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

Abstract

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2′, 5′-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134 kDa for G6PD, 107 kDa for 6PGD and 121 kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, K_M and V_max values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC₅₀ values in the range of 0.07–30.13 mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.

• Drug inhibition
• glucose 6-phosphate dehydrogenase (G6PD)
• glutathione reductase (GR)
• lung
• 6-phosphogluconate dehydrogenase (6PGD)
• protein purification and characterization
• rats (Sprague–Dawley)

Introduction

Both glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G6PD) and 6-phospho gluconate dehydrogenase (EC 1.1.1.44; 6PGD) are two main enzymes in the hexose monophosphate shunt, where ribose 5-phosphate (R5P) and NADPH are generated. R5P is required for the synthesis of nucleotides. NADPH has very important functions in the prevention of proteins, lipids, DNA, RNA and other molecules from oxidative damage in all cells1,2. NADPH also serves several vital functions including lipid synthesis, fatty acid chain elongation, some amino acid syntheses and cholesterol synthesis3.

Glutathione reductase is known as a substrate specific antioxidant enzyme (E.C.1.8.1.7; GR) that catalyzes the conversion of glutathione from oxide form to reduced form, an important cellular antioxidant, the presence of NADPH4. GSH is necessary in the decomposition of the hydrogen peroxide and other organic peroxides, the resistance of oxidative cases, bioremediation of xenobiotics, and defending of the disulfide bonds of cellular proteins5. Moreover, it is reported that GSH has a major role in the arrangement of apoptosis and is essential for the development of cells6.

G6PD, 6PGD and GR are housekeeping enzymes, present in the microorganisms, blood cells, plant and animal tissues7. They have been purified from several mammalian tissues with various techniques such as rat liver and kidney8–10, human erythrocyte11,12, rat small intestine13, grass carp hepatopancreases14, rainbow trout liver15 and lamb kidney16, and some of their characteristics and kinetic properties have been determined.

It is well known that many pharmaceutical agents have many adverse effects on the enzyme activities in vitro and in vivo17,18. These drugs may lead to important problems beyond its anticipated therapeutic effects during clinical use19,20. Inhibition of these enzymes will cause to decrease the amount of NADPH and GSH which are required in several biological functions including cleaning-up of oxidative stress products and xenobiotic detoxification. Accordingly, these shortages cause some problems, such as extended period disease, affect the quality of life of patients, increase costs of patient care, the triggering of other diseases and the like19,21,22.

There is no article available on the biochemical characterization and purification of three enzymes from rat lung, and on the effects of five drugs on the enzyme activities. This study was aimed to purify and characterize rat lung G6PD, 6PGD and GR, and to investigate effects of some antibiotics on the enzyme activities in laboratory conditions. In addition, effects of levofloxacin and furosemide on the activities of the enzymes were investigated in rat lung as in vivo.

Materials and methods

Materials

The protein standards and chemicals were obtained from Sigma Chem. (Munich, Germany) and Merck (Darmstadt, Germany). Affinity material was purchased from Pharmacia (Pittsburgh, PA). Blue Dextran 2000 were supplied from Sigma Chem. (St. Louis, MO).

Preparation of crude extract

Animals were sacrificed after anesthesia. Lungs removed were washed with a cold isotonic saline solution and then, preserved at −80 °C. Tissue was cut into small pieces and powdered by liquid nitrogen. The sample was transferred to the 1 mM EDTA + 2 mM DDT + 20 mM Tris–HCl (pH = 7.5) solution and centrifuged at 4 °C, 12 000 rpm for 20 min. Next experiments were executed with the clear supernatant.

Purification of the enzymes

2′, 5′-ADP Sepharose 4B was prepared according to the method previously described for affinity column23. The supernatant was applied onto the column with 10 ml column material and the column was washed with 50 mM phosphate buffer (1 mM DTT, 1 mM EDTA, pH 7.35) until the final absorbance difference became zero at 280 nm. Firstly, G6PD was eluted with 80 mM phosphate + 80 mM KCl + 0.5 mM NADP + 1 mM EDTA, pH 7.85. Secondly, 6PGD was eluted with a buffer of 80 mM KCl + 5 mM NADP + 1 mM EDTA + 80 mM phosphate, pH 7.85. At last, the solution (0.5 mM NADPH + 1 mM GSH + 1 mM EDTA + 50 mM phosphate, pH 7.35) was used to remove the GR from the column. For each enzyme, fractions with activity have been pooled. Then, the eluates were dialyzed against 20 mM cold phosphate buffer, pH 7.3 at +4 °C for 24 h.

Enzyme assay

The measurements of enzyme activity were done according to Carlberg and Mannervik’s methods24 for GR and Beutler’s method for G6PD and 6PGD25 with a Shimadzu Spectrophotometer UV-(1208) (Tokyo, Japan). All activity measurements were carried out at the saturated concentration of substrates and coenzymes in optimal pH and ionic strength determined experimentally.

