M. Cocucci

Effects of orthovanadate on H+ secretion, K+ uptake, electric potential difference and membrane ATPase activities of higher plant tissues.

Abstract Orthovanadate at concentrations between 5 and 20 μM inhibited the K + -stimulated, DCCD-sensitive ATPase activity of membrane preparations (fraction sedimenting between 13 000 and 80 000 g ) from radish seedlings while it had no effect on either the oligomycin-sensitive ATPase activity of mitochondrial preparations, or the phosphorylating activity of isolated mitochondria from radish seedlings or from pea internode segments. In whole germinating radish seeds orthovanadate inhibited the increase in fresh weight, net K + uptake and H + extrusion without inducing any detectable change of respiration nor of ATP and ADP levels, and it induced a rapid, partial depolarisation of the transmembrane electric potential difference. These effects in vivo were interpreted as indicating that orthovanadate inhibits an ATP-driven electrogenic proton pump corresponding to a plasmalemma ATPase.

Characteristics of fusicoccin-induced changes of transmembrane potential and ion uptake in maize root segments

Abstract The effects of fusicoccin (FC) on the transmembrane electric potential difference (PD) of cortical cells of isolated apical root segments was investigated under various conditions of osmotic pressure, temperature, ionic composition and pH of the incubation medium, and compared with the effects of this substance on the uptake of K + , Rb + , Na + and Cs + ions. FC induced a rapid hyper-polarization of the negative PD, ranging from 20 to 50 mV depending on the ionic composition of the medium. With 5·10 −5 M FC maximal PD value was reached in ca. 6 min. Changing the osmotic pressure of the medium from 0.01 to 0.25 osmolar (mannitol) progressively inhibited elongation without affecting the PD change thus showing that the effect of FC does not depend on the increase of cell enlargement. Low temperature (6°C) and the phosphorylation uncoupler ( p -trifluoromethoxy)-carbonyl cyanide-phenylhydrazone (FCCP) rapidly and severely decreased both normal and FC-induced PD. FC stimulated the rate of monovalent cation uptake. In the 1–10 mM concentration range this effect was much larger for K + and Rb + than for Na + and Cs + . K + and (to a lesser extent) Rb + at concentrations higher than 5·10 −4 M induced a rapid depolarization, larger in the presence than in the absence of FC, while Na + , Cs + and Li + at concentrations up to 10 −2 M did not significantly influence PD. No significant PD changes were observed when the anion Cl − was substituted with SO 4 2− or NO 3 2− . Increasing proton concentration in the medium from pH 6 to pH 4 induced a significant hyperpolarization, probably due to the inhibition of K + uptake by low pH. These data are compared with other results showing the remarkable activity of FC in stimulating proton secretion in root segments, and they are interpreted as indicating that FC activates in roots, as in a wide variety of other plant organs, an electrogenic mechanism of proton extrusion deeply involved in cation transport. The hypothesis is proposed that the differential activity of the monovalent cations in inducing depolarization of PD might depend on their different affinity for the proton/cation electrogenic exchange mechanism.

Effect of ABA, GA3 and FC on the development of potassium uptake in germinating radish seeds

Abstract Germination of Rhaphanus sativus seeds is accompanied in its early phase by a consistent loss to the medium of the main cations. During the first ten hours there is a general efflux of K + , Na + , Mg 2+ and Ca 2+ , while in the following period K + is actively taken up so that after eighteen hours almost no K + is detected in the external medium. When seed germination is stimulated by fusicoccin (FC) the development of uptake capacity is markedly anticipated. Moreover, inhibition of seed germination by abscisic acid (ABA) inhibits the development of K + uptake. The ABA-induced inhibition is reversed by FC and also, even if to a lesser extent, by gibberellic acid (GA 3 ). Preliminary data suggest that a K + activated ATPase present in membrane preparation is involved in the mechanism of active K + uptake in the seeds.

