different fromX_Ca. The inhibition of the response to Na⁺ by Ca²⁺ was examinedquantitatively on the assumption that the magnitude of the neuralresponse is proportinal to the amount of NaX_Na complex minusa constant (the threshold concentration of the NaX_Na complex).The results obtained indicate that Ca²⁺ competes with Na₊ forX_Na. The apparent dissociation constants for the NaX_Na complexand the CaX_Na complex obtained from the present study were 1.0M and 1.2 x 10^-3 M, respectively, X_Na as proposed here, doesnot represent simply a binding site for cations since therecan be competition for X_Na by an antagonistie cation. The highaffinity of X_Na for Ca²⁺ suggests that X_Na is a specific receptorsite involved in salt-taste reception. Since Mg²⁺ did not affectthe response to Na⁺, the affinity of X_Na for cations is notcharge-specific but is, rather, chemically specific. The presentresults indicate that both Ca²⁺ and Na⁺ have a dual action,being involved both in excitation and in inhibition, in waterfibers of the frog glossopharyngeal nerve.     

Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF-4-induced [Ca2+]i oscillations in rat pancreatic

AlF₄^-is known to generate oscillations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF₄^--evoked [Ca²⁺]_i oscillations were reported to be dependent on extracellular Ca²⁺, which contrasts with the [Ca²⁺]_i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF₄^- generates extracellular Ca²⁺-dependent [Ca²⁺]_i oscillations in rat pancreatic acinar cells. AlF₄^--induced [Ca²⁺]_i oscillations were stopped rapidly by the removal of extracellular Ca²⁺ and were abolished on the addition of 20 mM caffeine and 2 µM thapsigargin, indicating that Ca²⁺ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store is also required. The amount of Ca²⁺ in the intracellular Ca²⁺ store was decreased as the AlF₄^--induced [Ca²⁺]_i oscillations continued. Measurement of ⁴⁵Ca²⁺ influx into isolated microsomes revealed that AlF₄^-directly inhibited sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). The activity of plasma membrane Ca²⁺-ATPase during AlF₄^- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺. This study shows that AlF₄^- induces [Ca²⁺]_i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca²⁺ as a result of the partial inhibition of SERCA. cholecystokinin; plasma membrane adenosine 5'-triphosphatase; G proteins; caffeine     

Taste responses to divalent cations in the frog glossopharyngeal nerve: competitive inhibition of the Mg2+ response by Ca2+

In the frog glossopharyngeal nerve, single water fibers respondto low CaCl₂ (1–2 mM) and relatively high MgCl₂ (100 mM).In the present study, it was found that stimulation by a mixtureof low CaCl₂ and relatively high MgCl₂ led to a small response.This suggests that the Ca⁺ response is inhibited by the presenceof Mg²⁺ and the Mg²⁺ response is inhibited by the presence ofCa²⁺. Hence, it is suggested that there are different receptorsites for divalent cations in single water fibers of the frogglossopharyngeal nerve, a calcium receptor site (X_Ca) responsiblefor the Ca²⁺ response and a magnesium receptor site (X_Mg) responsiblefor the Mg²⁺ response. It has been reported that Mg²⁺ inhibitsthe Ca²⁺ response by competing with Ca²⁺ for X_Ca (Kitada andShimada, 1980). In the present study, the inhibition of theMg²⁺ response by Ca²⁺ was examined quantitatively under theassumption that the magnitude of the neural response is proportionalto the amount of MgX_Mg complex minus a constant (the thresholdconcentration of the MgX_Mg complex). The results obtained indicatethat Ca²⁺ competes with Mg²⁺ for X^Mg. The apparent dissociationconstants for MgX_Mg complex and CaX_Mg complex, which were obtainedfrom the present study, were 8.0 x 10^–2 M and 7.2 x 10^–4M, respectively. Thus, competition between Ca⁺ and Mg²⁺ forthe distinct receptor sites involved in taste reception wasdemonstrated by the results described in this paper. Since thedivalent cations do not always bring about activation of tastereceptors, the responses to salts in the frog glossopharyngealnerve cannot be explained in terms of changes in the surfacepotential outside the taste cells. The present results suggestthat there exist multiple specific receptor sites for cationsinvolved in salt taste responses, and only the binding of eachseparate cation to its appropriate receptor sites leads to activationof the receptor and the initiation of impulses in sensory nerveendings.     

