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Lactate dehydrogenase isoenzymes of human erythrocyte membranes
15
Clinica Chimica Acta, 79 (1977) 15-19 @ Elsevier/North-Holland Biomedical Press
CCA 8706
LACTATE DEHYDROGENASE ISOENZYMES ERYTHROCYTE MEMBRANES
OF HUMAN
S.S. ZAIL * and A.K. VAN DEN HOEK Department of Haematology, School of Pathology of the South African Institute for Medical Research and the University of the Witwatersrand, Johannesburg (South Africa) (Received
February
25th,
1977)
Summary
Human erythrocyte membranes and cytosol were fractionated in detergentcontaining polyacrylamide gels and stained for lactate dehydrogenase activity. A highly significant difference in the distribution of three isoenzymes of lactate dehydrogenase (LD-1, LD-2 and LD-3) between membranes and cytosol was found, a striking feature being the poor binding of LD-3 to the membrane fraction.
Introduction
The erythrocyte membrane contains certain enzymes, such as choline&erase, part of the membrane structure [l]. Others, which form an “integral” including several glycolytic enzymes, appear to be “peripherally” attached in that simple manoeuvres such as perturbation of ionic strength or pH can result in their elution [ 11. This may be related in part to methods used in preparing the membranes [ 21, but the recent demonstration of specific high affinity sites for two “peripherally” attached glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and aldolase, on the cytoplasmic pole of a 90 000 dalton trans-membrane polypeptide, indicates some probable physiological importance for this interaction [ 31. The attachment of lactate dehydrogenase (LDH) to erythrocyte membranes has been demonstrated by several workers [4-61, constituting about 2% of total erythrocyte LDH activity in membranes prepared by hypotonic lysis. The nature of this binding has not, however, been characterised. The binding of this enzyme to erythrocyte membranes is of particular interest as the adult red cell contains four easily demonstrable isoenzymes (LD-1, LD-2, LD-3 and LD-4) [ 71, which might show a specific distribution between the cytoplasm and the red * To
whom
correspondence
should
be addressed.
celf membrane. Such an association of specific isoenzymes with erythrocyte membranes has been unsuccessfully sought by others [Z] for glyceraldehyde-3phosphate dehydrogenase and phosphoglyceric kinase. We now report a significant difference in the distribution of the isoenzymes of LDH between the cytoplasm and membranes of human e~thro~ytes~ the striking feature being the poor binding of LD-3 to the membrane fraction. Material and methods Red cell membranes were prepared from freshly drawn heparinized venous blood by hypotonic lysis of washed red cells using 5 mM phosphate buffer, pH 8.0 as described by Fairbanks et al. (8). The membranes were stored overnight at -20°C. The membranes were solubihzed in Triton X-100 and separated in ~iton-~on~ning poiya~~l~ide gels and stained for lactate dehydrogenase activity as described by van den Hoek and Zail 191. Red cell cytosol obtained after the first hypotonic lysis of washed cells and centrifugation of membranes was treated in the same way as purified membranes. Approximately equivalent amounts of total LDH activities were loaded onto the gels, which were stained for lactate dehydrogenase activity and then scanned in a Eelman densitometer at 650 nm. Results and discussion In seven prep~atio~s obtained from different donors there was a s~~ifica~t difference in the distribution of LD-1 (P < 0.0005), LD-2 (P < 0.025) and LD-3 (P < 0.0005) between the human red cell membrane and the cytosol (two-tailed paired t-test) (Table I; Fig. 1). LD-4 was detected in trace amounts in some cytosol preparations but not in membranes, while LD-5 was not detectable in either cytosol UP membrane preparations. While LD-3 constitutes approximately a quarter of the total LDH activity of the red cell cytosol, it forms only a mean of 2.1% of the LDH activity of the membrane fraction. In two preparations there was no detectable binding of LD-3 to the membrane fraction. Associated with the poor binding of LD-3 to this fraction, there is a reciprocaI increase in the percentage binding of LD-1 to the membranes, such that the ratio LD-l/LD-2 in the cytosol of approximately 3 : 4 changes to approximately 2 : I in the membrane fraction. To exclude the poss~b~ity that the poor binding of LD-3 might be an artefact resulting from the preparation of the membrane fraction by hypotonic lysis, membranes were prepared after ultrasonication of washed cells in an isotonic medium [lo] 1 The distribution of LD-3 between the membrane fraction (4.5%) and the cytosol (27.5%) was similar to that found for membranes prepared in conventional fashion. Preparation of membranes by hypotonic lysis at pH 7,0,7.5 or 8.0, or the use of fresh membranes or cytosol (as opposed to those stored overnight at -20°C) resulted in the same dis~ibution of LDH isoenzymes as found for the conventions technique. Furthermore, the addition of 1 mM nicotinamide adenine dinucleotide (NAD) to the Iysing solution used for the preparation of the erythrocyte membranes, or the addition of 1 mM NAD to the final membrane and cytosol preparations and to the gel and electrode buffers also had no effect on the observed
17 TABLE I DISTRIBUTION
OF LDH ISOENZYMES
IN HUMAN ERYTHROCYTE
MEMBRANE
AND CYTOSOL
Distribution (46) LD1
LD-2
LD-Y
LD-3
LD-4
Cytosol 1 2 3 4 5 6 I Mean S.D.
