β-Glucosidase Activity in Corn Roots

The mechanism by which proton transport is coupled to ATP hydrolysis by vanadate-sensitive pumps is poorly understood. The effects of temperature on the activities of the vanadatesensitive ATPase from maize (Zea mays) roots were assessed to provide insight into the coupling mechanism. The initial rate of proton transport had a bell-shaped dependence on temperature with an optimal range between 20 and 300C. However, the rate of vanadate-sensitive ATP hydrolysis increased as the temperature was raised from 4 to 430C. The differential sensitivity of proton transport to temperatures above 300C was also observed when the ATPase was reconstituted into dioleoylphosphatidylcholine vesicles. Inhibition of proton transport with temperatures above 300C was associated with higher rates of proton leakage from the membranes. In addition, proton transport was more inhibited than ATP hydrolysis at temperatures below 100C. Reduced rates of proton transport at lower temperatures were not associated with higher rate of proton conductivity across the membranes. Therefore, the preferential inhibition of proton transport at temperatures below 100C may reflect an effect of temperature on the coupling between proton transport and ATP hydrolysis within the vanadate-sensitive ATPase.Plant cells contain two predominant classes ofproton translocating ATPase which energize solute transport (5,16 With vacuolar-type ATPase from maize (Zea mays) root tonoplast vesicles, the temperature dependence of the proton transport differed from that ofATP hydrolysis (17). The initial rate of proton transport declined as the temperature was increased above 25°C, whereas the rate of ATP hydrolysis by the pump continued to increase. These results supported the hypothesis that proton transport and ATP hydrolysis by the vacuolar ATPase were indirectly coupled. In this paper, the nature of the coupling mechanism of the plasma membranetype ATPase from maize root has been investigated in a similar manner by examining the temperature dependence of proton transport and ATP hydrolysis. MATERIALS AND METHODS Preparation of Membrane VesiclesMaize seedlings (Zea mays L., cv WF9 x Mo 17) were grown on filter paper moistened with CaCl2 (13). KI-washed microsomes were prepared from 3-d-old roots as described previously (3). The ATPase in these microsomes were reconstituted into di-18:1 PC' liposomes by a detergent dilution protocol using deoxycholate as described previously (3), using a lipid to protein ratio of 20 to 1 by weight. Protein concentration was determined by a modification of the Lowry method after precipitation by TCA in the presence of deoxycholate (2). Proton Transport and ATP Hydrolysis AssaysProton transport was followed primarily by changes in the absorbance of AO at 492.5 nm as described by de Michelis et al. (5). Assays were conducted with Beckman DU-702 spectrophotometer equipped with a thermoregulated cell holder attached to a circulating water bath. Temperature in a cuvette containing assay medium adjacent to the sample was monitored. Typi...

“…Maize seedlings (Zea mays L., cv WF9 x Mo 17) were grown on filter paper moistened with CaCl2 (13). KI-washed microsomes were prepared from 3-d-old roots as described previously (3).…”

Section: Materials and Methods Preparation Of Membrane Vesiclesmentioning

confidence: 99%

Effects of Temperature on the Coupled Activities of the Vanadate-Sensitive Proton Pump from Maize Root Microsomes

Bräuer

Loper²,

Schubert³

et al. 1991

“…Maize seedlings (Zea mays L. cv W7551) were germinated on filter paper moistened with 0.1 mm CaCl2 (27). Microsomes were isolated from 3-d-old roots by differential centrifugation as described previously (8), except that the 25 mm bis-trispropane (pH 7.8) in the homogenizing buffer was replaced with 50 mm Hepes (pH 7.5).…”

Section: Isolation Of Plasma Membranesmentioning

confidence: 99%

“…It is quite apparent that one subunit of the P-type ATPase, designated as a, is responsible for ATP binding, formation of the phosphorylated enzyme intermediate, and binding of the transport ions (11,28). However, several enzymes within this class of ATPase, including the (Na + K) and (H + K) ATPase, have at least a second subunit, designated as a (13,24,27). In the (Na + K) ATPase, the # subunit is responsible for binding a certain class of inhibitor (24), and as such is believed to play a role in enzyme regulation.…”

mentioning

confidence: 99%

Effects of Solubilization on the Inhibition of the P-Type ATPase from Maize Roots by N-(Ethoxycarbonyl)-2-Ethoxy-1,2-Dihydroquinoline

