Ex vivo studies of functional consequences of the causative mutations

3.4.1 Quantification of glucokinase enzyme activity

Since strategies for direct measurement of glucokinase enzyme have not yet been developed, an indirect fluorometric coupled assay was performed to quantify glucokinase activity in liver homogenate of 90-day-old GLS001 and GLS006 mice. Based on the phosphorylation of glucose by glucokinase enzyme to glucose-6- phosphate (G6P), which is in turn again metabolised by glucose-6-phosphate

PCR Master Mix Cycling conditions

Aqua bidest 3.65 µl Q-solution 4.00 µl 10x buffer 2.00 µl MgCl2 1.25 µl dNTPs (1 mM) 1.00 µl Primer 1 sense (2 µM) 0.50 µl Primer 1 antisense (2 µM) 0.50 µl Primer 2 sense (2 µM) 3.00 µl Primer 2 antisense (2 µM) 3.00 µl Taq Polymerase 0.10 µl 1. 5 min 94°C 2. 50 sec 94°C 3. 50 sec 57°C 4. 80 sec 72°C 5. 10 min 72°C 6. ∞ 4°C 36 cycles

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dehydrogenase (coupled to the reduction of nicotinamide adenine dinucleotidephosphate (NADP+)) into 6-phophogluconate (Fig. 3.4), glucokinase activity was evaluated by spectrophotometrical quantification of the turnover rate of NADP+ _{to NADPH in the presence of different glucose concentrations and excess}

glucose-6-phosphate dehydrogenase (G6PD) (Grupe et al. 1995; Hara et al. 1986; Trus et al. 1981).

Figure 3.4: Schematic diagram of the first step of glycolysis, catalysed by glucokinase enzyme and the following downstream reactions, enabling indirect quantification of glucokinase activity.

Complicating assay specifications represent the coincident presence of other hexokinases, catalysing the identical reaction and impeding therefore separate quantification of glucokinase activity. However, the high affinity of hexokinases for glucose leads to their saturation already at low glucose concentrations (Bell et al. 1996). The kinetic property of hexokinases can be availed to selectively quantify glucokinase activity by assay performance in the presence of either low glucose concentrations (0.03-0.5 mM; to quantify hexokinase phosphorylation activity) or by applying glucose concentrations in a range of 6-100 mM, where hexokinases are predicted to minor contribute to glucose phosphorylation activity of the low affinity glucokinase enzyme. In order to exclude hexokinase contribution, the maximal reaction velocity (Vmax) for hexokinases, calculated by means of the particular

reaction velocities in presence of the respective minor glucose concentrations, was subtracted from glucose phosphorylation velocities, determined by employing high glucose concentrations. The assay was performed according to a modified protocol adapted from Liang et al. (Liang et al. 1990).

51 3.4.1.1 Tissue sample preparation

Ad libitum fed mice were sacrificed under general anaesthesia (ketamine/xylacine mixture; 3.2.2) by cervical dislocation and liver samples of approximately 20 mg weight (weighed on a Mettler AE200 Electronic Analytical Balance, Mettler-Toledo Intl. Inc., Germany) were obtained. Tissue samples were immediately transferred into 2 ml reaction cups, containing 1 ml ice-cold 1x Homogenisation Buffer, and were homogenised for 1 minute with a Polytron PT 1200 E tissue homogeniser (Kinematica AG, Switzerland) at the highest level (25,000 rpm). Homogenates were subsequently sonicated by 7 strokes <1s each (Branson sonifier cell disruptor B15, Branson Ultrasonic Corporation, USA). Thereafter, tissue sonicates were centrifuged (75 minutes, 15,300 rpm at 4°C) and kept on ice until further assayed.

10x Homogenisation Buffer

KCl (Roth, Germany) 820 mg

K2HPO4 (Merck, Germany) 348 mg

DTT (Roth, Germany) 77 mg

EDTA (Sigma, Germany) 29 mg

Ad 10 ml aqua bidest.; stored at 4°C and diluted 1:10 to prepare ready-to-use 1x Homogenisation Buffer

3.4.1.2 Work flow of glucokinase activity determination

Glucose phosphorylation by glucokinase was initiated by addition of 500 µl Reaction Master Mix to 20 µl liver homogenate. The reaction mixture was incubated at 30°C for 35 minutes. A total number of 11 glucose concentrations were applied, covering either the low glucose range (0.5, 0,25, 0.125, 0.06, and 0.03 mM) or the high glucose spectrum (100, 50, 25, 12, and 6 mM). Each reaction series included a reaction blank with H2O instead of glucose. The reaction was finally stopped by pH

alteration via addition of 480 µl 1M NaHCO3 pH 9.4 and sample mixtures were

transferred into disposable semi-micro PMMA cuvettes (Neolab, Deutschland). Relative absorbance of each reaction was measured at a wavelength of 340 nm after reaction blank calibration, using a SPEKOL 1500 UV/vis spectrophotometer (Analytic Jena AG, Germany).

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Reaction Buffer (prepared freshly)

KCl (Roth, Germany) 298 mg

β-mercaptoethanol (Sigma, Germany) 40.3 µl

MgCl2 (Merck, Germany) 28 mg

BSA (Sigma, Germany) 20 mg

NADP+ (AppliChem, Germany) 15 mg

ATP (Sigma, Germany) 110 mg

Ad 20 ml 100mM Hepes pH 7.8; kept on ice until assayed

Glucose stock solutions

High glucose: 250 mM

α-D-glucose (Sigma, Germany) 0.45 g

Ad 10 ml aqua bidest.; stored at 4°C (short term) or at -20°C (long term) Low glucose: 1.25 mM

250 mM stock solution 250 µl

Ad 500 ml aqua bidest.

Ready-to-use glucose solutions were prepared by serial 1:2 dilution of the corresponding stock solutions with aqua bidest. (exception: 12 and 0.06 mM glucose solutions were prepared by 48:100 dilutions of 25 and 0.125 mM glucose solutions, respectively). All glucose containing reagents were stored at 4°C (short term) or at -20°C (long term).

Glucose-6-phosphate dehydrogenase (G6PD) solution (prepared freshly)

G6PD (1000 U/ml) (Roche, Germany) 20 µl

¾ Centrifuge at 10,000 rpm for 45 minutes, 4°C

¾ Discard the supernatant, resuspend with 250 µl ice-cold aqua bidest. and mix gently by pipetting

¾ Dilute 1:10 with ice-cold aqua bidest. (200 µl resuspended G6P + 1800 µl aqua bidest.). Assay dilution contains 8 U/ ml G6PD and is kept on ice until assayed

Reaction Master Mix

Reaction buffer 500 µl

High/Low glucose solution/aqua bidest. 400 µl

G6PD solution 100 µl

Immediately applied for assay complementation

The reaction velocities per time unit (minutes) in the presence of distinct glucose concentrations were calculated according to the Beer-Lambert law, assuming a coat thickness of 1 mm for the applied cuvettes and a molar decadic extinction coefficient (ε) for NADPH of 6.2 L×mmol-1. The reaction velocities V for glucokinase were obtained by subtracting the maximal velocity (Vmax) for hexokinase from the reaction

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velocities, observed at the 5 high glucose concentrations. As the high affinity hexokinases follow Michaelis Menten kinetics, the Vmax and Michaelis constant (Km)

for hexokinases were obtained after plotting reaction velocities at low glucose concentrations vs. the respective substrate concentrations. Using a non-linear regression curve fit tool, Km and Vmax were calculated according to the Michaelis-

Menten equation (GraphPad Prism 3.0 (GraphPad Software, USA)):

V

× [S]

V =