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Electrophoretic characterization of glutamic oxalacetic transaminase in human red cells of different ages
CLINICA CHIMICA ACTA
ELECTROPHORETIC TRANSAMINASE
P. M. MANNUCCI Institute
of Medical
(Received
21.5
CHARACTERIZATION
OF GLUTAMIC
IN HUMAN RED CELLS OF DIFFERENT
OXALACETIC AGES*
AND N. DIOGUARDI Pathology,
August z?th,
University
of Cagliari
(Italy)
1965)
SUMMARY
GOT isoenzymes of red cells from normal subjects, and from subjects with reticulocytosis have been analysed by cellogel electrophoresis. Hemolysates from normal and from relatively old red cells contain only the anodic fraction (GOT I) ; whereas hemolysates from subjects with reticulocytosis and from relatively young red cells contain, in addition, the cathodic fraction (GOT II). Since this isoenzyme is derived from mitochondria, it implies that these organelles are still present in reticulocytes and in young red cells.
Glutamic oxalacetic transaminase (GOT) has recently been demonstrated to exist in the different tissues in two isoenzymes which can be separated by means of electrophoresis and chromatographyl-S. It was shown that the anodal electrophoretic zone (GOT I) was derived from the cytoplasm and the cathodal zone from the mitochondrial fraction. In human erythrocytes only the anodal component was detected by Nisselbaum4. A simple method for the characterization of GOT isoenzymes has been performed in our laboratory by applying to cellogel electrophoresis the staining procedure developed by Decker and Raus. This report concerns a study of GOT isoenzymes in normal human hemolysates, in hemolysates obtained from red cells of different ages and from subjects with reticulocytosis. METHODS
Prefiaration of hemolysates 5 ml of heparinized venous blood were centrifuged at 170 g for IO min and the platelet-rich plasma was pipetted off and discarded. The blood cells, after resuspension at hematocrit levels in platelet-free plasma obtained by centrifuging at- 2000 g for 30 min, were poured into a glass column (20 x 1.2 cm), prewarmed at 37” and filled with loosely pressed ordinary cotton wool. The column was then inverted and * This work has been performed with the aid of the Assessorato Sanita Regione Autonoma Sarda. C&z. C&n.
Acta,
14 (1966) 215-218
216
P. M. MANUCCI,
N. DIOGUARDI
incubated at 37’ for 30 min. The elution was obtained using 0.15 M prewarmed saline, up to a final volume of nearly IO ml. The elution fluid contained erythrocytes, reticulocytes, lymphocytes and monocytes while granulocytes and platelets were absent. The removal of granulocytes is related to their property of phagocytosing the cotton wool; this material was as effective as glasswool, which was tested in preliminary experiments. The amount of saline used for the elution is fairly critical, because larger amounts tend to detach the granulocytes from the cotton wool. The few platelets unremoved in the platelet-rich plasma were retained by the cotton wool due to aggregation and adhesion. The elution fluid was then centrifuged twice at 350 g for IO min in a Modello Cagliari Centrifuge (Terzano, Milan). This allowed the lymphocytes and the monocytes to be removed with the supernatant whereas the red blood cells sedimented. The concentration of the lymphocytes and monocytes did not exceed 106 cells/ml. At this concentration the GOT activity of the lymphocytes is negligible. The packed erythrocytes were then washed three times with 0.15 M NaCl (I : 3 in volume) and hemolysis was obtained with distihed water in the proportion of I :I‘ and by freezing and thawing twice in an alcohol-ether mixture. The preparation was then submitted to an ultrasonic apparatus for 45 set and centrifuged at 2000 g in order to remove the stromata. Preparation of relatively old and reiatiuely young erythrocytes The erythrocytes of different ages were seperated by means of four centrifugations at 2000 g according to the technique of Brewer et aL.6.This method is based on the direct relationship existing between the age of the erythrocytes and their increase in density. The reticulocyte count showed a seven to tenfold increase in reticulocytes in the young (top) cell fraction as compared with the old (bottom) cell fraction. The quantitative level of GOT was three to fivefold higher in young erythrocytes. The two cell fractions were resuspended in their own plasma to an hematocrit level of approximately 50%. The same procedure described above was followed in order to obtain the hemolysates from these selected red cell preparations. The final preparations were thus completely free from granulocytes and platelets and contained only a negligible amount of lymphocytes and monocytes.