41.5 28.4 29.4 31.7 36.3 30.7 32.7 32.96 4.55
35.5 40.6 43.4 38.0 48.1 41.7 44.2 42.50 4.74
2.5
20.5
-
2.0 1.6
28.4 25.6
0.6 -
1.7 -
27.2 15.6 21.6 23.1 23.14 4.40
1.4 -
Membranes 1 2 3 4 5 6 7 Mean S.D.
67.5 67.8 72.5 54.5 67.7 57.7 62.4 64.30 6.38
32.5 30.2 27.0 39.5 32.3 38.5 35.4 33.63 4.47
-
2.0 0.5 6.0 -
-
3.8 2.2
-
2.01 2.22
distribution of LDH isoenzymes. The latter findings make it unlikely that instability of membrane-bound LD-3 is responsible for the observed isoenzyme distribution pattern [ 111. The possibility that Triton X-100 might differentially inactivate membrane-bound LD-3 in the solubilization process was excluded by comparing total membrane LDH activity in the presence and absence of 1% Triton X-100. No inhibitory effect of Triton was observed. The phenomenon of differential binding of LDH isoenzymes to red cell membranes is not confined to human erythrocytes. We found bovine erythrocyte cytosol LD-1 and LD-2 isoenzymes present in a ratio of approximately 7 : 3 with trace amounts of LD-3. However LD-1 forms almost 100% of the LDH bound to bbvine erythrocyte membrane8 with only trace binding of LD-2 (Fig. 2). Three other species tested thus far (pig, sheep, rabbit) have only LD-1 present in the red cell cytosol, and only LD-1 can be detected in these membranes. An additional finding of interest was the presence of an extra band staining for LDH activity in the human red cell cytosol fraction in a position just cathodal to LD-2 (labelled “y” in Fig. 1). This band consisted of about 2% of the stainable LDH activity and was never detected in plasma separations under the same conditions. Addition of 1 mM NAD to the cytosol fraction and to the gel and electrode buffers had no effect on the appearance of this band. Similarly a lo-fold increase in the concentration of gel and electrode buffers or the use of fresh preparations (as opposed to those stored overnight at -20°C) of red cell cytosol had no effect on the presence of band “y”. Electrophoresis in agarose gels [ 121 did not show the extra band. The exact nature of this band
---LD4
a
b
a
b
Fig. 1. Polyacrylamide gel electraphoresis cytosol LDH isoenzymes (b).
of human erythrocyte
membrane LDH isoenzymes (a). and
Fig. 2. Polyacrylamide LDH isoenzymes (b).
of bovine erythrocyte
membrane LDH isoenzymes (a). and
gel electrophoresis
is still under consideration, but it could represent a complex of LD-2 with a cytoplasmic component, or an artefact associated with the use of polyacrylamide as the separation medium. The physiological significance of the difference in the distribution of the isoenzymes of LDH between the red cell cytosol and membrane must remain speculative at present. It has been shown that the isoenzymes of LDH may interact differentially with various intracellul~ fractions in chicken skeletal muscle, that kinetic properties of the bound isoenzymes may be modified by this interaction and that NADH differentially solubilizes membrane bound LD-5 [13]. Similar interactions may affect metabolic control in the cell, particularly the mechanism whereby optimal NADH:NAD ratios are maintained in muscle cells under aerobic and anaerobic conditions [13,14]. whether such mechanisms are operative in red cells is unknown at present, but it is of interest that modification of some kinetic properties of LDH have been described in that fraction of enzyme bound to human erythrocyte membranes [6]. The differential binding of the isoenzymes of LDH to erythrocyte membranes may partly explain these findings. As far as we are aware, this is the first demonstration of binding of LD-1 (in contrast to LD-5) to cell membranes. The specific
19
advantages of the presence of LD-1 and LD-2 in the red cell cytosol and their binding to human red cell membranes, however, remains to be elucidated. Acknowledgements These Research
studies were supported by grants Council and Atomic Energy Board.
