H₂S production by CBS is regulated by Ca²⁺/calmodulin. A, A comparison of a consensus sequence of calmodulin binding domain of CBS with other calmodulin binding proteins. CaM-KII, Calmodulin-dependent kinase II; CaM-KI, calmodulin-dependent kinase I; MARCKS, myristoylated alanine-rich protein kinase C substrate; HSP84, heat shock protein 84 kDa. B, Immunoprecipitation assay for calmodulin binding to CBS. Ten milligrams of brain homogenate were immunoprecipitated with an antibody against CBS and analyzed by Western blotting with antibodies against CBS and calmodulin. Actin in the lysate served as a control. C, H₂S production from purified CBS is regulated by Ca²⁺and calmodulin. Protein (4.6 μg) of CBS purified by calmodulin Sepharose 4B column was incubated with 1 mm cysteine and 2 mm pyridoxal 5′-phosphate in the presence or absence of 6 μm Ca²⁺ and/or 1 μmcalmodulin, and H₂S production was measured.RM, Reaction mixture alone; EF, enzyme fraction alone. *p < 0.01; **p< 0.001; Student's t test. D,E, Ca²⁺ (D) or calmodulin (E) concentration-dependent H₂S production from purified CBS. Protein (4.6 μg) of CBS purified by calmodulin Sepharose 4B was incubated with 1 mmcysteine and 2 mm pyridoxal 5′-phosphate in the presence or absence of 1 μm calmodulin (D) or 6 μm Ca²⁺ (E), and the production of H₂S with different concentrations of Ca²⁺ (D) or calmodulin (E) was measured. F, Inhibition of H₂S production by calmodulin inhibitors. Purified CBS (4.6 μg) was incubated with 1 mm cysteine and 2 mmpyridoxal 5′-phosphate in the presence of 1 μm calmodulin and 6 μm Ca²⁺, and the effects of trifluoroperazine (●) or W-13 (○) on H₂S production were examined. All data from C–F represent the mean ± SEM of five experiments.