Glycine as a substrate for photorespiration

Substrates for photorespiration were examined by feeding ¹⁴Clabeled compounds to tobacco and corn leaf segments and by measuring¹⁴CO₂ evolution in light and darkness. CO₂ release in the darkwas rapid, but in light CO₂ release was slow due to refixationby photosynthesis. Carboxyl labeled glycine was more rapidlydecarboxylated than were glyoxylate, glycolate or serine. Hydroxypyridinemethanesulfonate, an inhibitor of glycolate oxidase, blocked CO₂ releasefrom glycolate but not from glycine. Isonicotynyl hydrazideblocked CO₂ release from both glycine and glycolate. DCMU blockedphotosynthetic refixation of the released CO₂, consequentlythe rates of CO₂ release in light and dark were about equal.It was concluded that CO₂ release during photo-respiration camefrom the conversion of 2 molecules of glycine to one serineand one CO₂. ¹⁴CO₂ release from glycine-l-¹⁴C in the dark or with DCMU inlight can be used as an assay for photorespiration ability. CO₂ release from glycine and glycolate by corn leaf segmentsin the dark proceeded at the rate of that in normal tobaccoleaf. This result, together with other work on O₂ exchange andenzymatic analysis, indicates that corn and other plants docarry on photorespiration, but it is not manifested by CO₂ releasein light. A yellow tobacco mutant, Consolation 402, had high rates ofphotorespiration by the ¹⁴CO₂ assay, nearly half (or more) asmany peroxisomes as chloroplasts, and high rates of CO₂ releasefrom glycine-l-¹⁴C or glycolate-l-¹⁴C. A common tobacco, BrightYellow, had lower rates of photorespiration, fewer visible peroxisomes,and slower decarboxylation of glycine and glycolate. The amount of ¹⁴CO₂ release from glycine-l-¹⁴C or glycolate-l-¹⁴Cincreased only slightly when the temperature was raised from25 to 35°C. ¹Parts of this work were abstracted at the Annual Meeting (April,1969) of Japanese Society of Plant Physiologists, Kanazawa ²Department of Biochemistry, Michigan State University, EastLansing, Michigan, U.S.A. (Received September 3, 1969; )     

Stomatal Responses to Applied ABA and CO2 in Epidermis Detached from Well-Watered and Water-Stressed Plants of Commelina communis L.

Epidermal strips from either well-watered or water-stressedplants of Commelina communis L. were subjected to a range ofABA concentrations (10^–6–10^–3 mol m^–3)in the presence (330 parts 10^–6 in air) or virtual absence(3 parts 10–6 in air) of CO₂. The stomatal response toCO₂ was greater in epidermis from water-stressed plants, althoughthere was a distinct CO₂ response in epidermis from well-wateredplants. Additions of ABA via the incubation medium had littleeffect on the relative CO₂ response. Stomata responded to ABAboth in the presence and virtual absence of CO₂, but the relativeresponse to ABA was greatest in the high CO₂ treatment. Whenwell-watered plants were sprayed with a 10^–1 mol m^–3ABA solution 1 d prior to use, the stomatal response of detachedepidermis to both CO₂ and ABA was very similar to that of epidermisdetached from water-stressed leaves. It is hypothesized thata prolonged exposure to ABA is necessary before there is anymodification of the CO₂ response of stomata.     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 3. Time-course Study on 13C Incorporation into Soluble Metabolites and Respiratory Evolution of 13CO2 from Roots and Nodules