from
the South
African
Medical
References 1 Juliano, R.L. (1973) Biochim. Biophys. Acta 300, 341-378 2 Schrier. S.L.. Ben-Bassat. I., Junga. I., Seeger. M. and Grumet. F.C. (1975) J. Lab. CIin. Med. 85, 797-810 3 Yu, J. and Steck. T.L. (1975) J. Biol. Chem. 250. 9176-9184 4 Duchon. G. and Collier, H.B. (1971) J. Membrane Biol. 6, 138-157 5 McDaniel, C.F. and Kirtley, M.E. (1974) J. Biol. Chem. 249.64784486 6 Wins, P. and Schoffeniels, E. (1969) Biochim. Biophys. Acta 185. 287-296 7 Wilkinson. J.H. (1970) Isoenzymes. p. 136, Chapman and Ha& London 8 Fairbanks, G.. Steck, T.J. and WaIIach. D.F.H. (1971) Biochemistry, 10. 2606-2617 9 Van den Hock. A.K. and ZaiI, S.S. (1977) Clin. Chim. Acta 79, 7-14 10 ZaiI. S.S. and van den Hoek, A.K. (1975) Clin. Chim. Acta 60,231-236 11 Zondag, H.A. (1963) Science 142.965-967 12 Starkweather, W.H., Cousineau. L., Schoch, H.K. and Zarafonetis, C.J. (1965) Blood 26, 63-73 13 Hultin. H.O. (1975) in Isoenzymes II, Physiological Function (Market, C.L., ed.). pp. 69-85, Academic Press, New York 14 Nadal-Ginard, B. and Market, C.L. (1975) in Isoenzymes II. Physiological Function (Market. C.L.. ed.), pp. 45-67. Academic Press, New York
Clinica Chimica Acta, 79 (1977) 15-19 @ Elsevier/North-Holland Biomedical Press
CCA 8706
LACTATE DEHYDROGENASE ISOENZYMES ERYTHROCYTE MEMBRANES
OF HUMAN
S.S. ZAIL * and A.K. VAN DEN HOEK Department of Haematology, School of Pathology of the South African Institute for Medical Research and the University of the Witwatersrand, Johannesburg (South Africa) (Received
February
25th,
1977)
Summary
Human erythrocyte membranes and cytosol were fractionated in detergentcontaining polyacrylamide gels and stained for lactate dehydrogenase activity. A highly significant difference in the distribution of three isoenzymes of lactate dehydrogenase (LD-1, LD-2 and LD-3) between membranes and cytosol was found, a striking feature being the poor binding of LD-3 to the membrane fraction.
Introduction
The erythrocyte membrane contains certain enzymes, such as choline&erase, part of the membrane structure [l]. Others, which form an “integral” including several glycolytic enzymes, appear to be “peripherally” attached in that simple manoeuvres such as perturbation of ionic strength or pH can result in their elution [ 11. This may be related in part to methods used in preparing the membranes [ 21, but the recent demonstration of specific high affinity sites for two “peripherally” attached glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and aldolase, on the cytoplasmic pole of a 90 000 dalton trans-membrane polypeptide, indicates some probable physiological importance for this interaction [ 31. The attachment of lactate dehydrogenase (LDH) to erythrocyte membranes has been demonstrated by several workers [4-61, constituting about 2% of total erythrocyte LDH activity in membranes prepared by hypotonic lysis. The nature of this binding has not, however, been characterised. The binding of this enzyme to erythrocyte membranes is of particular interest as the adult red cell contains four easily demonstrable isoenzymes (LD-1, LD-2, LD-3 and LD-4) [ 71, which might show a specific distribution between the cytoplasm and the red * To
whom
correspondence
should
be addressed.
