Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

Reconstituted proteoliposomes of tonoplast ATPase are formedon solubilization of tonoplast membranes from mung bean (Vignaradiata L.) with deoxycholate (DOC) in the presence of a mixtureof soybean phospholipids (asolectin), after removal of DOC bypassage through a PD-10 column (Pharmacia). This method is idealbecause of its simplicity and rapidity. Selective insertionof sets of tonoplast H⁺-ATPase polypeptides (68 kDa, 60 kDa,16 kDa and several minor polypeptides) into liposomes usingthis method was confirmed by SDS-PAGE and immuno-blotting withantibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomeswas demonstrated by quinacrine-fluorescence quenching in thepresence of ATP-Mg²⁺. ATP-Mg²⁺ was shown to be the preferredsubstrate in both reconstituted and native tonoplast vesicles,and its optimum concentration was 0.75 to 3.0 mM. Quenchingwas completely abolished by a channel-forming ionophore, gramicidinD, and an inhibitor of tonoplast H⁺-ATPase, KNO₃. Antibodiesto 68-kDa and 60-kDa peptides partially inhibited the pumpingof protons. The rate of pumping of protons increased with thenumber of proteoliposomes, the maximal concentration of whichwas equivalent to 250 µg of protein per reaction mixture.The optimum pH for pumping was 6.5 when inside of proteoliposomeswere loaded pH at 7.2. The rate of pumping of protons was reducedwhen proteoliposomes were made using asolectin and cholesterolat 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibitedby KNO₃, with half-maximal inhibition at approximately 7 mM.The enzyme actively hydrolyzed ATP in preference to GTP, CTP,UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodiesagainst the 60-kDa polypeptide strongly inhibited ATPase activityas compared to antibodies against the 68-kDa polypeptide. Theresults obtained in this study demonstrate directly that functionaltonoplast H⁺-ATPase can be inserted selectively into liposomes. (Received August 31, 1990; Accepted April 18, 1991)     

Effects of Cerebroside and Cholesterol on the Reconstitution of Tonoplast H+-ATPase Purified from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

For an examination of the effects of cholesterol and cerebrosideon the rate and extent of proton-pumping across the membranesof proteoliposomes prepared with tonoplast H⁺-ATPase, the tonoplastH⁺-ATPase of mung bean (Vigna radiata L.) was purified by fastprotein liquid chromatography (FPLC) and incorporated into liposomesprepared from asolectin and cholesterol or cerebroside. Proteoliposomeswere formed after the removal of Triton X-100 from a mixtureof Triton X-100, asolectin and purified tonoplast H⁺-ATPaseby passage through an Ampure DT column. Proteoliposomes preparedfrom cholesterol and asolectin at a ratio of 45 : 55 (w/w) andat a ratio of lipid to protein of 200 : 1 (w/w) gave the largestpH gradient, as determined by the ATP-generated quenching ofquinacrine fluorescence. In the presence of cholesterol, thepH gradient formed across the membranes of proteoliposomes andthe average diameter of proteoliposomes increased about two-fold.The initial rate of proton-pumping decreased to 20% of thatobserved with proteoliposomes prepared from asolectin alone.The addition of cerebroside to asolectin at a ratio of 5 : 95(w/w) caused a 1.6-fold increase in the maximum pH gradientwithout any significant change in the initial rate of proton-pumpingor the diameter of proteoliposomes, but the maximum pH gradientdecreased greatly at ratios above 20 : 80 (w/w). The maximumpH gradient was transient and decreased spontaneously when onlyasolectin was used to prepare proteoliposomes, or when cerebrosideand asolectin were used together. Disappearance of the protongradient once it had formed and/or leakage of protons were suppressedby cholesterol at ratios above 30 : 70 (w/w). It was clear,therefore, that cholesterol and asolectin at ratios 30 : 70(w/w) to 45 : 55 (w/w) formed larger and more stable proteoliposomesthan did asolectin alone. ¹Present address: Laboratory of Climatic Stress Control, TohokuNational Agricultural Experiment Station, 4 Shimokuriyagawa,Morioka, Iwate, 020-01 Japan     

