There are many intrinsic membrane ATPases, which can transport ions in one direction across a membrane without being accompanied by an ion of opposite charge giving rise to an electrical potential difference. These intrinsic membrane proteins were variously named as translocases, transferases or penetrases. They depend on ATP as energy source for transport.
The allosteric transition of the transport ATPases from one state to another results in the movement of the ion from one boundary of the cell membrane to other. The intrinsic ATPase proteins have amphipathic properties which allow them to bridge the hydrophobic region of the lipid bilayer. This property makes them potential channels for transport. The proteins actually protrude into the cytoplasm and periplasm.
The mechanism states that the ions induce a change in the configuration of the protein when they are bound to it, and as a result the active catalytic site is exposed to its substrate, Mg-ATP. So, the ions act as cofactors in the enzymatic hydrolysis of ATP.
These plasma membrane ATPases are called P-type ATPases. They perform a variety of physiological functions along with ion transport. These enzymes contain a single polypeptide of about 100kDa that both binds ATP and catalyzes ion transport. This P-type ATPase is encoded by a multi-gene family that exhibits tissue-specific expression.
In Arabidopsis, plasma membrane H+ – ATPases are encoded by genes of the AHA family. At least 10 AHA genes have been identified, each encoding a distinct plasma membrane H+ -ATPase isoform. Studies using the GUS reporter gene fused to the promoter regions of specific AHA genes have revealed that isoform expression is tissue specific.
Plasma membrane ATPase pumps a single H+ out of the cell for each MgATP hydrolyzed.
During hydrolysis, the γ-phosphate of ATP becomes transiently bound to an aspartyl residue on the enzyme, forming an acyl-phosphate bond, hydrolysis of which provides the driving force for the pump reaction cycle. In this cycle different conformations of the enzyme (E1 and E2 states) expose the H+ -binding site to alternate sides of the membrane.
Ca2 + -ATPases, another group of P-type ATPases, are distributed among various plant membranes including plasma membrane, the ER, the chloroplast membrane, and vacuolar membranes. These enzymes pump Ca2+ out of the cytosol to decrease the cytosolic free Ca2+ concentration, which is essential in all cells to prevent precipitation of phosphates.
All Ca2 +-ATPases are P-type ion-motive ATPases. They vary considerably depending on the membrane type in which they occur. The plasma membrane (PM) type having a calmodulin binding domain is activated by calmodulin. The ER-type, on the other hand, contains no calmoduIin-binding domain.
In Arabidopsis, an ER-type Ca2 + -ATPase identified by molecular cloning has been localized immunologically to the ER. In cell membranes, Ca2+ -ATPase activity is enhanced by calmodulin. Such enzymes have been identified not only in the plant cell membrane but also in the inner chloroplast membrane and vacuolar membrane.
The magnitude of electrochemical potential against which Ca2 + must be pumped by these enzymes is very large. This is not only due to the concentration of free Ca2+ on one side of the membrane, but also due to the cytosol-negative membrane potential opposes the export of divalent cations.
The free energy of the Ca2+ electrochemical potential difference across the plasma membrane is about – 60 kJ.mol-1.
It exceeds the free energy input available from ATP hydrolysis (-50 kJ.mol-1). In some cases the enzyme has been found to catalyze Ca2 +/H+ exchange, which can offset the large opposing driving force from Ca2 +. Vacuolar and other membranes are energized through vacuolar H + -ATPases (V-type H + -ATPases).
These enzymes are distant relatives of F-type H+ -ATPases (found in inner mitochondrial and thylakoid membranes), but V-type ATPases operate solely in the direction of ATP hydrolysis. So, the pumps catalyze transport of ions or complex organic molecules against their thermodynamic gradients.
At membranes other than ATP-synthesizing membranes of mitochondria and chloroplasts, pumps are generally driven by ATP hydrolysis. At all membranes, H+ pumps dominate the transport characteristics, removing H+ from the cytosol and generating a p.m.f. across such membrane.
