GCK mutations detected in MODY2 children from South Italy.

a<p>GenBank: accession number (AH005826). ^bThe reference cDNA sequence was obtained from GenBank (NM_000162) and +1 corresponds to the A of the ATG translation initiation codon. ^cPolyphen prediction: probably damaging (1), benign (2), possibly damaging (3). SIFT score: <0.05 deleterious variant, ≥0.05 tolerated variant. ^dSwissprot accession number: P35557. ^eSibling pairs (MD19/20: two sisters; MD69/70: brother/sister).</p

Kinetic constants of human recombinant wild type-GCK and mutant β-cell GST-GCK fusion proteins.

<p>Data represent means ± SEM of 3 separate enzyme expressions each tested in duplicate. Note that the Hill coefficient (nH) and the relative activity index (I_a) are unit less. Kcat: GCK catalytic constant; S_0.5: affinity constant for glucose; nH: Hill coefficient; Km for ATP: affinity constant for ATP; I_a: GCK activity index. (*)<i>p</i><0.05, <i>t</i> test; (**)<i>p</i><0.005, <i>t</i> test.</p

Effect of temperature on the stability of GST-GCK mutants

<p>. Stock enzyme solutions were diluted to 250 µg/ml in storage buffer containing 30% glycerol, 50 mM glucose, 10 mM glutathione, 5 mM DTT, 200 mM KCl and 50 mMTris/HCl, pH 8.0. Panel A: The enzyme solutions were incubated for 30 min at temperatures ranging from 30 to 55°C and then assayed at 30°C as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038906#s4" target="_blank">Methods</a> section. Panel B: The enzyme solutions were incubated for periods of time from 5 to 60 min at 50°C. Results are means and SEM of three independent enzyme preparations for each mutant except for GST-GCK (Phe150Tyr) which corresponds to two independent enzyme preparations. (*) <i>p</i>≤0.03, (†) <i>p</i><0.008.</p