Functional Reconstitution of Plasma Membrane H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes Prepared with Various Molecular Species of Phospholipids

The activity of solubilized plasma membrane ATPase is affectedby the nature of exogenously added molecular species of phospholipids.To examine the role of the polar head group and of the molecularspecies of phospholipids in H⁺-pumping, the ATPase solubilizedfrom plasma membranes of mung bean (Vigna radiata L.) hypocotylswas reconstituted in liposomes prepared with a variety of phospholipids. The extent of activation of solubilized plasma membrane ATPasedue to the addition of 1-palmitoyl 2-oleoyl-phospholipids (PO-phospholipids)and asolectin decreased in the following order: POPS POPC asolectin POPG > POPE > POPA (see List of Abbreviations). H⁺-pumpinginto proteoliposomes reconstituted with asolectin and plasmamembrane ATPase was demonstrated by quinacrine fluorescencequenching in the presence of ATP-MgSO₄. H⁺-pumping was inhibitedby VO₄ and gramicidin D. When plasma membrane ATPase was reconstitutedin liposomes prepared with various PO-phospholipids, the abilityof PO-phospholipids to support H⁺-pumping into the proteoliposomesdecreased in the following order: POPG POPS > asolectin POPC. POPE and POPA failed to support any H⁺-pumping. A remarkablyhigh rate of H⁺-pumping was observed in proteoliposomes preparedwith 1-saturated 2-unsaturated fatty acids, such as POPC, butH⁺-pumping could hardly be detected in proteoliposomes preparedwith 1-, 2-unsaturated or 1-, 2-saturated fatty acids, suchas PSPC or DLPC. ATPase activity in proteoliposomes was dependenton the species of PO-phospholipids used for reconstitution anddecreased in the following order: POPS > POPG > POPC asolectin > POPA > POPE. DLPC (see List of Abbreviations)which includes a 1-, 2-unsaturated fatty acid supported onlymarkedly depressed activity. Both H⁺-pumping and the hydrolysis of ATP by the plasma membraneATPase are strongly affected by the polar head group and compositionof the fatty acyl chain of phospholipids used to prepare liposomesfor reconstitution of the ATPase. (Received May 31, 1991; Accepted September 18, 1991)     

茉莉酸甲酯和脱落酸对绿豆下胚轴质膜H+-ATPase水解活性的影响

茉莉酸甲酯(MeJA)促进绿豆下胚轴质膜H+-ATPase水解活性.活体条件下,50μmol·L-1 MeJA处理7 h的酶活性提高30%;离体条件下,10 μmol·L-1 MeJA处理2 h的酶活性最大,即提高30%.壳梭孢素(FC)和MeJA在离体条件下对H+-ATPase活性的促进效应相同,均提高30%左右,无协同效应;活体条件下,FC促进质膜H+-ATPase水解活性可达70%,而MeJA仅为30%.离体条件下,脱落酸(ABA)对H+-ATPase水解活性无明显促进;而活体条件下则有一定的抑制.     

茉莉酸甲酯处理对绿豆下胚轴质膜H+-ATPase水解活力及磷酸化水平的影响

运用γ-32P示踪、蛋白激酶和磷酸酶抑制剂药理实验探讨茉莉酸甲酯(MeJA)对质膜H -ATP酶水解活力及磷酸化水平的影响.结果如下:MeJA可促进H -ATP酶水解活力30%;斑蝥素和岗田酸促进了MeJA对质膜H -ATP酶的刺激作用;星形孢菌素和白屈菜红碱削弱了MeJA对质膜H -ATP酶的刺激作用.H -ATP酶活力变化同时,其上的γ-32P标记量发生变化.Ca2 对H -ATP酶水解活力有很大的刺激作用,但对MeJA促进H -ATP酶活力的作用没有进一步的影响.根据这些结果可以得出结论:MeJA刺激质膜H -ATP酶水解活力的变化与H -ATP酶磷酸化水平呈正相关,并且催化这一作用的蛋白激酶可能不依赖于Ca2 ,而蛋白磷酸酶依赖于Ca2 .     

