Modification of the Red Beet Plasma Membrane H-ATPase by Diethylpyrocarbonate.

Incubation of the red beet (Beta vulgaris L.) plasma membrane H(+)-ATPase with micromolar concentrations of diethylpyrocarbonate (DEPC) resulted in inhibition of both ATP hydrolytic and proton pumping activity. Enzyme activity was restored when DEPC-modified protein was incubated with hydroxylamine, suggesting specific modification of histidine residues. Kinetic analyses of DEPC inhibition performed on both membrane-bound and solubilized enzyme preparations suggested the presence of at least one essential histidine moiety per active site. Inclusion of either ATP (substrate) or ADP (product and competitive inhibitor) in the modification medium reduced the amount of inhibition observed in the presence of DEPC. However, protection was not entirely effective in returning activity to noninhibited control values. These results suggest that the modified histidine does not reside directly in the ATP binding region of the enzyme, but is more likely involved in enzyme regulation through subtle conformational effects.

Modification of an essential arginine residue associated with the plasma membrane ATPase of red beet (Beta vulgaris L.) storage tissue.

The dicarbonyl compounds, phenylgloxyl and 2,3-butanedione were used to demonstrate the presence of an essential arginine residue in the mechanism of the red beet (Beta vulgaris L.) plasma membrane ATPase. Treatment of the red beet ATPase with either of these reagents resulted in an inhibition of ATP hydrolytic activity protectable by the inclusion of either ATP or ADP during inhibitor incubation. Ligands of the ATP hydrolytic reaction also protected against phenylglyoxyl inhibition and affected the ability of ADP to protect against inhibition by this reagent. Kinetic analysis of 2,3-butanedione and phenylglyoxyl inhibition suggested the presence of a single arginine residue susceptible to attack by these reagents. As similar results with these arginine modification reagents were found for both the plasma membrane-associated and solubilized forms of the ATPase, it is apparent that the function of this arginyl moiety is not affected by detergent treatment and removal of the enzyme from the membrane.

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : I. Characterization of a Ca-Pumping ATPase Associated with the Endoplasmic Reticulum.

Calcium transport was examined in microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue using chlorotetracycline as a fluorescent probe. This probe demonstrates an increase in fluorescence corresponding to calcium accumulation within the vesicles which can be collapsed by the addition of the calcium ionophore A23187. Calcium uptake in the microsomal vesicles was ATP dependent and completely inhibited by orthovanadate. Centrifugation of the microsomal membrane fraction on a linear 15 to 45% (w/w) sucrose density gradient revealed the presence of a single peak of calcium uptake which comigrated with the marker for endoplasmic reticulum. The calcium transport system associated with endoplasmic reticulum vesicles was then further characterized in fractions produced by centrifugation on discontinous sucrose density gradients. Calcium transport was insensitive to carbonylcyanide m-chlorophenylhydrazone indicating the presence of a primary transport system directly linked to ATP utilization. The endoplasmic reticulum vesicles contained an ATPase activity that was calcium dependent and further stimulated by A23187 (Ca(2+), A23187 stimulated-ATPase). Both calcium uptake and Ca(2+), A23187 stimulated ATPase demonstrated similar properties with respect to pH optimum, inhibitor sensitivity, substrate specificity, and substrate kinetics. Treatment of the red beet endoplasmic reticulum vesicles with [gamma-(32)P]-ATP over short time intervals revealed the presence of a rapidly turning over 96 kilodalton radioactive peptide possibly representing a phosphorylated intermediate of this endoplasmic reticulum associated ATPase. It is proposed that this ATPase activity may represent the enzymic machinery responsible for mediating primary calcium transport in the endoplasmic reticulum linked to ATP utilization.

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue.

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.