5.2.1 PR E P A R AT I ON O F CE LL E XT R A CT S
Cell extracts were prepared from Pseudomonas sp. strains Chol1, G12, and R1 (irrespective of substrates used for growth) as well as strains 1 and 9 as described earlier (Birkenmaier et al., 2007). For some experiments, cell extracts were prepared from strains Chol1, R1 and G12, grown with cholate + succinate (and kanamycin), by harvesting the cells from the mid-exponential phase and transferring them to cholate for 1 hr to induce the set of enzymes responsible for cholate oxidation. For preparation of cell extracts from Dietzia sp. strain 2, cells were harvested from early-, mid- or late-exponential phase by centrifugation (5,900 x g for 10 min at 4°C), washed twice with ice-cold potassium-phosphate buffer (50 mM, pH 7.1), and resuspended in a small volume of the same buffer. Cells in this cell suspension were finally broken by fifteen to twenty passages through a pre-chilled French press cell (Aminco) at 138 MPa. Homogenates were centrifuged at 20,000 x g for 20 min at 4°C to separate cell extracts from cell debris. Cell extracts were further separated into soluble (cytosolic proteins) and particulate fractions (membrane-bound proteins) by ultracentrifugation at 45,000 rpm for 60 min at 4°C.
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The membrane fraction was washed with the aforementioned buffer and submitted to a further ultracentrifugation. All cell extracts were immediately used for enzyme assays or stored at -20°C.
5.2.2 EN Z Y M E A S S AY S
5.2.2.1 DE T E CT I O N O F EN Z YM E A C TI V IT IE S IN T H E C EL L E XT R A CT S OF
PS E U D OM O NA S S P. ST R AI N CH O L1, R1 A N D G12
All enzyme assays were carried out at 30°C and 1 unit of enzyme activity is defined as the amount of enzyme which catalyzes the conversion of 1 µmol of substrate per minute. The activities of the enzymes of tri-carboxylic acid cycle (TCA) were measured as described (Brune and Schink, 1990) with minor modifications. Except citrate synthase, the activities of all the other enzymes of CAC were also measured in the presence of propionyl-CoA (100 µM) in the assay.
a) Aconitase (EC 4.2.1.3) was measured by monitoring the citrate-dependent formation of NADPH from NADP+ at 365 nm using the extinction coefficient 3.4 mM-1cm-1. This activity was determined by coupling citrate conversion to isocitrate with the isocitrate dehydrogenase reaction. The reaction mixture (1 ml) contained Tris-HCl (100 mM, pH 8.0), MgCl2 (10 mM), NADP+ (2.5 mM) and cell extract (0.9 - 1.0 mg proteins), and the reaction was started by the addition of sodium citrate (20 mM).
b) Citrate synthase (EC 2.3.3.1) activity was measured by monitoring the oxaloacetate-dependent formation of thio-nitrobenzoate (TNB2-) at 412 nm using the extinction coefficient 13.6 mM-1cm-1 (Munoz-Elias et al., 2006). In this assay, the free CoA released from acetyl-CoA upon addition of oxaloacetate reacted with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) and led to the formation of TNB-.
The reaction mixture (1 ml) contained HEPES-NaOH (50 mM, pH 8.0), DTNB (1 mM), acetyl-CoA (0.1 mM), and cell extract (0.5 - 0.6 mg proteins), and the reaction was started by addition of oxaloacetate (2 mM) The background hydrolysis of CoA-esters and thioesterase activity was also recorded for ~ 2 min before the addition of oxaloacetate and subtracted later.
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c) Isocitrate dehydrogenase (EC 1.1.1.42) was measured by monitoring the d-isocitrate-dependent formation of NADPH from NADP+ at 365 using the extinction coefficient 3.4 mM-1cm-1. The reaction mixture (1 ml) contained Tris-HCl (100 mM, pH 8.0), MgCl2 (10 mM), NADP+ (2.5 mM), and cell extract (0.5 - 0.6 mg proteins) and the reaction was started by the addition of d-isocitrate (1 mM).
d) Isocitrate lyase (EC 4.1.3.1) was measured by a continuous (Giachetti et al., 1984) and a discontinuous assay (Serrano et al., 1998) as described earlier. The activity of isocitrate lyase was also measured in presence of propionyl-CoA (100 µM).
In this assay, the isocitrate-dependent formation of glyoxylate phenylhydrazone was monitored spectrophotometrically at 325 nm using the extinction coefficient 17 mM-1cm-1. Isocitrate was cleaved into glyoxylate and succinate, the resulting glyoxylate reacted with phenylhydrazine hydrochloride and led to the formation of glyoxylate phenylhydrazone. The reaction mixture (1 ml) contained HEPES (50 mM, pH 7.0), MgCl2 (2.5 mM), phenylhydrazine hydrochloride (5 mM), and cell extract (0.4 - 0.5 mg proteins), and the reaction was started upon addition of d-isocitrate (5 mM).