Protein determination

The amount of protein in each step was detected with the method stated by Bradford at 595 nm26 and bovine serum albumin was used to draw a standard graph.

Electrophoresis

In order to determine the subunit molecular weights and control the enzyme purity, SDS-polyacrylamide gel electrophoresis was made based on the method described by Laemmli27, and 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) were used as markers.

Gel filtration

Sephadex G-150 was used to determine the native molecular mass of enzymes. The column was prepared in the 40 ml bed volume and equilibrated with 50 mM Tris–HCl (pH 7.5) + 100 mM KCl buffer. Standard graph was drawn using the molecular mass standards (12.4–200 kDa) whose elution volumes were detected by measuring the protein absorbance at 280 nm and the void volume of column was calculated with Blue Dextran 2000. The elution volume of each enzyme was identified by surveying the enzyme activity.

Biochemical characteristics of enzymes

To determine the optimum conditions for the enzymes, the activities of enzymes were tested in 0.05 M H₃PO₄–H₃BO₃ buffer within the pH range of 5.5–9.5 for optimum pH, in the range from 20 mM to 1 M Tris–HCl buffer for optimum ionic strength, between 0 °C and 90 °C for optimum temperatures and in 0.2 M Tris–HCl buffer in pH of 8.0, 8.5 and 9.0, and in 0.2 M phosphate buffer in pH of 5.5, 6.0, 6.5, 7.0 and 7.5 for stable pH at the equal units of enzyme and buffer solutions.

Lineweaver–Burk graphs were used to determine both K_M and V_max values of substrates/coenzymes and the K_i constants and types of inhibition for product inhibition28. The activities of the enzymes were tested at five different substrate/coenzyme concentrations with a changeless concentration of another substrate. Effect of NADPH on G6PD was determined by two different concentrations of NADPH (0.1–0.2 mM) with 0.2 mM G6P and NADP⁺ concentrations varied from 4 μM to 20 μM. To determine inhibition type and K_i value, NADPH on 6PGD were tested by two different inhibitor concentrations (NADPH: 0.16 and 0.26 mM) with 0.2 mM G6P and NADP⁺ concentrations varying from 4 μM to 20 μM. For NADP⁺ and GSH, product inhibitions on the GR were investigated. For each product, varying two concentrations were examined (GSH: 15 and 25 mM; NADP⁺: 0.4 and 0.8 mM). All of the kinetic studies on the enzymes were made at 25 °C in optimal pH and ionic strength.

In vitro drug effects

To determine the effects of five antibiotics on rat lung, enzymes purified were put in the reaction solution by varying the five different drug concentrations. The enzyme activities without drugs were taken as 100%. For each drug (levofloxacin between 0.05 and 0.12 mM, furosemide between 0.1 and 0.3 mM, ceftazidime between 0.7 and 3 mM, between cefuroxime 3 and 15 mM, and gentamicin between 5 and 25 mM for G6PD; levofloxacin between 0.05 and 0.12 mM, furosemide between 0.1 and 0.3 mM, ceftazidime between 0.7 and 3 mM, cefuroxime between 3 and 15 mM, and gentamicin between 5 and 25 mM for 6PGD; levofloxacin between 0.01 and 0.04 mM, furosemide between 0.05 and 0.17 mM, ceftazidime between 0.3 and 1 mM, cefuroxime between 0.5 and 3 mM, and gentamicin between 15 and 42 mM for GR), Activity%–[Drug] graphs were drawn and used to calculate the drug concentrations (IC₅₀ values) causing a 50% decrease in enzyme activity. The types of inhibition and K_i constants were determined via Lineweaver–Burk graphs.

In vivo drug effect

Twelve young Sprague–Dawley rats with a weight of 140–180 g were supplied from Ataturk University Experimental Medical Application and Research Centre. Animals were maintained under standard laboratory conditions before the experiment. Drug dosages were determined according to the literature29. Levofloxacin and furosemide were administered as 10 mg/kg via intraperitoneal. After 25 min of the injection, rats were anesthetized with ethanol and sacrificed. The lungs were hastily taken, washed with cold isotonic saline (0.9 NaCl) and stored at −80 °C until use. This study received ethical approval from the Ethics Committee on Animal Research of Ataturk University.

Analysis of kinetic data

In vitro data were analyzed by Microsoft Office Excel 2010 and was given as ± SD. One-way analysis of variance (ANOVA) with post hoc least significant difference (LSD) test was used to compare the group means for the results of the in vivo studies and p < 0.05 was noted statistically considerable. Statistical analyses were made using SPSS 15.0 (Armonk, NY).