celf membrane. Such an association of specific isoenzymes with erythrocyte membranes has been unsuccessfully sought by others [Z] for glyceraldehyde-3phosphate dehydrogenase and phosphoglyceric kinase. We now report a significant difference in the distribution of the isoenzymes of LDH between the cytoplasm and membranes of human e~thro~ytes~ the striking feature being the poor binding of LD-3 to the membrane fraction. Material and methods Red cell membranes were prepared from freshly drawn heparinized venous blood by hypotonic lysis of washed red cells using 5 mM phosphate buffer, pH 8.0 as described by Fairbanks et al. (8). The membranes were stored overnight at -20°C. The membranes were solubihzed in Triton X-100 and separated in ~iton-~on~ning poiya~~l~ide gels and stained for lactate dehydrogenase activity as described by van den Hoek and Zail 191. Red cell cytosol obtained after the first hypotonic lysis of washed cells and centrifugation of membranes was treated in the same way as purified membranes. Approximately equivalent amounts of total LDH activities were loaded onto the gels, which were stained for lactate dehydrogenase activity and then scanned in a Eelman densitometer at 650 nm. Results and discussion In seven prep~atio~s obtained from different donors there was a s~~ifica~t difference in the distribution of LD-1 (P < 0.0005), LD-2 (P < 0.025) and LD-3 (P < 0.0005) between the human red cell membrane and the cytosol (two-tailed paired t-test) (Table I; Fig. 1). LD-4 was detected in trace amounts in some cytosol preparations but not in membranes, while LD-5 was not detectable in either cytosol UP membrane preparations. While LD-3 constitutes approximately a quarter of the total LDH activity of the red cell cytosol, it forms only a mean of 2.1% of the LDH activity of the membrane fraction. In two preparations there was no detectable binding of LD-3 to the membrane fraction. Associated with the poor binding of LD-3 to this fraction, there is a reciprocaI increase in the percentage binding of LD-1 to the membranes, such that the ratio LD-l/LD-2 in the cytosol of approximately 3 : 4 changes to approximately 2 : I in the membrane fraction. To exclude the poss~b~ity that the poor binding of LD-3 might be an artefact resulting from the preparation of the membrane fraction by hypotonic lysis, membranes were prepared after ultrasonication of washed cells in an isotonic medium [lo] 1 The distribution of LD-3 between the membrane fraction (4.5%) and the cytosol (27.5%) was similar to that found for membranes prepared in conventional fashion. Preparation of membranes by hypotonic lysis at pH 7,0,7.5 or 8.0, or the use of fresh membranes or cytosol (as opposed to those stored overnight at -20°C) resulted in the same dis~ibution of LDH isoenzymes as found for the conventions technique. Furthermore, the addition of 1 mM nicotinamide adenine dinucleotide (NAD) to the Iysing solution used for the preparation of the erythrocyte membranes, or the addition of 1 mM NAD to the final membrane and cytosol preparations and to the gel and electrode buffers also had no effect on the observed
17 TABLE I DISTRIBUTION
OF LDH ISOENZYMES
IN HUMAN ERYTHROCYTE
MEMBRANE
AND CYTOSOL
Distribution (46) LD1
LD-2
LD-Y
LD-3
LD-4
Cytosol 1 2 3 4 5 6 I Mean S.D.
41.5 28.4 29.4 31.7 36.3 30.7 32.7 32.96 4.55
35.5 40.6 43.4 38.0 48.1 41.7 44.2 42.50 4.74
2.5
20.5
-
2.0 1.6
28.4 25.6
0.6 -
1.7 -
27.2 15.6 21.6 23.1 23.14 4.40
1.4 -
Membranes 1 2 3 4 5 6 7 Mean S.D.