Bräuer

Gurriel²,

Tu³

1992

The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electrochemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time-and concentration-dependent manner consistent with a firstorder reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.Enzymes that link the transport of ions to either the hydrolysis or synthesis of ATP have been classified into three broad categories of transport ATPase, designated as P-, V-, and F-type (28). These ATPases can be distinguished by their biochemical, physical, and physiological properties. The Ftype ATPase couples the flux of ions, usually protons, down their electrochemical gradient to the synthesis of ATP. The other two classes of ATPases couple the transport of ions against their electrochemical gradient to the release of free energy from ATP hydrolysis.The mechanism by which the energy from ATP is converted into the movement of ions by the V-and P-type ATPases is poorly understood. A generalized reaction scheme has been proposed for the P-type ATPases in which transport reactions are tightly coupled to the reactions, leading to phosphorylation and dephosphorylation of the enzyme (24,28). Soon after the discovery of the V-type ATPase, it was apparent that a reaction mechLanism in which ATP hydrolysis and ion transport were tightly linked could not account for the differential inhibition and activation of the activities of

“…Nagahashi and Baker (20) performed differential centrifugation and centrifugation time course studies on maize j-glucosidase activity and found that the enzyme migrated past the ER marker only after a long centrifugation time. Nagahashi et al (19) reported that the association of,-glucosidase activity with the cell wall in maize roots was pH-dependent, about sixfold greater at alkaline (7.7) than at acidic (6.0) pH values.…”

mentioning

confidence: 99%

Purification and Partial Characterization of Maize (Zea mays L.) β-Glucosidase

Esen

1992

100

Maize (Zea mays L.) ,-glucosidase (ft-d-glucoside glucohydrolase, EC 3.2.1.21) was extracted from the coleoptiles of 5-to 6-day-old maize seedlings with 50 millimolar sodium acetate, pH 5.0. The pH of the extract was adjusted to 4.6, and most of the contaminating proteins were cryoprecipitated at 0°C for 24 hours. The pH 4.6 supematant from cryoprecipitation was further fractionated by chromatography on an Accell CM column using a 4.8 to 6.8 pH gradient of 50 millimolar sodium acetate, which yielded the enzyme in two homogeneous, chromatographically different fractions. Purified enzyme was characterized with respect to subunit molecular weight, isoelectric point, amino acid composition, NH2-terminal amino acid sequence, pH and temperature optima, thermostability, and activity and stability in the presence of selected reducing agents, metal ions, and alkylating agents. The purified enzyme has an estimated subunit molecular mass of 60 kilodaltons, isoelectric point at pH 5.2, and pH and temperature optima at 5.8 and 500C, respectively. The amino acid composition data indicate that the enzyme is rich in Glx and Asx, the sum of which approaches 25%. The sequence of the first 20 amino acids in the N-terminal region was H2N-Ser-AIa-Arg-ValGly-Ser-Gln-Asn-Gly-Val-Gln-Met-Leu-Ser-Pro-(Ser?)-Glu-llePro-Gin, and it shows no significant similarity to other proteins with known sequence. The enzyme is extremely stable at 0 to 40C up to 1 year but loses activity completely at and above 550C in 10 minutes. Likewise, the enzyme is stable in the presence of or after treatment with 500 millimolar 2-mercaptoethanol, and it is totally inactivated at 2000 millimolar 2-mercaptoethanol. Such metal ions as Hg2+ and Ag+ reversibly inhibit the enzyme at micromolar concentrations, and inhibition could be completely overcome by adding 2-mercaptoethanol at molar excess of the inhibitory metal ion. The alkylating agents iodoacetic acid and iodoacetamide irreversibly inactivate the enzyme and such inactivation is accelerated in the presence of urea.in any other plant (8,28). Some maize inbreds lack the enzyme and are thus thought to be homozygous for a null allele of this locus (28). The recent data (4) show that the null phenotype is not due to the lack of enzyme activity; the enzyme is not detected in zymograms because it occurs as large quaternary structures and does not enter the gel.Maize f.-glucosidase was isolated by Huber and Nevins (10) as a buffer-soluble fraction and shown not to be active in the hydrolysis of,3-glucans. Nagahashi and Baker (20) performed differential centrifugation and centrifugation time course studies on maize j-glucosidase activity and found that the enzyme migrated past the ER marker only after a long centrifugation time. Nagahashi et al. (19) reported that the association of,-glucosidase activity with the cell wall in maize roots was pH-dependent, about sixfold greater at alkaline (7.7) than at acidic (6.0) pH values. They concluded that the cell wall-bound activity was not associated with the wall it...