The Role of Phospholipids in Plasma Membrane ATPase Activity in Vigna radiata L. (Mung Bean) Roots and Hypocotyls

Root and hypocotyl plasma membrane H⁺-ATPases were partially purified from deoxycholate-solubilized fractions of microsomes in mung bean (Vigna radiata L.) plants in the presence of glycerol. Certain properties of the ATPases and the manner in which phospholipids affect their activity were compared. Root ATPase was similar to hypocotyl ATPase with respect to substrate specificity, salt stimulation, pH dependence, K_m for ATP·Mg²⁺ and inhibitor sensitivity, except for inhibition by vanadate. Both purified ATPases required phospholipids for their activation. Optimum concentrations of exogenously added phospholipid mixture (asolectin) to hypocotyl and root ATPase mixture were 0.03% and 1.0%, respectively. Root ATPase activation did not decrease if more than 1.0% asolectin was added. Qualitatively, phosphatidylserine and phosphatidylcholine brought about greater ATPase activation than other phospholipids. The hypocotyl ATPase was activated by phosphatidylinositol, phosphatidylserine and phosphatidylglycerol to a greater extent than the root ATPase. Root, but not hypocotyl ATPase, was slightly inhibited by the addition of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. The hypocotyl plasma membrane contained phosphatidylinositol + phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and unsaturated fatty acids in greater abundance than the root plasma membrane. The differential activation of the plasma membrane ATPases may arise from these differences.     

Binding of 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole to an Essential Cysteine Residue(s) in the Tonoplast H-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls

Vacuolar-type H⁺-ATPase was solubilized from tonoplasts of mung bean (Vigna radiata L.) and purified on a Mono Q anion-exchange column by fast protein liquid chromatography. The purified enzyme was inactivated by the reactive adenine analog, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). This inactivation was reversed by addition of dithiothreitol (DTT). Inactivation by NBD-Cl was prevented by Mg-ADP, a competitive inhibitor of ATPase. [¹⁴C]NBD-Cl predominantly modified the 68-kilodalton subunit and the degree of ¹⁴C incorporation was decreased in the presence of Mg-ADP or upon subsequent addition of DTT. The loss of activity followed pseudo first-order kinetics with respect to NBD-Cl concentration, and double log plots of pseudo first-order rate constants versus reagent concentration yielded a straight line with a slope of 0.957. The NBD-modified/inactivated enzyme showed an absorbance maximum at 418 nanometers and a fluorescence emission peak at 515 nanometers. The absorption and fluorescence emission spectra of the NBD-modified enzyme were essentially the same as those of the model compound, N-acetyl-S-NBD cysteine. Absorbance by the modified enzyme at 418 nanometers disappeared upon addition of DTT, which coincided with the restoration of ATPase activity and the decrease in bound [¹⁴C]NBD-Cl. These findings show that NBD-Cl modifies an essential cysteine residue(s) at or near the catalytic site in the 68-kilodalton subunit of tonoplast H⁺-ATPase and that the modification closely correlates with the loss of ATPase activity.     

Isolation of Smooth Endoplasmic Reticulum and Tonoplast from Mung Bean Hypocotyls (Vigna radiata [L.] Wilczek) Using a Ficoli Gradient and Two-Polymer Phase Partition

