G818EER51 ± 1363 ± 5111 ± 825 ± 312 ± 32Moderate/Severe
When expressed in transfected HEK293T cells and quantified by Western blotting, this variant was in the range of 40%-52%. This variant has a basal ATPase activity of 63% ± 5% compared to WT (100%) and was categorized as class 2 (partial reduction in expression and basal ATPase activity that was modestly stimulated by N-Ret-PE) with a moderate/severe predicted severity.
Analysis of the rates of hydrolysis of ATP and CTP in the mutant protein demonstrated that they were significantly reduced (Fig. 3). The results presented in Fig. 3A indicated that the ATPase function of G863A mutant protein was reduced ∼3-fold as compared with NBD1wt, indicating ∼70% of inhibition of the ATPase activity. TheVmax (ATPase) for G863A mutant was 128 pmol/min/mg and that of the wild-type NBD1 was 584 pmol/min/mg (Table II). A time-course analysis of ATP hydrolysis using 2.5 μg of protein (Fig. 3C) suggested the actual rates of ATP hydrolysis were attenuated 3-fold as a consequence of the mutation. We have earlier reported that the NBD1wt has significantly higher CTPase than ATPase activity (1717.Biswas, E.E.Biochemistry. 2001; 40:8181-8187CrossrefScopus (25)PubMedGoogle Scholar). However, in the mutant G863A protein the CTPase activity was reduced (Fig.3B). In this case, the CTP hydrolysis of G863A was reduced to ∼30% of the activity of NDB1wt. The Vmaxfor G863A mutant was 104 pmol/min/mg and that of the wild-type NBD1 was 376 pmol/min/mg (Table II).
ATPase function of G863A mutant protein was reduced ∼3-fold as compared with NBD1wt, indicating ∼70% of inhibition of the ATPase activity (Fig. 3). TheVmax (ATPase) for G863A mutant was 128 pmol/min/mg and that of the wild-type NBD1 was 584 pmol/min/mg (Table II).