Well-nodulated soya bean (Glycine max L.) plants were allowedto assimilate ¹³CO₂ for 10 h in the light, under steady-stateconditions in which CO₂ concentration and ¹³C abundance wereboth strictly controlled at constant levels. The respiratoryevolution of ¹³CO₂ from roots and nodules and ¹³C incorporationinto various metabolic fractions were measured during the ¹³CO₂feeding and subsequent 48 h chase period. CO₂ respired from nodules was much more rapidly labelled with¹³C than that from roots. The level of labelling (percentageof carbon currently assimilated during the ¹³COM₂ feeding period)of CO₂ respired from nodules reached a maximum of about 87 percent after 4 h of steady-state ^l3CO₂ assimilation and thereafterremained fairly constant. The absolute amount of labelled carbonevolved by the respiration of the nodules during the 10 h ¹³CO₂feeding period was 1·5-fold that of root respiration.These results demonstrated that the currently assimilated (labelled)carbon was preferentially used to support nodule respiration,while root respiration relied considerably on earlier (non-labelled)carbon reserved in the roots. Sucrose pools were mostly composed of currently assimilatedcarbon in all tissues of the plants, since the levels of labellingaccounted for 86–91 per cent at the end of the ¹³CO₂ feeding.In the nodules, the kinetics and levels of sucrose labellingwere in fairly good agreement with those of respired CO₂, whilein the roots, the level of labelling of respired CO₂ was significantlylower than that of sucrose. Succinate and malate were highly labelled in both roots andnodules but they were labelled much more slowly than sucroseand respired CO₂. The kinetics and levels of labelling of theseKrebs cycle intermediates resembled those of major amino acidswhich are derived directly from Krebs cycle intermediates. Itis suggested that large fractions of organic acids in noduleswere physically separate from the respiration site. Glycine max L., Soya bean, ¹³CO₂ assimilation, respiratory evolution of ¹³CO₂, carbon metabolism in root nodules     

The Influence of Ca2+ and K+ on H14CO3-Influx in Internodal Cells of Chara corallina

The influences of Ca²⁺-free solutions and increasing K⁺ concentrationson the H¹⁴CO^–₃ influx capacity of Chara corallina wereinvestigated. It was found that contact with Ca^2–freesolutions resulted in a gradual reduction in the H¹⁴CO^–₃influx capacity of these cells. Recovery of this influx capacity,following the return of Ca²⁺ to the experimental solution, followeda ‘mirror-image’ of the time course of decay. Potassium concentrations above a certain critical value (2 mM)induced a rapid reduction in H¹⁴CO^–₃ influx capacity.Normal activity was recovered within 60–90 min followingthe return to 0.2 mMK⁺ solutions. It was also shown that 10mM K⁺ can be used to determine the relative contribution of¹⁴C supplied by diffusion of ¹⁴CO₂ and transport of H¹⁴CO^–₃.The Ca²⁺ and K⁺ results are discussed in relation to the effectsof these treatments on the electrical properties of the plasmalemma.     

Proton Translocation Associated with Denitrification in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

H⁺ translocation driven by NO₃^–, NO₂^– and N₂O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H⁺ extrusion, addition of KNO₃,KNO₂ and N₂O caused only a rapid alkalinization. The H⁺ consumptionstoichiometries, H⁺/2e^– ratios for NO₃^– reductionto NO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were –1.90, –3.18 and –2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H⁺ consumption in the periplasm, the H⁺ extrusionstoichiometries, H⁺/2e^– ratios with endogenous substratesin the presence of K⁺/valinomycin for NO₃^– reduction toNO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983)     

Compartmental analysis of 35SO2-4exchange kinetics in roots and leaves of a tropical legume Macroptilium atropurpureum cv. Siratro

Compartmental analysis of ³⁵SO^2-₄ tracer exchange kinetics hasbeen used to estimate unidirectional fluxes and compartmentcontents in excised root and leaf tissue of the tropical legume,Macroptilium atropurpureum. In excised root tissue only 5% ofthe sulphate taken up across the plasmalemma was reduced toorganic forms whereas in excised leaf tissue approximately 20%was reduced. It was necessary, therefore, to incorporate themetabolism of sulphate during the course of the experiment intothe compartmental models. In root tissue, wash-out data was fitted by three exponentials,assumed to correspond to exchange in the extracellular spaces,cytoplasm and vacuole, but in leaf tissue two large, slowlyexchanging compartments have been postulated in order to achievea fit to the data. It is likely that differences in leaf cellpopulations cause the ‘anomalous’ tracer exchangekinetics and the justification of this assumption is discussed. The fluxes of sulphate at the plasmalemma were greater thanthe corresponding fluxes at the tonoplast in both roots andleaves. The flux of SO^2-₄ from the cytoplasm to the externalsolution did not appear to limit the loss of SO^2-₄ from thevacuole. At an external SO^2-₄concentration of 0.25 mol m^-3 therate constants for exchange in the vacuole were two orders ofmagnitude greater in roots than in the slowest exchanging leafcell population. It is possible, therefore, that the slow lossof SO^2-₄ from leaf cell vacuoles may limit the redistributionof sulphate during S-stress. Key words: Compartmental analysis, sulphate, deficiency, Macroptilium atropurpureum     

