Regulation of plasma membrane Ca(2+)-ATPase activity by acetylated tubulin: influence of the lipid environment.

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300µg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50µg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300µg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50µg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.

Brain plasma membrane Na+,K+-ATPase is inhibited by acetylated tubulin.

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the alpha-subunit of Na+,K+-ATPase. In the present work we show that the interaction of tubulin with Na+K+-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na+K+-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.

Na+,K+-ATPase was found to be the membrane component responsible for the hydrophobic behavior of the brain membrane tubulin.

We have previously described that the tubulin isolated from brain membranes as a hydrophobic compound by partitioning into Triton X-114 is a peripheral membrane protein [corrected]. The hydrophobic behavior of this tubulin is due to its interaction with membrane protein(s) and the interaction occurs principally with the acetylated tubulin isotype. In the present work we identified the membrane protein that interacts with tubulin as the Na+,K+-ATPase alpha subunit by amino acid sequencing. Using purified brain Na+,K+-ATPase we were able to isolate part of the total hydrophilic tubulin as a hydrophobic compound which contains a high proportion of the acetylated tubulin isotype.

Carnitine resembles choline in the induction of cholinesterase, acid phosphatase, and phospholipase C and in its action as an osmoprotectant in Pseudomonas aeruginosa.

The present study demonstrates that under conditions of iso or hyperosmolarity, P. aeruginosa utilized carnitine as the carbon, nitrogen or carbon and nitrogen sources. As occurred in the case of choline, the bacteria synthesized cholinesterase (ChE), acid phosphatase (Ac.Pase) and phospholipase C (PLC) under any of these conditions and in the presence of high or low Pi concentrations. Carnitine acted as an osmoprotectant when the cells were grown in the presence of preferred carbon and nitrogen sources and high NaCl concentrations. Under these conditions the three enzyme activities were not produced. The osmotically stressed bacteria grown under any of the above conditions accumulated betaine. Its presence indicated that carnitine may be metabolized by P. aeruginosa to produce betaine which could account for the induction of the three enzyme activities or its action as an osmoprotectant. The phosphatidylcholine encountered in the host cell membranes allows the bacteria to obtain free choline by the coordinated action of PLC and Ac.Pase. Since the consequence of this action may be cell disruption, the increase of free carnitine in the natural environment of the bacteria is also possible. These two compounds, choline and carnitine, acting in conjunction or separately, may increase the production of PLC and Ac.Pase activities by P. aeruginosa and thus enhance the degradative effect upon the host cells.