Electrophoretic separations were carried out on cellogel strips (17.5 x 2.5 cm) supplied by Chemetron, Milan. Phosphate K buffer of 0.04 M strength, of pH 7.5 and with a constant voltage of 8 V/cm for 3 h, was employed. The hemolysates were applied at a distance of 3 cm from the cathode in quantities of 0.001-0.003 ml. The isoenzymatic zones separated by electrophoresis were detected by staining the cellogel strips according to the method of Decker and Rau6. RESULTS
AND
DISCUSSION
GOT isoenzymatic pattern of normal hemolysates is shown in Fig. I. 22 individuals with normal reticulocyte count are all characterized by the presence of one zone alone which is anodic and faster than hemoglobin (GOT I). The same pattern was demonstrated in preparations of relatively old erythrocytes whereas all the preparations of young erythrocytes, containing a large amount C&L Chiw.
Acta,
14 (1966) 215-218
GOT TRANSAMINASE
IN RED CELLS
217
of reticulocytes, showed the presence of a small cathodal component (GOT II), moving slower than hemoglobin (Fig. 2). In studies carried out on 8 subjects recovering from acute hemolysis due to sensitivity to fava beans, and with a reticulocytic crisis; on 4 subjects with posthemorrhage reticulocytosis; and on 9 subjects with reticulocytosis related to various types of hemolytic anemias, the cathodal isoenzyme was always detectable when
Fig. I. Electrophoretic Reticulocytes I %.
separation of GOT isoenzymes in normal red cells. Normal hemolysate.
Fig. 2. Electrophoretic separation of GOT isoenzymes in relatively old red cells (a, reticulocytes 1.5%) and relatively young red cells (b, reticulocytes 1274)
Fig. 3. Electrophoretic separation of GOT isoenzymes in cases with reticulocytosis. (a) reticulocytes 3.5%, (b) reticulocytes 8.7%. (c) reticulocytes 12.3%, (d) reticulocytes 15.3%.
reticulocytes were up to 2.5-30/O. The intensity of the staining appeared to be related to the number of reticulocytes present in the preparation. Since the cathodal GOT isoenzyme has been shown to be derived from the mitochondrial fraction, the detection of a small cathodal zone in the preparations of yound red cells may be related to the presence of residual mitochondria in reticulocytes. The same observation made in hemolysates rich in reticulocytes gives a further support to this hypothesis. Otherwise, only the anodic isoenzyme of cytoplasmic origin was detected in normal hemolysates and in old red cells, where the reticulocytes are present in very small amounts. The derivation of GOT II from contaminating white cells must be excluded because all the hemolysates were completely free from granulocytes and platelets and the lympho-monocytes present in the preparations were not sufficient to interfere with their own GOT activity. Clin. Chim. Acta,
14 (1966) 215-218
P. M. MANUCCI,
218
N. DIOGUARDI
We are grateful to Miss Marianne Schweitzer, Dr. G. IdCo and Dr. G. Fiorelli for helpful criticism and advice. REFERENCES I 2 3 4 5 6
G. A. FLEISHER, C. S. POTTER AND K. G. WAKIM, Proc. Sot. Exptl. Biol. Med., 103 (1960) 229. J. W. BOYD, C&n. Chim. Acta, 7 (1962) 424. J. S. NISSELBAUM AND 0. BODANSKY, J. Biol. Chem., 239 (1964) 4232. J. S. NISSELBAUM, Federation Proc., 24 (1965) 365. L. E. DECKER AND E. M. RAU, Proc. Sot. Expl. Biol. Med., 112 (1963) 144. J. G. BREWER, R. D. POWELL, T. R. TARLOV AND A. S. ALVING, J. Lab. Clin. Med., 63 (1964) 106.
