Comp. Biochem. Physiol., 1975, VoL 51B, pp. 463 to 466. Pergamon Press. Printed in Great Britain

RED CELL ENZYMES--I. GLYCOLYTIC ENZYMES AND REDUCTION OF GLUTATHIONE IN BOVINE RED BLOOD CELLS N. S. AGAR, MARGARETA. GRUCA, JOAN DUNSAR AND J. D. HARLEY Children's Medical Research Foundation, P.O. Box 61, Camperdown, N.S.W. 2050, Australia (Received 3 May 1974)

Abstract--1. Spectrophotometric assays of twelve different enzymes of red blood cells were conducted in six breeds of cattle. 2. Activity of fructose diphosphate aldolase was significantly higher in Sahiwal and crossbred [(Bos indicus) Sahiwal x (Bos taurus) Red Polls] than in the other breeds. 3. The rates of regeneration of reduced glutathione (GSH) and the activity of the enzymes associated with the regeneration of GSH (hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase) were not significantly different between the breeds studied. INTRODUCTION GLUTATHIONE(GSH) is widely distributed in living tissues. It is maintained in relatively large amounts and functions in oxidation-reduction reactions. GSH appears to protect protein sulphydryl groups within the red cell, primarily those of haemoglobin against oxidation either directly or by detoxifying hydrogen peroxide through the glutathione peroxidase reaction. In addition to this important role that GSH plays in the maintenance and normal functioning of the red cell, the concentration of this tripeptide in these cells has been shown to bear a relationship to such productive traits in animals as fleece weight in sheep (Agar et al., 1972), and milk yield, growth rate and body size in cattle (Kidwell et al., 1955; Boriskenko, 1961; Slepkov, 1961). Studies on GSH metabolism in relation to energy metabolism of the red blood cell have recently been carried out in sheep (Agar & Smith, 1973). No such report appears to have been made in cattle (Bos taurus). We now report the results of such an investigation aimed to evaluate the glutathione metabolism in relation to energy metabolism in the red blood cells of this species. MATERIALS AND METHODS Twenty-four adult cattle of six different breeds, Friesian, Jersey, Red Polls, Sindi, Sahiwal and a crossbred (Sahiwal × Red Polls) were used from the C.S.I.R.O. Division of Animal Genetics, Sydney. A single blood sample was taken for the determination of GSH regeneration rate and red cell enzyme activities. GSH Regeneration

Regeneration of GSH by intact red cells was determined using a slight modification of the original method of

Kosower et aL (1967). Red cells were washed three times with 0.15 M saline and were then suspended in the ratio of 1 : 1. in 0'1 M NaCI-0.1 M glycylglycine buffer, pH 7.1. The red cell GSH was oxidized with diamide (Kosower e t a L , 1969). After allowing the mixture to stand for 10 rain at 4°C, 1 M dextrose was added in the final concentration of 0.01 ml/ml red cell suspension. The samples were then incubated at 37°C so that the cellular metabolism could reduce the oxidized glutathione. Aliquots of this mixture were removed for the determination of GSH at zero time and at every 5 rain. GSH was measured by the method of Beutler et aL (1963). The control values were obtained from an aliquot of original washed red cell suspension lacking the diamide and the dextrose. The regeneration rates were calculated by a least square fit and were expressed as tzM GSH regenerated per min per g of haemoglobin. Enzymic assay

To 0-1 ml of washed red cells was added 1.0 ml of a haemolysing solution described by Smith et al. (1970). The solution was mixed for 20 rain at 4°C and centrifuged at 20,000 g for 20 rain. The stroma-free haemolysate was used for enzymic assays. Red cell enzymes were assayed at 37°C in a cuvette having a light path of 1 cm and a final volume of 1.0 ml. All enzymes were measured in 50 mM imidazol buffer, pH 7.2. Previously described methods (Smith et aL, 1970, 1972; Beutler, 1971) were employed. The concentrations of the reactants in the cuvette are given below for each enzyme assay. Hexokinase. Imidazol buffer 50 mM, NADP 0,2 mM, NaF 2.0raM, MgCI~ 0.2raM, G6PD 0.5EU/ml, neutralized ATP 0"4 raM, glucose 2.0 mM and haemolysate (1 to 5 dilution of the original) 0.05 ml. Glucose phosphate isomerase. Imidazol buffer 50 mM, NADP 0.2 mM, G6PD 0-5 EU/ml, F6P 4"0 mM and haemolysate (1-20) 0.05 ml. Phosphofructokinase. Imidazol buffer 50 raM, NADH 0-2raM, MgCI~ 0.2raM, neutralized ATP 0.4mM, neutralized AMP 0.06 mM, FDA 0.60 EU/rnl, GDH