Results

As seen in Figure 1, by delivering NADP⁺ and NADPH gradient elution with 2′, 5′-ADP Sepharose 4B affinity chromatographic techniques, G6PD enzyme was eluted in tubes 5–9, 6PGD enzyme in tubes 15–19 and GR enzyme in tubes 24–28. G6PD was eluted with 0.5 mM NADP⁺, while 6PGD was eluted with 5 mM NADP⁺ and GR with 0.5 mm NADPH. The enzymes were pure and showed a single band (Figure 2). Table 1 shows that G6PD enzyme with 181.38 EU/mg protein specific activity was purified 580 times with 96% efficiency, 6PGD enzyme with 133.976 EU/mg protein specific activity 784 times with 90% efficiency, GR enzyme with 34.88 EU/mg protein specific activity 940 times with 95% efficiency. SDS-PAGE and gel filtration chromatography was implemented for molecular mass calculation of natural and subunits of the enzymes. R_f–log MW graph was drawn with the help of Figure 2 and subunit molecular mass of G6PD, 6PGD and GR enzymes were respectively calculated as 66, 52 and 63 kDa. Natural molecular mass of same enzymes were respectively calculated as 134, 110 and 121 kDa via Kav–log MW graph plotted according to the results of gel filtration chromatography. Studies made for optimal conditions (pH, ionic strength, temperature and stable pH) have been represented in Table 2.

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

V_max and K_M values of the substrate and coenzymes were obtained from the Lineweaver–Burk graphs. K_M values of G6P, 6PGA and GSSG were 0.028, 0.030 and 0.230 mM, and V_max value of the same substrate were 0.431, 1.398 and 0.263 EU/ml, respectively. K_M values calculated for NADP⁺ at rat lung G6PD and 6PGD were 0.0053 and 0.030 mM, and V_max values at same enzymes were 0.446 and 1.398 EU/ml. V_max and K_M values were found as 0.020 mM and 0.125 EU/ml for NADPH at rat lung GR.

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.

The product inhibition studies showed that NADPH markedly inhibited the activity of G6PD enzyme as uncompetitive with a K_i of 27 ± 2 μM and the activity of 6PGD as noncompetitive with a K_i of 147 ± 5 μM. Also, rat lung GR were noncompetitively inhibited by GSH with K_i constant with 16.66 ± 1.11 mM and NADP⁺ with K_i constant with 0.531 ± 0.04 mM.

Inhibition effect of drugs on enzymes is expressed in IC₅₀ values and K_i constants. IC₅₀ and K_i graphs plotted to determine effects on enzymes activities of levofloxacin are shown in Figure 3. IC₅₀ values and K_i constants in Table 3 show that furosemide and levofloxacin are the drugs that inhibit G6PD, 6PGD and GR enzymes the most. Other drugs are ranked in terms of inhibition effects from largest to smallest respectively as ceftazidime, cefuroxime and gentamicin. In addition to in vivo studies, for levofloxacin and furosemide drugs with the highest inhibition effects, in vitro studies were conducted on rats. 6PDG enzyme with p values smaller than 0.05 is highly inhibited by levofloxacin and GR enzyme by furosemide. G6PD enzyme is activated by furosemide (Table 4).

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

Discussion

Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme catalyzing the first step of the phosphogluconate pathway. Similarly, 6-phosphogluconate dehydrogenase (EC.1.1.1.44; 6PGD) catalyzes the third reaction in the same pathway, and in the existence of NADP⁺, converts from 6-phosphogluconate molecule to D-ribulose 5-phosphate. The hexose monophosphate shunt is the essential source for NADPH generation in all cells, and deficiency of both enzymes significantly reduces the amount of NADPH playing a vital role for the cell30,31. As a member of oxidoreductase enzymes, glutathione reductase (GR) enzyme (NADPH; GSSG oxidoreductase EC 1.6.4.2) converts oxidized glutathione (GSSP) into reduced glutathione (GSH). For this reason, this enzyme is significant as an antioxidant32,33.

2′, 5′-ADP Sepharose 4B material is used effectively to purify the NADPH/NADP⁺ dependent enzymes for years. Erat purified G6PD and GR enzymes in a single step with human erythrocyte-gradient elution34. In another study by us, G6PD, 6PDG and GR enzymes were purified from rat kidney with gradient elution for the first time. We applied the same method here with success and these three enzymes were separately eluted in the same step. These specific activities, % efficiency and purification coefficients are optimal values for kinetic studies. Moreover, compared to the literature, the purification process was fairly successful and suitable for scientific studies13,35–37.

These enzymes have been purified from several mammalian tissues. It has been reported that they were homodimer protein with native molecular mass in the range of 100–125 kDA. The active forms of the purified enzymes may be homodimer since natural molecular masses of enzymes are almost twice as much as subunit molecular masses. These molecular masses and subunit numbers show parallelism with the literature findings9,23,36,38–41.