In the coupled-assay, isocitrate lyase activity was measured by following the lactate dehydrogenase (LDH)-mediated reduction of glyoxylate to glycolate with concomitant oxidation of NADH. The d-isocitrate-dependent oxidation of NADH was measured at 340 nm using the extinction coefficient 6.2 mM-1cm-1. The reaction mixture (1 ml) contained HEPES (50 mM, pH 7.0), MgCl2 (2.5 mM), NADH (0.3 mM), LDH (1 U) and cell extract (0.4 - 0.5 mg proteins), and the reaction was started upon addition of d-isocitrate (5 mM).
e) α-Ketoglutarate dehydrogenase (EC 1.2.4.2) was measured by monitoring the α-keto glutarate-dependent formation of NADH from NAD+ at 365 nm using the extinction coefficient 3.4 mM-1cm-1. The reaction mixture (1 ml) contained Tris-HCl (100 mM, pH 8.5), Li3-CoA (0.5 mM), NAD+ (4 mM) and cell extract (0.4 - 0.5 mg proteins), and the reaction was started upon addition of α-keto glutarate (10 mM).
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f) Malate dehydrogenase (EC 1.1.1.82) was measured by monitoring the malate-dependent formation of NADPH at 365 nm using the extinction coefficient 3.4 mM-1cm-1. The reaction mixture (1 ml) contained Tris-HCl (100 mM, pH 8.3), MgCl2 (2.5 mM), NADP+ (1 mM), and cell extract (0.5 - 0.6 mg proteins), and the reaction was started upon addition of malate (5 mM).
g) 2-Methylcitrate synthase (EC 2.3.3.5) activity was measured by monitoring the oxaloacetate-dependent formation of TNB2- at 412 nm using the extinction coefficient 13.6 mM-1cm-1. In this assay, the free CoA released from propionyl-CoA upon addition of oxaloacetate reacted with DTNB and led to the formation of TNB2-. Except using propionyl-CoA, the reaction conditions and principles were similar to the reaction of citrate synthase (see above). Propionyl-CoA was used instead of acetyl-CoA in the reaction mixture.
h) Pyruvate carboxylase (EC 6.4.1.1) was measured by following malate dehydrogenase (MDH)-mediated reduction of oxaloacetate to malate with concomitant oxidation of NADH. Initially, the carboxylation of pyruvate led to the formation of oxaloacetate.
The pyruvate-dependent formation of NAD+ was monitored at 340 nm with extinction coefficient 6.2 mM-1cm-1 as described earlier (Lai et al., 2006) with minor modifications.
Membrane-free cytosolic fraction was used to monitor the activity of PYC. The reaction mixture (1 ml) contained Tris-HCl (50 mM, pH 7.8), MgCl2 (5 mM), NaHCO3 (50 mM), ATP (2 mM), NADH (0.3 mM), MDH (1 U), and cell extract (0.4 - 0.5 mg proteins), and the reaction was started upon addition of sodium pyruvate (5 mM).
i) Pyruvate dehydrogenase (EC 1.2.4.1) was measured by the pyruvate-dependent formation of NADH at 365 nm using the extinction coefficient 3.4 mM-1cm-1 as described earlier (Jeyaseelan et al., 1980) with minor modifications. The reaction mixture (1 ml) contained Tris-HCl (50 mM, pH 7.8), MgCl2 (2 mM), dithiothreitol (2.5 mM), thiamine-pyrophosphate (0.4 mM), Li3-CoA (0.1 mM), NAD+ (5 mM) and cell extract (0.4 - 0.5 mg proteins), and the reaction was started upon addition of sodium pyruvate (5 mM).
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j) Succinate dehydrogenase (EC 1.3.99.1) was measured by monitoring the succinate-dependent reduction of dichlorophenolindophenol (DCPIP), an artificial electron acceptor at 522 nm using the extinction coefficient 8.6 mM-1cm-1. The reaction mixture (1 ml) contained Tris-HCl (100 mM, pH 7.6), DCPIP (0.1 mM), and cell extract (0.2 - 0.3 mg proteins), and the reaction was started upon addition of succinate (10 mM).
5.2.3 ME A S UR I N G T H E AC TI V IT IE S O F EN Z YM E S O X I D IZ I NG C H O LA T E I N S T R A I N S 1, 2 AN D 9
Cell extracts from strain 1 and 9 were prepared and used to measure the activity of 3α-hydroxy steroid dehydrogenase (3-Hsd) and 3-keto steroid dehydrogenase (3-Ksdh), enzymes responsible for oxidation of cholate as described earlier (Birkenmaier et al., 2007). Cell extract from Dietzia sp. strain 2 was used to measure the activity of enzymes responsible for oxidation of cholate as described earlier (Birkenmaier et al., 2007) with minor and major modifications (different buffers at wide range of pH).
5.2.4 ME A S UR I N G T H E AC TI V IT IE S O F EN Z YM E A C T IV A TI N G C H OL A TE IN S T R A I N 2
The activity of cholyl-CoA ligase was measured in the cell extracts of Dietzia sp. strain 2 as described earlier (Birkenmaier et al., 2007). The reaction mixture (0.5 ml) contained MOPS (50 mM, pH 7.8), Li3-CoA (1 mM), ATP (1 mM), MgCl2 (2.5 mM) and cell extract (0.25 to 0.30 mg proteins) from Dietzia sp. strain 2, and the reaction was started upon addition of cholate (1 mM). Samples were withdrawn immediately after the addition of cholate (time 0) and at regular intervals thereafter, acidified by adding 10 µl HCl (1 M) to cease the reaction, centrifuged (5900 x g for 5 min at room temperature) and stored at -20°C until HPLC analysis.
5.2.5 PR O T E I N Q UA N TI FI C AT I O N
Protein was quantified by using bichinonic acid (BCA) protein assay kit (Pierce, USA) with bovine serum albumin as standard.
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5.3 ANAL YT ICAL ME T H O DS