Kinetics of the H+-ATPase from Dry and 5-Hours-Imbibed Maize Embryos in Its Native, Solubilized, and Reconstituted Forms

Abstract

Membranes undergo recovery upon rehydration in seed germination. Previous work has described that the plasma membrane H+-ATPase from maize embryos adopts two different forms at 0 and 5 h of imbibition. We investigated how the kinetics of these two forms could be affected by alterations in the plasma membrane (PM). In comparison to the 0-h, PMs from the 5-h imbibed embryos showed changes in glycerophospholipid composition, decrease in leakage, and increase in fluidity. Kinetics of the PM H+-ATPase from 0 and 5-h imbibed embryos showed negative cooperativity. With the removal of the membrane environment, the activity of the enzymes shifted to a more complex kinetics, displaying two enzyme components. Lipid reconstitution produced one component with positive cooperativity. In all cases, enzymes from 0 and 5-h imbibed embryos presented similar kinetics with some quantitative differences. These results indicate that the two enzyme forms have the potential ability to respond to changes in the membrane environment, but the fact that they do not show differences in the native membranes at 0 or 5 h implies that modifications in the membrane are not drastic enough to alter their kinetics, or that they are able to preserve their boundary lipids or associated proteins and thus retain the same kinetic behavior.

Early carbon mobilization and radicle protrusion in maize germination

“…3C). In this regard, it has been previously shown that the enzyme is already present at 0 h of imbibition with an activity differentially distributed throughout the embryo regions and that it is gradually replaced by different enzyme forms during the first 5 h of maize imbibition (Sánchez-Nieto et al , 1998; Enríquez-Arredondo et al , 2005; Sánchez-Nieto et al , 2011). Therefore, the increase in H + -ATPase activity at 18 h of imbibition was probably related to either the expression of a different isozyme, such as the MHA1 and MHA2 isoforms described in maize coleoptiles (Frías et al , 1996), or to post-translational regulatory mechanisms (Sondergaard et al , 2004).…”

“…However, this apparent dissimilarity could be explained by the appearance of new transporter isoforms. This is a common event that occurs in the progress of germination at early times (Andrianarison and Beneytout, 1992; Waterworth et al , 2000; Enríquez-Arredondo et al , 2005; Sánchez-Nieto et al , 2011). In the case of glucose (Fig.…”

“…In the case of sucrose, the 0 h transporter, present in lower amount, could be relayed by another form that, although in larger amounts, has the same basal activity as the initial form. However, it can be acknowledged that the different forms of transporters may correspond either to different isoenzymes or to the same proteins regulated by factors that are produced in the course of imbibition (Andrianarison and Beneytout, 1992; Waterworth et al , 2000; Enríquez-Arredondo et al , 2005; Sánchez-Nieto et al , 2011). …”

ModulatoryATPbinding to the E2 state of maize plasma membrane H+‐ATPase indicated by the kinetics of vanadate inhibition

“…A,B. Nevertheless, as several researchers have found that the kinetics of this enzyme may resemble cooperativity , our data were also analyzed with Eadie–Scatchard plots (Fig. C,D), which are ‘most sensititive to detect deviations from Michaelian behavior’ .…”

“…They suggested that, under physiological conditions, this site may be inaccessible to the substrate. Since then, several reports have provided evidence for the existence of different ATP‐binding sites in maize PM H + ‐ATPase, because the kinetic data suggested positive or negative cooperativity . In 1997, Roberts and Beaugé presented four models of substrate binding to PM H + ‐ATPase of maize, one of which they called a ‘minimal’ model, which does not require two binding sites to explain cooperativity.…”