67.5 67.8 72.5 54.5 67.7 57.7 62.4 64.30 6.38
32.5 30.2 27.0 39.5 32.3 38.5 35.4 33.63 4.47
-
2.0 0.5 6.0 -
-
3.8 2.2
-
2.01 2.22
distribution of LDH isoenzymes. The latter findings make it unlikely that instability of membrane-bound LD-3 is responsible for the observed isoenzyme distribution pattern [ 111. The possibility that Triton X-100 might differentially inactivate membrane-bound LD-3 in the solubilization process was excluded by comparing total membrane LDH activity in the presence and absence of 1% Triton X-100. No inhibitory effect of Triton was observed. The phenomenon of differential binding of LDH isoenzymes to red cell membranes is not confined to human erythrocytes. We found bovine erythrocyte cytosol LD-1 and LD-2 isoenzymes present in a ratio of approximately 7 : 3 with trace amounts of LD-3. However LD-1 forms almost 100% of the LDH bound to bbvine erythrocyte membrane8 with only trace binding of LD-2 (Fig. 2). Three other species tested thus far (pig, sheep, rabbit) have only LD-1 present in the red cell cytosol, and only LD-1 can be detected in these membranes. An additional finding of interest was the presence of an extra band staining for LDH activity in the human red cell cytosol fraction in a position just cathodal to LD-2 (labelled “y” in Fig. 1). This band consisted of about 2% of the stainable LDH activity and was never detected in plasma separations under the same conditions. Addition of 1 mM NAD to the cytosol fraction and to the gel and electrode buffers had no effect on the appearance of this band. Similarly a lo-fold increase in the concentration of gel and electrode buffers or the use of fresh preparations (as opposed to those stored overnight at -20°C) of red cell cytosol had no effect on the presence of band “y”. Electrophoresis in agarose gels [ 121 did not show the extra band. The exact nature of this band
---LD4
a
b
a
b
Fig. 1. Polyacrylamide gel electraphoresis cytosol LDH isoenzymes (b).
of human erythrocyte
membrane LDH isoenzymes (a). and
Fig. 2. Polyacrylamide LDH isoenzymes (b).
of bovine erythrocyte
membrane LDH isoenzymes (a). and
gel electrophoresis
is still under consideration, but it could represent a complex of LD-2 with a cytoplasmic component, or an artefact associated with the use of polyacrylamide as the separation medium. The physiological significance of the difference in the distribution of the isoenzymes of LDH between the red cell cytosol and membrane must remain speculative at present. It has been shown that the isoenzymes of LDH may interact differentially with various intracellul~ fractions in chicken skeletal muscle, that kinetic properties of the bound isoenzymes may be modified by this interaction and that NADH differentially solubilizes membrane bound LD-5 [13]. Similar interactions may affect metabolic control in the cell, particularly the mechanism whereby optimal NADH:NAD ratios are maintained in muscle cells under aerobic and anaerobic conditions [13,14]. whether such mechanisms are operative in red cells is unknown at present, but it is of interest that modification of some kinetic properties of LDH have been described in that fraction of enzyme bound to human erythrocyte membranes [6]. The differential binding of the isoenzymes of LDH to erythrocyte membranes may partly explain these findings. As far as we are aware, this is the first demonstration of binding of LD-1 (in contrast to LD-5) to cell membranes. The specific
19
advantages of the presence of LD-1 and LD-2 in the red cell cytosol and their binding to human red cell membranes, however, remains to be elucidated. Acknowledgements These Research
studies were supported by grants Council and Atomic Energy Board.
from
the South
African
Medical
References 1 Juliano, R.L. (1973) Biochim. Biophys. Acta 300, 341-378 2 Schrier. S.L.. Ben-Bassat. I., Junga. I., Seeger. M. and Grumet. F.C. (1975) J. Lab. CIin. Med. 85, 797-810 3 Yu, J. and Steck. T.L. (1975) J. Biol. Chem. 250. 9176-9184 4 Duchon. G. and Collier, H.B. (1971) J. Membrane Biol. 6, 138-157 5 McDaniel, C.F. and Kirtley, M.E. (1974) J. Biol. Chem. 249.64784486 6 Wins, P. and Schoffeniels, E. (1969) Biochim. Biophys. Acta 185. 287-296 7 Wilkinson. J.H. (1970) Isoenzymes. p. 136, Chapman and Ha& London 8 Fairbanks, G.. Steck, T.J. and WaIIach. D.F.H. (1971) Biochemistry, 10. 2606-2617 9 Van den Hock. A.K. and ZaiI, S.S. (1977) Clin. Chim. Acta 79, 7-14 10 ZaiI. S.S. and van den Hoek, A.K. (1975) Clin. Chim. Acta 60,231-236 11 Zondag, H.A. (1963) Science 142.965-967 12 Starkweather, W.H., Cousineau. L., Schoch, H.K. and Zarafonetis, C.J. (1965) Blood 26, 63-73 13 Hultin. H.O. (1975) in Isoenzymes II, Physiological Function (Market, C.L., ed.). pp. 69-85, Academic Press, New York 14 Nadal-Ginard, B. and Market, C.L. (1975) in Isoenzymes II. Physiological Function (Market. C.L.. ed.), pp. 45-67. Academic Press, New York