Kinetic characterization of P-type membrane ATPase from Streptococcus mutans.

The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F(1)F(o)-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K(0.5)=0.27 mM) and low (K(0.5)=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg(2+) saturating condition (high affinity site, K(0.5)=0.10 mM, and low affinity site, K(0.5)=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.

Use of visible light-based photodynamic therapy to bacterial photoinactivation.

The main focus of this laboratory exercise was to investigate the photodynamic therapy (PDT) acting over Streptococcus mutans. A handheld photopolymerizer and a classical photosensitizer (Rose Bengal) were used to induce photodynamic response. In this way, a suspension of S. mutans was treated with different concentrations of Rose Bengal (0-10 µmol/liter), irradiated with a light (400-600 nm) for 20 s, and then cell viability was evaluated. It was observed that the light (per se) is not toxic, and in the dark, Rose Bengal is toxic only to the cells tested at concentrations above 5.0 µmol/liter. Under light exposure, concentrations of Rose Bengal above 0.5 µmol/liter killed all S. mutans. Therefore, for the purpose of our work, the photoactivation of Rose Bengal using the handheld photopolymerizer was efficient in bacteria inactivation.

A 100 kDa vanadate and lanzoprazole-sensitive ATPase from Streptococcus mutans membrane.

The cariogenic potential of Streptococcus mutans is due to the production of organic acids derived from energy metabolism, which implies the need of mechanisms for the organism to tolerate this acidic environment. The F(1)F(o)-ATPase is generally considered as the main enzyme responsible for cytoplasmic proton extrusion, but mutations that resulted in a 50% reduction in F(1)F(o)-ATPase activity in S. mutans still allowed the micro-organism to grow and extrude acid, keeping the intracellular pH one pH unit above the extracellular ambient. This finding suggests the existence of other enzymatic (or cellular) mechanisms that keep the cytosolic pH neutral during micro-organism growth. This paper describes a membrane protein in S. mutans, with a molecular weight of 100 kDa, which exhibits ATPase activity inhibited by classic inhibitors of P-type ATPases (orthovanadate) and H(+),K(+)-ATPase (lanzoprazole), has an optimum pH comparable to other H(+)-ATPases and undergoes phosphorylation during the catalytic reaction, like that of H(+)-ATPases described in yeast and plant plasma membrane. Together, these results strongly suggest that the enzyme we describe here is a P-type H(+)-ATPase or H(+),ion-ATPase that can act in association with F(1)F(o)-ATPase during the growth of the S. mutans.

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study.

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper, we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4h of incubation at 25 degrees C. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degrees C. Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate. The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle.