Comparison of plasma membrane H+-ATPase activity in vesicles obtained from dry and hydrated maize embryos.

ATP hydrolysis from H+-ATPase of plasma membrane was measured in vesicles from maize embryos imbibed at times between 0 and 5 h. The activity had a maximum at 2 h of imbibition. In order to detect whether the enzyme had the same characteristics through the first 5 h of imbibition, vanadate and lysophophatydilcholine sensitivities, as well as trypsin, pH and temperature effects on the activity of the H+-ATPase from plasma membrane vesicles isolated from embryos imbibed at 0 or 5 h were studied. The results indicate that the activity expressed at 0 h is very different from the activity at 5 h. The activity from embryos imbibed for 5 h was less sensitive to vanadate, trypsin and lysophosphatidylcholine, more sensitive to denaturing temperatures and with a broader pH dependence, as compared to the activity from embryos that were not imbibed. When vanadate-sensitive ATPase activity was purified by anion exchange chromatography, the peaks obtained from the 0 and 5 h imbibed embryos were different and non-overlapping. These data could be interpreted in terms of different enzyme structures from dry and imbibed embryos due to either different primary structures or covalent modifications, or differences in membrane vicinities.

Effect of phytotoxic resin glycoside on activity of H(+)-ATPase from plasma membrane.

A resin glycoside mixture isolated fromIpomoea tricolor inhibited radicle growth ofEchinochloa crusgalli. The effect of the resin was tested on the activity of the plasma membrane H(+)-ATPase fromE. crusgalli. For this purpose, plasma membrane vesicles were purified by the method of aqueous two-phase partitioning. The resin glycoside inhibited by 30% the activity of the plasma membrane ATPase. The same result was obtained with the purified main component of the resin. This indicates that the plasma membrane ATPase can be one of the cellular targets of the resin. Hence it is possible that the mechanism of action of the resin involves an inhibition of the plasma membrane ATPase.

A modified colorimetric method for the determination of orthophosphate in the presence of high ATP concentrations.

We describe a modified colorimetric method that quantitates inorganic phosphate linearly up to 60 nmol, with high stability of the developed color and with a low interference by ATP concentration (up to 30 mM). This method is very suitable for use in ATPase enzymatic assays, especially with enzymes that have low specific activities and (or) high Km values for ATP.

Effect of diacetyl piquerol on H(+)-ATPase activity of microsomes fromIpomoea purpurea.

The effect of an allelopathic compound, diacetyl-piquerol on the H(+) -ATPase activity of the microsomal fraction from the radicles of a common weedIpomoea purpurea was studied. The diacetyl-piquerol inhibited the germination and radicle growth fromI. purpurea; the radicle growth was increasingly inhibited (10% to 100%) as piquerol concentrations were raised (10 µM to 1000 µM). The H(+)-ATPase activity was inhibited (48%) by 500 µM diacetyl-piquerol, and this inhibition was higher in plasma membrane ATPase (67.2%) than in tonoplast membrane ATPase (31.4%). Additional studies of the precise physiological mechanisms of interference caused by allelopathic compounds are needed.

Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion.

Trypsin cleavage has been used to probe structure-function relationships of the Escherichia coli ATP synthase (ECF1F0). Trypsin cleaved all five subunits, alpha, beta, gamma, delta, and epsilon, in isolated ECF1. Cleavage of the alpha subunit involved the removal of the N-terminal 15 residues, the beta subunit was cleaved near the C-terminus, the gamma subunit was cleaved near Ser202, and the delta and epsilon subunits appeared to be cleaved at several sites to yield small peptide fragments. Trypsin cleavage of ECF1 enhanced the ATPase activity between 6- and 8-fold in different preparations, in a time course that followed the cleavage of the epsilon subunit. This removal of the epsilon subunit increased multisite ATPase activity but not unisite ATPase activity, showing that the inhibitory role of the epsilon subunit is due to an effect on cooperativity. The detergent lauryldimethylamine oxide was found to increase multisite catalysis and also increase unisite catalysis more than 2-fold. Prolonged trypsin cleavage left a highly active ATPase containing only the alpha and beta subunits along with two fragments of the gamma subunit. All of the subunits of ECF1 were cleaved by trypsin in preparations of ECF1F0 at the same sites as in isolated ECF1. Two subunits, the beta and epsilon subunits, were cleaved at the same rate in ECF1F0 as in ECF1 alone. The alpha, gamma, and delta subunits were cleaved significantly more slowly in ECF1F0.(ABSTRACT TRUNCATED AT 250 WORDS)