A new method for the isolation of smooth endoplasmic reticulumand tonoplast from etiolated mung bean hypocotyls (Vigna radiata[L.] Wilczek) has been developed. After centrifugation in aFicoli density gradient [5.5% (w/w) in 15% (w/w) sucrose] ofa crude microsomal membrane fraction (10,000–156,000?gpellet) which had been prepared and resuspended in buffer systemsthat contained 0.25 M sorbitol, more than 80% of the total amountsof smooth endoplasmic reticulum and tonoplast were co-bandedat the interface between the sample load and the Ficoll layer,while most of the other cellular membranes, including plasmamembrane, Golgi membranes and yellow-colored membrane materials,which were presumably the etioplast envelopes, were sedimentedthrough the Ficoli layer. Smooth endoplasmic reticulum and tonoplastwere separated from each other to a high degree of enrichmentby a subsequent two-polymer phase partitioning. The separationis based on the principle that mung bean tonoplast has a highpartition coefficient for the polyethylene glycol-enriched upperphase and the smooth endoplasmic reticulum has a high partitioncoefficient for the Dextran-enriched lower phase. Assessed interms of degree of contamination by activities of membrane markerenzymes, the isolated smooth endoplasmic reticulum and tonoplastfractions were found to be highly purified. An ATPase sensitiveto neutral detergent and vanadate was found to be specificallyassociated with the smooth endoplasmic reticulum. ¹Contribution No. 3172 from the Institute of Low TemperatureScience (Received April 22, 1988; Accepted September 28, 1988)     

Biosynthesis of Phosphatidylglycerol in Isolated Mitochondria of Etiolated Mung Bean (Vigna radiata L.) Seedlings

Phosphatidylglycerophosphate synthase (sn-glycerol-3-phosphate:CDP-diacylglycerol phosphatidyltransferase) and phosphatidylglycerophosphate phosphatase were characterized in mung bean (Vigna radiata L.) mitochondria. The synthase has a rather broad pH optimum between 7 and 9, whereas the phosphatase has one of about 7. Both enzymic activities are stimulated by Triton X-100 and require divalent cations but differ in their cation specificities. The synthase shows apparent Km values of 9 and 3 [mu]M for sn-glycerol-3-phosphate and CDP-diacylglycerol, respectively. Phosphatidylglycerophosphate, in contrast to lysophosphatidic and phosphatidic acid, is effectively dephosphorylated by the phosphatase, which exhibits an apparent Km value of 12 [mu]M for its substrate. Each enzyme shows higher activities with the dipalmitoyl species of its substrate than with the dioleoyl species. These substrate specificities of both enzymes are predominantly based on differences in apparent Vmax values.     

Characterization of ATPases Associated with Various Cellular Membranes Isolated from Etiolated Hypocotyls of Vigna radiata (L.) Wilczek

Cellular membrane fractions, including endoplasmic reticum (ER),Golgi-enriched membrane, plasma membrane and tonoplasts, wereisolated from Vigna radiata seedlings. Each of these membranefractions was associated with specific ATPases which were highlydependent on Mg²⁺. ATPases of ER, Golgi-enriched membrane andplasma membrane were sensitive to vanadate but the tonoplastATPase was not. ATPases were mostly dependent on Cl¹, but aslight stimulation by K⁺ was observed in the case of ATPasesof Golgi-enriched membrane and plasma membrane. KNO₃ inhibitedtonoplast ATPase but stimulated the other ATPases. ER ATPasecan be distinguished from other ATPases by the following characteristics:specific inhibition by KNO₂ and Triton X-100, stimulation bylow concentrations of diethylstilbestrol and 4,4'-diisothiocyanostilbene-2,2'-disulfonicacid, and high sensitivity to heat. The ATPases showed typicalMichaelis-Menten kinetics and had K_m values of 0.5 to 0.6 ITIMMg²⁺-ATP for ER, Golgienriched-membrane and tonoplast ATPases,and 2.27 msi Mg²⁺-ATP for plasma membrane ATPase. ATPases ofGolgi-enriched membranes and plasma membranes had similar properties,but they were still distinguishable by the differences in theirK_m values and their responses to Triton X-100. Based on theseresults, it is postulated that each cellular membrane is associatedwith a specific ATPase in cells of V. radiata. ¹Contribution No. 3171 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 28, 1988)     

Lipid Composition of Plasma Membranes and Tonoplasts Isolated from Etiolated Seedlings of Mung Bean (Vigna radiata L.)