Hydroxyl Radicals and a Thylakoid-Bound Endopeptidase are Involved in Light- and Oxygen-Induced Proteolysis in Oat Chloroplasts

The hypothesis that light- and oxygen-induced proteolysis inchloroplasts is mediated by active oxygen species was examined.In order to determine whether or not H₂O₂ and/or ^{dot}OH radicalsare involved in these degradative processes we compared thedegradation of proteins in isolated oat chloroplasts exposedto white light at 80 W m^-2 with that in chloroplasts incubatedin darkness in the absence or presence of H₂O₂ or a ^{dot}OH-generatingsystem composed by ascorbic acid, FeCl₃ and H₂O₂ (Asc-Fe-H₂O₂).Light enhanced the rate of degradation of at least 18 polypeptides,while proteolysis was almost negligible in darkness in the abscenceof additives. H₂O₂ had a very small effect. However, Asc-Fe-H₂O₂-treatedchloroplasts in darkness showed a pattern of protein degradationalmost identical to that observed in the light. A thylakoid-boundendopeptidase (EP), the activity of which increased under photooxidativeenvironmental conditions and treatment with an ^{dot}OH-generatingsystem, was partially purified and characterized as a serinetypeprotease. Treatments with inhibitors of serine-type proteaseprevented both light- and Asc- Fe-H₂O₂-induced proteolysis.EP was more active against both soluble and membranous proteinsthat had been pretreated with Asc-Fe-H₂O₂ than against untreatedproteins. It is proposed that a high dose of light irradiationpromotes proteolysis by increasing the formation of ^{dot}OH,which may modify proteins such that they become more susceptibleto EP-catalyzed hydrolysis. ¹Fisiología Vegetal, Dept. de Biología Vegetal,Universidad de Alcalá de Henares, Present address: 28871Alcalá de Henares (Madrid), España.     

Enhancing effects of transition metals on the salt taste responses of single fibers of the frog glossopharyngeal nerve: specificity of and similarities among Ca2+, Mg2+ and Na+ taste responses

Fibers of the frog glossopharyngeal nerve (water fibers) thatare sensitive to water also respond to CaCl₂, MgCl₂ and NaCl.In the present study, interaction among cations (Ca²⁺, Mg²⁺and Na⁺) on taste cell membrane in frogs was studied using transitionmetals (NiCl₂, CoCl₂ and MnCl₂), which themselves are barelyeffective in producing neural response at concentrations below5 mM. Unitary discharges from single water fibers were recordedfrom fungiform papillae with suction electrode. Transition metalions (0.05–5.0 mM) had exclusively enhancing effects onthe responses to 50 mM Ca²⁺, 100 mM Mg²⁺ and 500 mM Na⁺. Theeffects of transition metal ions were always reversible. Therank order of effectiveness of transition metals at 1 mM inthe enhancement of the responses to 50 mM CaCl₂, 100 mM MgCl₂and 500 mM NaCl was NiCl₂ > CoCl² > MnCl₂. The concentrationof transition metal ions effective to enhance salt responsewas almost the same among Ca²⁺, Mg²⁺ and Na⁺ responses. Theresults suggest that a common mechanism is involved in the enhancementof Ca²⁺, Mg²⁺ and Na⁺ taste responses. The enhanced Mg²⁺ responseand the enhanced Na⁺ response were greatly inhibited by theaddition of Ca²⁺ ions, and the enhanced Ca²⁺ response was inhibitedby the addition of Mg²⁺ or Na⁺ ions, suggesting that competitiveantagonism occurs between Ca²⁺ and Mg²⁺ ions and between Ca²⁺and Na⁺ ions in the presence of Ni²⁺ ions. Ni²⁺ ions had a dualeffect on the Ca²⁺ response induced by low concentration (0.1mM) of CaCl₂: enhancement at lower concentrations (0.02–0.1mM) of NiCl₂ and inhibition at higher concentrations (0.5–5mM)of NiCl₂. The present results suggest that transition metalions do not affect the receptor-antagonist complex, but affectonly the receptor-agonist complex.