Abstract
Rubratoxin B, a lactone-containing bisanhydride metabolite of certain toxigenic molds, inhibited (Na+-K+)-stimulated ATPase activity of mouse brain microsomes in a dose-dependent manner with an estimated IC50 of 6 x 10(-6) M. Hydrolysis of ATP was linear with time and enzyme concentration, with or without rubratoxin in reaction mixtures. Altered pH and activity curves for (Na+-K+)-ATPase demonstrated comparable inhibition by rubratoxin in buffered acidic, neutral, and alkaline pH ranges. Kinetic studies of cationic-substrate activation of (Na+-K+)-ATPase indicated classical competitive inhibition for Na+ and K+. Results also showed competitive inhibition for K+ activated p-nitrophenyl phosphatase as demonstrated by altered binding site parameters without change in the catalytic velocity of dephosphorylation of the enzyme . phosphoryl complex. Noncompetitive inhibition with regards to activation by ATP and p-nitrophenyl phosphate was indicated by altered Vmax values with no change in Km values. Inhibition was partially restored by repeated washings. Preincubation with sulfhydryl agents protected the enzyme from inhibition. Cumulative inhibition studies with rubratoxin and ouabain indicated possible interaction between the two inhibitors of (Na+-K+)-ATPase. Rubratoxin appeared to exert its effects on (Na+-K+)-ATPase by interacting at Na+ and K+ sites.
Highlights
Medical Center, Rubratoxin B, a la&one-containing bisanhydride metabolite of certain toxigenic molds, inhibited (Na+-K+)-stimulated ATPase activity of mouse brain microsomes in a dosedependent manner with an estimated I& of 6 x lo-” M
Kinetic Analysis-Analysis of the effect of rubratoxin B on various substrate activation parameters of the brain microsomal ATPase preparation was undertaken to determine the nature of inhibition
Our results indicate that rubratoxin B is a potent inhibitor of membrane ATPase because this toxin significantly inactivated brain microsomal (Na+-K+)-stimulated adenosine triphosphatase at low concentrations (I&, 6.0 x lo+ M)
Summary
Rubratoxin B was isolated from P. rubrum according to the procedure described by Hayes and Wilson [17]. Mg of protein/h CAP,); the remaining 20 to 30% of the activity was the basal Mgy+ component (mostly oligomycin-insensitive) determined after the addition of 1 /rl of oligomycin (5 x lo-” M) in ethanol [20,21,22]. Determination ofATPase Activity- The microsomal ATPase activity of the mouse brain preparation was measured using endpoint phosphate analyses [23]. Analysis of p-Nitrophenyl Phosphatase Activity- K+-stimulated phosphatase activity of brain microsomal enzyme preparation was measured using methods described by Ahmed and Judah [25] and Albers and Koval [26]. Incubation time was 15 min after which trichloroacetic acid was added at a final concentration of 5% (w/v) to stop the reaction
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