The lipid composition of plasma membranes and tonoplasts from etiolated mung bean hypocotyls was examined in detail. Phospholipids, sterols, and ceramide monohexoside(s) were the major lipid classes in both membranes. The content of phospholipids on a protein basis was higher in the tonoplast, but the content of total sterols was similar in both membranes. Accordingly, the sterol to phospholipid molar ratio in the plasma membrane was higher than that of the tonoplast. Phosphatidylethanolamine and phosphatidylcholine comprised the major phospholipids in both membranes. Phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were identified as minor phospholipid components. The content of phosphatidylinositol and phosphatidylglycerol was relatively high in the tonoplast, comprising 11 and 5% of the total phospholipids, respectively. Although special care was taken against the degradative action of phospholipase D and phosphatidic acid phosphatase during the isolation of these membranes, by adding EDTA, EGTA, KF, choline, and ethanolamine to the homogenizing medium, significant amounts of phosphatidic acid, about 15% of the total phospholipids, were detected in the plasma membrane. On the other hand, the content of phosphatidic acid in tonoplasts and other membrane fractions was very low. This fact may indicate that high levels of phosphatidic acid occur naturally in plasma membranes. Phosphatidylglycerol in both membranes and phosphatidylinositol in the tonoplast contained high levels of palmitic acid, which comprised more than 50% of the total fatty acids. Significant differences were observed in the sterol compositions of plasma membranes and tonoplasts. More than 90% of the sterols in the plasma membrane were unesterified, while the tonoplast was enriched in glycosylated sterols, especially acylated sterylglycosides. Ceramide monohexoside was found to be specifically located in these membranes, in particular, in the tonoplast, in which it comprised nearly 17% of the total lipids.     

Reversible and Irreversible Reduction of ACC-dependent Ethylene Formation in Mung Bean (Vigna radiata [L] Wilczek) Hypocotyls Caused by Chilling

Etiolated seedlings of mung bean (Vigna radiata [L] Wilczek)grown at 26?C in the dark were chilled at 0?C for various periodsand then returned to 26?C to follow the development of chillinginjury and changes in ACC-dependent ethylene-forming activity(EFA). After chilling for one day, they could grow normallyat 26?C, but not after chilling for more than two days. Thelonger chilling caused a remarkable increase in electrolyteleakage from the hypocotyl tissues, suggesting plasma membraneinjury. The ACC-dependent EFA in vivo in hypocotyls was foundto be sensitive to chilling and progressively declined as afunction of the chilling period. After 1-day chilling, the activitydeclined to less than 50 per cent of the unchilled tissues,but it returned to the normal level within 3 h after the returnto 26?C. However, chilling more than two days caused irreversibleloss of the activity. Thus, there seems to be an intimate relationshipbetween irreversible loss of the EFA and chilling injury ofthe tissues. The recovery of EFA after chilling for one daycould be markedly inhibited by cycloheximide and monensin, suggestingthat the recovery requires protein synthesis and, presumably,includes repair of partially injured cellular membranes, mediatedthrough the Golgi apparatus. ¹Contribution No. 2894 from the Institute of Low TemperatureScience (Received June 23, 1986; Accepted November 7, 1986)     

Effects of Gamma-Irradiation on the Activity of Tonoplast H+-ATPase from Potato Tubers (Solanum tuberosum L.)

