view article

Figure 3
In vitro validation of the inhibitory mechanism. (a) Activity (%) of astacin (at 1 nM concentration), meprin β (at 1 nM concentration) and ovastacin (at 5.6 nM) in the presence of fetuin-B variants at 300 nM (CPDC and CPRC) or 12.5 nM (other variants). Full astacin, meprin β and ovastacin activities (100%) correspond to substrate-turnover rates of 35.4 ± 5.7, 7.7 ± 0.2 and 10.7 ± 0.8 nM s−1, respectively; w/o, without fetuin; hFB, human fetuin-B; bFB, bovine fetuin-B; mFB, murine fetuin-B; mFBCY2, CY2 domain of mFB; mFBCTR, CTR domain of mFB; mFBC39S,C374S, double point mutant of murine fetuin-B; CPDC and CPRC, cyclic peptides comprising the respective sequences. Experiments were performed in triplicate; error bars indicate standard deviations. (b) Plot of fractional velocity (logarithmic scale) with different fetuin-B variants: mFB, murine fetuin-B; mFBABA, variant with CY1 and CTR from mouse fetuin-A and CY2 from mFB; mFBH1-swap, mutant replacing CY2 hairpin I with CY1 hairpin I; mFBP155A, mFBD156A, mFBP155A,D156A and mFBC154S,C157S are point mutants of murine fetuin-B. Enzyme and substrate concentrations were 1.0 nM and 195 µM for astacin, 0.5 nM and 26 µM for meprin β and 0.7 nM and 26 µM for ovastacin, respectively. Error bars indicate standard deviations. (c) Ki values determined from (b) using Morrison's equation with the respective standard errors (SE).

Volume 6| Part 2| March 2019| Pages 317-330
ISSN: 2052-2525