Tests carried out to calculate the effect of ionic strength on enzymes, the most suitable ionic strength environment was determined as 200 mM Tris–HCl for G6PD, 200 mM phosphate for 6PGD and 80 mM Tris–HCl buffer for GR enzyme. Calculations show that G6PD, 6PGD and GR enzymes show the highest activity with pH values respectively as 8.0 (50 mM H₃PO₄–H₃BO₃), 8.0 (50 mM H₃PO₄–H₃BO₃) and 8.5 (80 mM Tris–HCI). Hence, these pH values are accepted as optimal pH values. Research showed that these results are highly parallel to other results in the literature8,37,42–44.

For optimal temperature calculation of the enzymes, activities of optimal ionic strength and optimal pH enzymes were measured at 10 °C intervals within 10–70 °C range. Calculations show that G6PD, 6PDG and GR enzymes show the highest activity respectively at 55 °C, 40 °C and 65 °C values. Therefore, this temperature is accepted as the optimal temperature. According to these results, GR is the most resistant enzyme against temperature. This result is also in parallel with similar results in the literature16,35,45–48.

For calculation of stable pH of enzymes, 200 mM phosphate buffers with 5.5, 6.0, 6.5, 7.0 and 7.5 pH, and 200 mM Tris–HCl buffers with 8.0, 8.5 and 9.0 pH were preserved at +4 °C together with the equal amount of enzyme solution, and activity measurements were performed for 72 h with 12 h intervals. The most stable pH of G6PD, GR and 6PDG enzymes are determined as 6.0 (0.2 mM phosphate buffer), 7.0 (0.2 M phosphate buffer) and 8.5 (200 mM Tris–HCl), respectively. Different stable pH profiles observed with buffalo erythrocyte (G6PD)35, chicken liver (6PGD)37 and rainbow trout (Oncorhynchus mykiss) liver (GR)15.

The K_M value for NADP⁺ was smaller than that for G6P and indicated that the enzyme had a strong affinity towards the coenzyme. Similar conclusions were reported for G6PD from human placental49 and rainbow trout (Oncorhynchus mykiss) erythrocytes50.

G6PD enzyme is inhibited by NADPH and metabolically controlled with NADPH inhibition. We observed that NADPH efficiently inhibited the G6PD enzyme and inhibition type was determined as uncompetitive. The inhibition type of NADPH on G6PD enzyme from different origin was reported as noncompetitive and competitive, but, the inhibition type on rat lung G6PD is unlike to that in the previous studies13,16,50,51.

6PG has the lower affinity towards 6PGD when compared with NADP⁺ since the K_M for 6PGA is higher than that for NADP⁺. Similar results were obtained for 6PGD of rat small intestine and rabbit mammary gland36,52. The rat lung 6PGD was noncompetitively inhibited by NADPH with a K_i of 0.147 ± 0.05 mM. K_i values obtained for NADPH are lower than that in other53 and higher than that reported in some studies8,36,52. The inhibition type was same as that obtained from rat erythrocytes36, but different from maize and human brain54,55.

According to K_M values found for GSSG and NADPH, the K_M value for NADPH is smaller than the one for GSSG. Thus, the affinity of NADPH to the enzyme is greater than GSSG. Similar values were reported for GR from human erythrocyte and bovine liver56,57.

NADP⁺ has a higher rate of inhibition on the enzyme activities than GSH. Similar results have been reported by others58,59. Rat lung GR was noncompetitively inhibited by GSH and NADP⁺. Inhibition type determined for GSH was similar to previous studies, but, inhibition type determined for NADP⁺ was different59,60.

The antibiotics are commonly utilized clinically for the treatment of many diseases throughout the world. However, it is well known that drugs have a remarkable toxic effect on the human and experimental animal61. It has been reported that various drugs inhibited as in vivo and in vitro the enzyme activities of G6PD, 6PGD and GR30,43,62. Particularly, IC₅₀ values and K_i constants of levofloxacin and furosemide drugs are significantly low. In some studies, it was reported that two drugs inhibited on the sheep and chicken liver glutathione reductase37,63 and on human erythrocyte 6-phosphogluconate dehydrogenase64. Our results detected for furosemide are similar to previous results, but levofloxacin exhibited further higher inhibitory effect than previous reports.

We selected levofloxacin and furosemide with the most effective drugs in vitro experiments for in vivo studies. The results of in vivo experiments showed that levofloxacin exhibited an inhibitory effect on the activities of rat lung 6PGD. The GR activity was inhibited by furosemide, but this drug demonstrated an activation effect on the G6PD activity. The reason that levofloxacin did not show a strong inhibition in vivo might be due to its protein binding property which may result in a decrease in the amount of the drug that reaches to the lung65. It is an important finding that the two drugs did not exhibit a strong inhibitory effect in vivo as they did under in vitro conditions on the enzyme activities essential for both antioxidant defense system and the other metabolic processes.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.