Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics

Figures & data

Table 1. Purification scheme of G6PD, 6PGD and GR from rat lung.

Enzymes	Purification step	Total volume (ml)	Activity (U/ml)	Total activity (U)	Protein (mg/ml)	Specific activity (EÜ/mg)	Yield %	Purification fold
G6PG	Homogenate	16	0.92	14.75	2.95	0.31	100	1
	Affinity chromatography	6	2.36	14.15	0.013	181.38	96	580
6PGD	Homogenate	16	0.50	8.06	2.95	0.171	100	1
	Affinity chromatography	4.5	1.61	7.23	0.012	133.976	90	784
GR	Homogenate	16	0.110	1.758	2.96	0.04	100	1
	Affinity chromatography	4.5	0.370	1.664	0.0106	34.88	95	940

Table 2. Optimum conditions for G6PD, 6PGD and GR the rat lung.

Enzymes	Optimal pH	Optimum ionic strength (mM)	Optimal temperature (°C)	Stable pH
G6PD	8.0	160	55	6.0
6PGD	8.0	5–40	40	8.5
GR	8.5	80	65	7.0

Figure 1. Purification of rat lung G6PD, 6PGD and GR enzymes by 2′, 5′-ADP sepharose 4B affinity column.

Figure 2. SDS-PAGE bands of enzymes (Lane 1: rat lung 6PGD, Lane 2: GR rat lung G6PD; Lane 3: rat lung, Lane 4: Standard proteins: bovine carbonic anhydrase (30 kDa), chicken ovalbumin (45 kDa) and bovine albumin (66 kDa), rabbit phosphorylase B (97 400) and E. coli P-galactosidase (116 000) and rabbit myosin (205 000)).

Figure 3. Graphs drawn to calculate the K_i and IC₅₀ values of levofloxacin on the rat lung G6PD, 6PGD and GR.

Table 3. IC₅₀ values, K_i constants and inhibition type obtained from regression analysis graphs for rat lung G6PD, 6PGD and GR in the presence of different drugs (NC: noncompetitive; C: competitive).

G6PD				6PGD			GR
Inhibitor	IC50 (mM)	Ki (mM)	Inhibition type	IC50 (mM)	Ki (mM)	Inhibition type	IC50 (mM)	Ki (mM)	Inhibition type
Levofloxacin	0.07	0.07 ± 0.03	NC	0.07	0.08 ± 0.03	NC	0.02	0.03 ± 0.01	NC
Furosemide	0.13	0.17 ± 0.05	NC	0.14	0.17 ± 0.05	NC	0.08	0.08 ± 0.03	NC
Ceftazidime	1.90	2.03 ± 1.18	NC	1.93	1.79 ± 0.91	NC	0.68	0.91 ± 0.42	NC
Cefuroxime	8.15	7.21 ± 3.63	NC	9.24	15.64 ± 7.48	NC	1.56	1.57 ± 0.31	NC
Gentamicin	17.34	22.06 ± 3.38	C	5.77	7.03 ± 1.74	C	30.13	43.37 ± 17.91	NC

Table 4. In vivo effects of levofloxacin and furosemide on rat lung G6PD, 6PGD and GR activities.

Enzymes	Control X±SD (n = 6)	Drugs	X±SD (n = 6)	p
Lung G6PD	0.500 ± 0.031	Furosemide	0.584 ± 0.023	0.000
		Levofloxacin	0.510 ± 0.039	0.591
Lung 6PGD	0.650 ± 0.038	Furosemide	0.617 ± 0.021	0.098
		Levofloxacin	0.603 ± 0.035	0.025
Lung GR	0.309 ± 0.014	Furosemide	0.232 ± 0.120	0.000
		Levofloxacin	0.305 ± 0.018	0.605