C&z. Chim.
Acta,
14 (1966)
215-218
ELECTROPHORETIC TRANSAMINASE
P. M. MANNUCCI Institute
of Medical
(Received
21.5
CHARACTERIZATION
OF GLUTAMIC
IN HUMAN RED CELLS OF DIFFERENT
OXALACETIC AGES*
AND N. DIOGUARDI Pathology,
August z?th,
University
of Cagliari
(Italy)
1965)
SUMMARY
GOT isoenzymes of red cells from normal subjects, and from subjects with reticulocytosis have been analysed by cellogel electrophoresis. Hemolysates from normal and from relatively old red cells contain only the anodic fraction (GOT I) ; whereas hemolysates from subjects with reticulocytosis and from relatively young red cells contain, in addition, the cathodic fraction (GOT II). Since this isoenzyme is derived from mitochondria, it implies that these organelles are still present in reticulocytes and in young red cells.
Glutamic oxalacetic transaminase (GOT) has recently been demonstrated to exist in the different tissues in two isoenzymes which can be separated by means of electrophoresis and chromatographyl-S. It was shown that the anodal electrophoretic zone (GOT I) was derived from the cytoplasm and the cathodal zone from the mitochondrial fraction. In human erythrocytes only the anodal component was detected by Nisselbaum4. A simple method for the characterization of GOT isoenzymes has been performed in our laboratory by applying to cellogel electrophoresis the staining procedure developed by Decker and Raus. This report concerns a study of GOT isoenzymes in normal human hemolysates, in hemolysates obtained from red cells of different ages and from subjects with reticulocytosis. METHODS
Prefiaration of hemolysates 5 ml of heparinized venous blood were centrifuged at 170 g for IO min and the platelet-rich plasma was pipetted off and discarded. The blood cells, after resuspension at hematocrit levels in platelet-free plasma obtained by centrifuging at- 2000 g for 30 min, were poured into a glass column (20 x 1.2 cm), prewarmed at 37” and filled with loosely pressed ordinary cotton wool. The column was then inverted and * This work has been performed with the aid of the Assessorato Sanita Regione Autonoma Sarda. C&z. C&n.
Acta,
14 (1966) 215-218
216
P. M. MANUCCI,
N. DIOGUARDI
incubated at 37’ for 30 min. The elution was obtained using 0.15 M prewarmed saline, up to a final volume of nearly IO ml. The elution fluid contained erythrocytes, reticulocytes, lymphocytes and monocytes while granulocytes and platelets were absent. The removal of granulocytes is related to their property of phagocytosing the cotton wool; this material was as effective as glasswool, which was tested in preliminary experiments. The amount of saline used for the elution is fairly critical, because larger amounts tend to detach the granulocytes from the cotton wool. The few platelets unremoved in the platelet-rich plasma were retained by the cotton wool due to aggregation and adhesion. The elution fluid was then centrifuged twice at 350 g for IO min in a Modello Cagliari Centrifuge (Terzano, Milan). This allowed the lymphocytes and the monocytes to be removed with the supernatant whereas the red blood cells sedimented. The concentration of the lymphocytes and monocytes did not exceed 106 cells/ml. At this concentration the GOT activity of the lymphocytes is negligible. The packed erythrocytes were then washed three times with 0.15 M NaCl (I : 3 in volume) and hemolysis was obtained with distihed water in the proportion of I :I‘ and by freezing and thawing twice in an alcohol-ether mixture. The preparation was then submitted to an ultrasonic apparatus for 45 set and centrifuged at 2000 g in order to remove the stromata. Preparation of relatively old and reiatiuely young erythrocytes The erythrocytes of different ages were seperated by means of four centrifugations at 2000 g according to the technique of Brewer et aL.6.This method is based on the direct relationship existing between the age of the erythrocytes and their increase in density. The reticulocyte count showed a seven to tenfold increase in reticulocytes in the young (top) cell fraction as compared with the old (bottom) cell fraction. The quantitative level of GOT was three to fivefold higher in young erythrocytes. The two cell fractions were resuspended in their own plasma to an hematocrit level of approximately 50%. The same procedure described above was followed in order to obtain the hemolysates from these selected red cell preparations. The final preparations were thus completely free from granulocytes and platelets and contained only a negligible amount of lymphocytes and monocytes.