Tonoplast vesicles were prepared from potato tubers (Solariumtuberosum L.) on a step gradient (0% and 6%, w/w) of dextranT-70 to clarify the mechanism by which the tonoplast H⁺-ATPaseis inactivated by gamma-irradiation. H⁺-ATPase activity andH⁺ -pumping were examined after irradiation of tubers (in vivoirradiation) and of isolated tonoplast vesicles (in vitro irradiation)at doses up to 1.0 kGy. Both in vivo irradiation and in vitroirradiation resulted in significant decreases in ATPase andH⁺-pumping activities. The ATPase and H⁺-pumping activities12 h after irradiation were much lower than those 2 h afterirradiation. Solubilized H⁺-ATPase was inactivated, in a dose-dependentmanner, by irradiation (enzyme irradiation) to a greater extentthan was observed after in vitro irradiation or in vivo irradiation.The activity of ATPase 12 h after enzyme irradiation was almostthe same as it was 2 h after enzyme irradiation. The free fattyacid content of vacuolar membranes was increased by in vivoirradiation and by in vitro irradiation with an accompanyingdecrease in tonoplast H⁺-ATPase activity. Lipids from irradiatedtonoplasts had a considerable inhibitory effect on the activityof solubilized H⁺-ATPase. This result suggests that the directinactivation of H⁺-ATPase in potato tonoplast by gamma-irradiationis augmented by the effects of deterioration of membrane lipidsthat is induced by the irradiation. (Received December 21, 1994; Accepted May 16, 1994)     

Kinetics of Inactivation of NADP+-Linked Isocitrate Dehydrogenase from Germinating Mung Bean (Vigna radiata)

Metal chelating agent EDTA inhibits the activity of mung-bean NADP⁺-linked isocitrate dehydrogenase (ICDH) in a competitive manner. The activity of the Apo-enzyme was restored by divalent metal ions with the order of effectiveness found to be Mn ²⁺> Mg²⁺ > Zn²⁺ > Co²⁺ > Cu²⁺. here appeared to be a single type of metal binding site that was saturated either with 0.5 mM of Mn²⁺ or with 2.5 mM of Mg²⁺. ADP, ATP and NADPH inhibit the enzyme in competitive manner. On titration with 5, 5’-dithiobis (2-nitrobenzoate), i.e. DTNB, the mung bean isocitrate dehydrogenase showed 4.0 reactive -SH groups per molecule. The denatured ICDH enzyme of mung bean possess 8.1-SH groups per molecule. The blocking of this group with -SH reagents, lead to the inactivation of mung bean ICDH enzyme. Time-dependent inactivation of ICDH with iodoacetamide and Nethylmaleimide (NEM) revealed decay in the activity in a single exponential manner.     

A Study of NADP+- linked Isocitrate Dehydrogenase from Germinating Mung Bean (Vigna radiata)

NADP⁺- linked isocitrate dehydrogenase has been purified to apparent homogeneity from 36 h germinated mung beans by ammonium sulphate fractionation, heat treatment, acid treatment, and DEAE — Cellulose column chromatography. The enzyme was purified to 150 fold with 15% recovery. The preparation showed single protein band on native PAGE and was free from bound nucleotides and coloured pigments (A₂₈₀/A₂₆₀ = 1.4). The molecular weight was found to be 141,000 and was made of four identical subunits (mol wt 36,000). Thermal inactivation at 50, 53, and 55 °C revealed simple first order kinetics and t_1/2 was found to be 38, 10, and 3 min, respectively. The enzyme exhibited absolute specificity for NADP⁺ and substrate. The K_m for isocitrate and NADP⁺ was 28.57 µM and 70 µM, respectively. The enzyme appeared to be regulated by various metabolites of Krebs’ cycle intermediates.     

Substrate-Induced Stability of Glyceraldehyde 3-Phosphate Dehydrogenase from Mung Beans (Vigna radiata L.)

Time-dependent thermal inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) present in the extract of mung beans at different periods of germination showed biphasic kinetics in the 12-h germinated seeds but single exponential decay at 24 h of germination. The glyceraldehyde 3-phosphate (G-3-P) concentration in the deproteinated extracts was found to increase with period of germination up to 36 h, parallel to that of GAPDH activity. G-3-P was found to offer protection of the enzyme against thermal inactivation and trypsin digestion. It is suggested that accumulation of G-3-P in germinating mung beans may be of physiological significance and it might offer protection to the enzyme in vivo against thermal inactivation and proteolysis.     