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N . S . AGAR, MARGARET A. GRUCA, JOAN DUNBAR AND J. D. HARLEY

0"32 EU/ml, TPI 0.25 EU/ml, F6P 4.0 mM and haemolysate (1-5) 0.05 ml. Fructose diphosphate aldolase. Imidazol buffer 50 raM, NADH 0.2 mM, GDH 0.32 EU/ml, TPI 0-25 EU/ml, FDP 4.0 mM and haemolysate (1-5) 0-05 ml. Glyceraldehyde-3-phosphate dehydrogenase. Imidazol buffer 50 raM, NADH 0"2 raM, neutralized ATP 0.4 raM, MgCI2 0.2 raM, dithioerythritol 0"1 mM, PGK 0"31 EU/ ml, 3PGA 7.0 mM and haemolysate (1-20) 0.05 ml. 3-Phosphoglycerate kinase. Imidazol buffer 50 mM, MgCI2 0.2raM, neutralized ATP 0.4mM, NADH 0.2raM, dithioerythritol 0.1 mM, GAPD 0"3 EU/ml, 3PGA 7-0 mM and haemolysate (1-20) 0.05 ml. Monophosphoglyceratemutase. Imidazol buffer 50 mM, enolase 0-2 EU/ml, 2,3-DPG 0'01 raM, MgCI2 0"02 mM, 3PGA 7"0 mM and haemolysate (1-5) 0.05 ml. Enolase. Imidazol buffer 50mM, MgCI2 0.2mM, 2PGA 0"6 mM and haemolysate (1-5) 0.05 ml. Pyruvate kinase. Imidazol buffer 50mM, NADH 0"2 raM, MgCI~ 0.2 mM, KCI 75 mM, LDH 4.4 EU/ml, neutralized ADP 1"2 raM, PEP 3"0 mM and haemolysate (1-5) 0.05 ml. Lactate dehydrogenase. Imidazol buffer 50mM, NADH 0"2 raM, sodium pyruvate 3.8 mM and haemolysate (1-20) 0.05 ml. Glucose-6-phosphate dehydrogenase. Imidazol buffer 50 mM, NADP 0-2 mM, G6P 0.6 mM and haemolysate (1-5) o.o5 rid. 6-Phosphogluconate dehydrogenase. Imidazol buffer 50 raM, NADP 0-2 mM, 6PGA 0-6 mM and haemolysate (1-5) 0"05 ml. Glutathione reductase. Imidazol buffer 50 mM, NADPH 0.2raM, neutralized EDTA 30raM, GSSG 3.3 mM and haemolysate (1-5) 0-05 ml. Absorbance (at 340 nm for NAD or NADP-linked enzyme assays and at 240 nm for PEP-linked enzyme assays) was recorded in a Gilford Model 2400 recording spectropbotometer. Enzyme activity was expressed as ~M of substrate used per min per g haemolgobin. The following abbreviations are used in the text: HK, hexokinase; GPI, glucose phosphate isomerase; PFK, phosphofructokinase; FDA, fructose diphosphate aldolase; GAPD, glyceraldehyde-3-phosphate debydrogenase; PGK, phosphoglyeerate kinase; MPGM, monophosphate-glycerate mutase; enolase, phosphopyruvate hydrase; PK, pyruvate kinase; LDH, lactate dehydrogenase; G6PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate debydrogenase; GSH-R, glutathione reductase; TPI, triosephosphate isomerase; GDH, 8-glycerophosphate dehydrogenase; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FDP, fructose-l,6-diphosphate; 2 PGA, 2-phosphoglycerate; 3 PGA, 3-pbosphoglycerate; 6 POA, 6phosphogluconate; PEP, phosphoenol pyruvate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monopbosphate; NADH, nicotinamide adenine dinucleotide reduced; NADPH, nicotinamide adenine dinucleotide phosphate reduced; HAl)P, nicotinamide adenine dinucleotide phosphate; GSSG, oxidized glutathione. RESULTS The GSH regeneration rates in the red cells of cattle of six different breeds are shown in Table 1. The regeneration rate was lowest in the red cells

Table 1. GSH Levels and regeneration rates in the red blood cells of different breeds of cattle (mean+_S.E.M.)

Friesian Red Polls Jersey Sindi Sahiwal Cross (Sahiwal x Red Polls) All breeds

GSH*

GSH regenerationt

9"51 _+0.084 8"48 _ 0.558 9.87+_0.908 9.47+_0.539 8.55 _+0.608 9.10+-0.280

0"148 -+0-022 0.091_+0"020 0.112+_0.035 0.124+_0.034 0.210_+0.034 0.161_+0-008

9.16+_0.229

0.141+_0.013

* Expressed as #M/g Hb.

I Expressed as/~M GSH/min per g Hb. obtained from Red Polls (0-091 +0.020/~M GSH/ min per g Hb) and highest in those of Sahiwal (0.210_+ 0.034/~M GSH/min per g Hb). However there were no statistically significant differences between the breeds. The overall rate of GSH regeneration for six breeds was 0.141 +-0.013. Since there were no statistically significant differences among the breeds, the results obtained with one animal (Friesian) have been presented in Fig. 1 to

2.0

0") L9

I-5

0-5

4

8

12 rrm

16

20

Fig. 1. Rates of GSH regeneration in bovine red blood cells. The regeneration rate is expressed as pM GSH/min per g Hb. represent the GSH regeneration. A progressive increase in the concentration of GSH occurred during 20 rain incubation. Continuing the incubation for more than 20 rain did not lead to a further increase. During this period more than 90 per cent of the original GSH was regenerated. It has been shown previously that the application of diamide sufficient to oxidize all of the intracellular GSH of the red cell leads to the regeneration of GSH which reaches the control value in approximately 30 rain in man (Kosower et al., 1969) and 20 rain in many other mammalian species including sheep, goat, horse, dog, rabbit and red kangaroo (Agar et al., 1974).

Enzymes in cattle red cells Activity of only one enzyme, fructose diphosphate aldolase, was found to be different between the breeds; it was significantly higher in Sahiwal (P

Red cell enzymes--I. Glycolytic enzymes and reduction of glutathione in bovine red blood cells.

Comp. Biochem. Physiol., 1975, VoL 51B, pp. 463 to 466. Pergamon Press. Printed in Great Britain RED CELL ENZYMES--I. GLYCOLYTIC ENZYMES AND REDUCTIO...
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