Electrophoretic separations were carried out on cellogel strips (17.5 x 2.5 cm) supplied by Chemetron, Milan. Phosphate K buffer of 0.04 M strength, of pH 7.5 and with a constant voltage of 8 V/cm for 3 h, was employed. The hemolysates were applied at a distance of 3 cm from the cathode in quantities of 0.001-0.003 ml. The isoenzymatic zones separated by electrophoresis were detected by staining the cellogel strips according to the method of Decker and Rau6. RESULTS
AND
DISCUSSION
GOT isoenzymatic pattern of normal hemolysates is shown in Fig. I. 22 individuals with normal reticulocyte count are all characterized by the presence of one zone alone which is anodic and faster than hemoglobin (GOT I). The same pattern was demonstrated in preparations of relatively old erythrocytes whereas all the preparations of young erythrocytes, containing a large amount C&L Chiw.
Acta,
14 (1966) 215-218
GOT TRANSAMINASE
IN RED CELLS
217
of reticulocytes, showed the presence of a small cathodal component (GOT II), moving slower than hemoglobin (Fig. 2). In studies carried out on 8 subjects recovering from acute hemolysis due to sensitivity to fava beans, and with a reticulocytic crisis; on 4 subjects with posthemorrhage reticulocytosis; and on 9 subjects with reticulocytosis related to various types of hemolytic anemias, the cathodal isoenzyme was always detectable when
Fig. I. Electrophoretic Reticulocytes I %.
separation of GOT isoenzymes in normal red cells. Normal hemolysate.
Fig. 2. Electrophoretic separation of GOT isoenzymes in relatively old red cells (a, reticulocytes 1.5%) and relatively young red cells (b, reticulocytes 1274)
Fig. 3. Electrophoretic separation of GOT isoenzymes in cases with reticulocytosis. (a) reticulocytes 3.5%, (b) reticulocytes 8.7%. (c) reticulocytes 12.3%, (d) reticulocytes 15.3%.
reticulocytes were up to 2.5-30/O. The intensity of the staining appeared to be related to the number of reticulocytes present in the preparation. Since the cathodal GOT isoenzyme has been shown to be derived from the mitochondrial fraction, the detection of a small cathodal zone in the preparations of yound red cells may be related to the presence of residual mitochondria in reticulocytes. The same observation made in hemolysates rich in reticulocytes gives a further support to this hypothesis. Otherwise, only the anodic isoenzyme of cytoplasmic origin was detected in normal hemolysates and in old red cells, where the reticulocytes are present in very small amounts. The derivation of GOT II from contaminating white cells must be excluded because all the hemolysates were completely free from granulocytes and platelets and the lympho-monocytes present in the preparations were not sufficient to interfere with their own GOT activity. Clin. Chim. Acta,
14 (1966) 215-218
P. M. MANUCCI,
218
N. DIOGUARDI
We are grateful to Miss Marianne Schweitzer, Dr. G. IdCo and Dr. G. Fiorelli for helpful criticism and advice. REFERENCES I 2 3 4 5 6
G. A. FLEISHER, C. S. POTTER AND K. G. WAKIM, Proc. Sot. Exptl. Biol. Med., 103 (1960) 229. J. W. BOYD, C&n. Chim. Acta, 7 (1962) 424. J. S. NISSELBAUM AND 0. BODANSKY, J. Biol. Chem., 239 (1964) 4232. J. S. NISSELBAUM, Federation Proc., 24 (1965) 365. L. E. DECKER AND E. M. RAU, Proc. Sot. Expl. Biol. Med., 112 (1963) 144. J. G. BREWER, R. D. POWELL, T. R. TARLOV AND A. S. ALVING, J. Lab. Clin. Med., 63 (1964) 106.
C&z. Chim.
Acta,
14 (1966)
215-218