Lysophosphatidylcholine-activated, vanadate-inhibited, Mg2+-ATPase from radish microsomes

Abstract An orthovanadate-inhibited, nitrate-insensitive, phospholipid-requiring Mg2+-ATPase has been partially purified (approx. 40-fold) from microsomal preparations from 24 h germinated radish seedlings. The specific activity obtained was 10–13 μmol Pi · min−1 per mg protein, namely by 4- to 10-fold higher than that reported for the known similar enzyme preparations from corn and oat roots, and by 3- to 10-fold lower than that of the extensively purified plasmalemma enzymes from Neurospora and yeast. The partially purified activity was fairly specific for ATP, other nucleotide triphosphates being hydrolysed at less than 10% the rate with ATP; no activity was present towards ADP, AMP, p-nitrophenyl phosphate and other phosphate esters. The activity was strongly dependent on the presence of phospholipids with a marked preference for lysophosphatidylcholine, and showed an absolute requirement for Mg2+ or some other divalent cations (CO2+, Mn2+, Mg2+, Ni2+, Zn2+ in order of decreasing effectiveness); Ca2+ could not substitute for Mg2+ and was strongly inhibitory in its presence. K+, Rb+ and Na+ and also to a lesser extent NH4+ and Li+ were significantly stimulatory, while the anions NO3−, H2PO4−, Cl− and SO42− were ineffective. Orthovanadate, N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzensulfonate, tetraiodofluorescein and tetrachlorotetraiodofluorescein were strongly inhibitory. The coincidence of the Km for ATP with that for Mg2+ suggested that ATP-Mg is the true substrate. Accordingly, the enzyme showed a normal Michaelis-Menten kinetics for ATP-Mg with an apparent Km of approx. 0.5 mM. The similarity of the characteristics of this enzyme with those of the plasmalemma enzymes from lower plants suggests its location at the plasma membrane, while some data ‘in vivo’ and in native sealed vesicle systems indicate its involvement in active proton transport.

Inhibition of plasma membrane and tonoplast ATPases by erythrosin B

In microsomal preparations from radish seedlings erythrosin B (EB) and rose bengal (RB) inhibited both the vanadate and the nitrate-sensitive ATPases, by about 85% at 100 μM and by about 50% at 10 μM. Mitochondrial and thylakoid ATPases were much less affected by EB (100 μM EB inhibiting by about 30% and 10 μM being ineffective), while they are markedly inhibited by 100 μM RB. Also a microsomal ADPase activity was inhibited by 100 μM EB by about 40% and strongly inhibited by RB. Both EB and RB were ineffective on acid phosphatases. Fluorescein (FL) was ineffective on all the membrane-associated enzymes tested. Partial purification of the vanadate-sensitive plasma membrane ATPase markedly enhanced its sensitivity to EB, I50 being shifted from approx. 10 μM to 0.1 μM, when soybean phospholipids or lysophosphatidylcholine were added to the assay after the inhibitor; however, when lysophosphatidylcholine was added before the inhibitor, the sensitivity to EB markedly decreased (I50 = approx. 10 μM, namely the value found for the microsomes). The Lineweaver-Burk plots showed that EB affects both Km and Vmax of the vanadate-sensitive ATPase. These data suggest a possible utilization of EB as an useful inhibitor of H+-transport membrane ATPases in ‘in vivo’ as well as in subcellular membrane preparations.

The effects of cycloheximide on respiration, protein synthesis and adenosine nucleotide levels in Rhodotorula gracilis

Abstract In Rhodotorula gracilis cyclohemixide (CHI) depresses O 2 uptake by about 45% and inhibits the incorporation of leucine into proteins almost completely. The effect of CHI on respiration reaches its maximum about 40 min after feeding the inhibitor and is followed by the inhibition of protein synthesis. The inhibition of respiration appears to be closely correlated with that of amino acid incorporation into protein over a wide range of CHI concentration and is accompanied by a significant increase in the level of ATP and a decrease in the levels of ADP and AMP. The addition of 2,4-dinitrophenol (DNP), which decreases the ATP:(ADP + AMP) ratio, completely prevents the inhibition of respiration. These results suggest that the inhibition of respiration by CHI is a consequence of a shift of the high energy ∼ P system toward a higher phosphorylation state, following the block of ∼ P utilization in protein synthesis and other ATP-consuming processes.