Essential Arginyl Residues in the Plasma Membrane H-ATPase from Vigna radiata L. (Mung Bean) Roots

Proton-translocating ATPase (H⁺-ATPase) was purified from mung bean (Vigna radiata L.) roots. Treatment of this enzyme with the arginine-specific reagent 2,3-butanedione in the presence of borate at 37°C (pH 7.0), caused a marked decrease in its activity. Under this condition, half-maximal inhibition was brought about by 20 millimolar 2,3-butanedione at 12 minutes. MgATP and MgADP, the physiological substrate and competitive inhibitor of the ATPase, respectively, provided partial protection against inactivation. Loss of activity followed pseudo-first order kinetics with respect to 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration gave a curve with a slope of 0.984. Thus, inactivation may possibly result from reaction of one arginine residue at each active site of the enzyme. The results obtained from the present study indicate that at least one arginyl residue performs an essential function in the plasma membrane H⁺-ATPase, probably at the catalytic site.     

Enzymology of l-Tyrosine Biosynthesis in Mung Bean (Vigna radiata [L.] Wilczek)

The enzymes of the 4-hydroxyphenylpyruvate (prephenate dehydrogenase and 4-hydroxyphenylpyruvate aminotransferase) and pretyrosine (prephenate aminotransferase and pretyrosine dehydrogenase) pathways of l-tyrosine biosynthesis were partially purified from mung bean (Vigna radiata [L.] Wilczek) seedlings. NADP-dependent prephenate dehydrogenase and pretyrosine dehydrogenase activities coeluted from ion exchange, adsorption, and gel-filtration columns, suggesting that a single protein (52,000 daltons) catalyzes both reactions. The ratio of the activities of partially purified prephenate to pretyrosine dehydrogenase was constant during all purification steps as well as after partial inactivation caused by p-hydroxymercuribenzoic acid or heat. The activity of prephenate dehydrogenase, but not of pretyrosine dehydrogenase, was inhibited by l-tyrosine at nonsaturating levels of substrate. The K(m) values for prephenate and pretyrosine were similar, but the specific activity with prephenate was 2.9 times greater than with pretyrosine.Two peaks of aromatic aminotransferase activity utilizing l-glutamate or l-aspartate as amino donors and 4-hydroxyphenylpyruvate, phenylpyruvate, and/or prephenate as keto acid substrates were eluted from DEAE-cellulose. Of the three keto acid substrates, 4-hydroxyphenylpyruvate was preferentially utilized by 4-hydroxyphenylpyruvate aminotransferase whereas prephenate was best utilized by prephenate aminotransferase. The identity of a product of prephenate aminotransferase as pretyrosine following reaction with prephenate was established by thin layer chromatography of the dansyl-derivative.     

Impairment of Tonoplast H-ATPase as an Initial Physiological Response of Cells to Chilling in Mung Bean (Vigna radiata [L.] Wilczek)

Biochemical alterations of cellular membranes in chilling-sensitive mung bean (Vigna radiata [L.] Wilczek) hypocotyls were investigated with reference to chilling injury. Reversible decreases in activities of tonoplast H⁺-ATPase and in vivo respiration became manifest within 24 hours of chilling when tissues suffered no permanent injury as assessed by electrolyte leakage and regrowth capacity. These changes were found to be the earliest cellular responses to chilling. A density-shift on a sucrose density gradient was observed in Golgi membranes early in the chilling treatment, suggesting that Golgi function and/or membrane biogenesis via the Golgi may have been altered upon chilling. After chilling more than 2 days, irreversible changes were generally produced in cellular membranes including the plasma membrane, endoplasmic reticulum, and mitochondria. Respiratory functions remained intact in mitochondria isolated from tissues prechilled for 24 hours, but were impaired after prechilling for 3 days. Given the important role of the tonoplast H⁺-ATPase in the active transport of ions and metabolites, the early decline in the tonoplast H⁺-ATPase activity may give rise to an alteration of the cytoplasmic environment and, consequently, trigger a series of degenerative reactions in the cells.