Changes in the potato (Solanum tuberosum L.) tuber at the onset of dormancy and during storage at 23 °C and 3 °C. I. Biochemical and physiological parameters

SummaryIn the last 30 d of potato (Solanum tuberosum L.) tuber growth metabolic activity decreased. Levels of glucose-6-P and sucrose in whole tuber tissues declined and in tuber slices there was a decrease in the uptake from the medium and in the incorporation into macromolecules of [U-14C]sucrose. During storage at 23°C only the uptake of [U-14C]sucrose increased concomitant with tuber sprouting, indicating a possible involvement of the transport mechanisms in dormancy breaking. At 3°C, levels of reducing sugars and sucrose increased in response to the low temperature and increased release of K+ and malondialdehyde levels indicated cell membrane damage. The cell membrane functionality was restored at sprouting. The sprouting potential of the tubers was evaluated using the sprouting ability of single-bud explants (“seedcores”) in response to water, GA3 or ABA dips. This sprouting potential of tubers changed with stage of tuber growth and storage duration and temperature, indicating that the tissue hormonal state changed strongly throughout tuber life, probably in relation with the “sink” to “source” transition.

Reconstitution of proton pumping activity of a plasma membrane ATPase purified from radish

Abstract Plasma membrane ATPase partially purified from radish seedlings ( Raphanus sativum L.) (2.4–3.5 μmol P i min −1 mg −1 protein) has been reconstituted in proteoliposomes by the cholate-dialysis technique. Proteoliposomes are able to acidify their internal volume in the presence of Mg:ATP. Mg:ATP-dependent proton pumping is prevented by N , N ′-dicyclohexylcarbodiimide (DCCD) and by vanadate at the same concentrations which are effective on the phosphohydrolyzing activity of the plasma membrane ATPase.

Effects of ophiobolin A on potassium permeability, transmembrane electrical potential and proton extrusion in maize roots

Abstract In maize seedling roots ophiobolin (OPH) A at higher concentration (5 × 10−5 M) promoted a depolarization of the transmembrane electrical potential (PD) and increased the depolarizing effect of K+; at lower concentration (7.5 × 10−6 M) it promoted a hyperpolarization of PD completely counteracted by K+ and still detectable in the presence of the uncoupler (p-trifluoromethoxy)-carbonyl cyanide-phenyl-hydrazone (FCCP). OPH A also increased both the leakage and the efflux of K+; moreover, all the concentrations of OPH A promoted the alkalinization of the medium; in the absence of K+, the capacity to acidify the incubation medium was restored by adding K+ to the medium. These data suggest that the lack of proton extrusion is not linked to a direct action of OPH A on the proton pump, but is linked to an effect of OPH A on the plasma-membrane permeability to K+. The above data are also discussed in relation to the regulation of proton extrusion by a chemical coupling with K+ or by an electrical coupling with PD.

Co-sedimentation of one form of plasma membrane H+-ATPase and of the fusicoccin receptor from radish microsomes

Abstract The fusicoccin (FC) receptor has been partially purified (4–5 times) to a specific activity of about 30 pmol · mg protein −1 from 24 h radish seedlings microsomes in conditions in which the vanadate-sensitive H + -ATPase activity was preserved (final ATPase specific activity about 3.5 μmol P i · min −1 · mg protein −1 ). The purification involved the washing of microsomes with 0.5% Triton X-100, the solubilization of the residue with lysophosphatidylcholine (lyso-PC) and the fractionation of lyso-PC extract on a glycerol-sucrose density gradient. Since the two detergents lowered the FC-binding capacity of the microsomes already at concentration below their critical micellar concentration (c.m.c.), microsomes were incubated with 10 −6 M FC in order to saturate all the binding sites before the treatments. The ATPase activity distributed on glycerol-sucrose gradient in two distinct peaks, one at the low, another at the high density region. The ATPase activity in the two peaks showed an identical sensitivity to inhibitors, but significant differences as far as the pH optimum and the sensitivity to heat-inactivation are concerned. [ 3 H]FC was present in two regions: one at the top, another at the high density; the distribution of [ 3 H]FC in the high density region was coinciding with the peak of ATPase in this region and was, after dilution, pelletable, indicating the presence of a FC-protein complex. These findings are interpreted as suggesting that the FC receptor co-sediments with one form (isoenzyme?) of the plasma membrane H + -ATPase.