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High-performance liquid chromatographic purification of the H4 and MH3 isoenzymes of lactate dehydrogenase from beef heart
Journal of Chromatography, 408 (1987) 372-377 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands CHROM. 19 835
Note
High-performance MH3 isoenzymes
liquid chromatographic of lactate dehydrogenase
Z. SKABRAHOVA*, J. KOVkR and Z. GLATZ Department of Biochemistry, Purkynje University, Kotlihkh
purification of the from beef heart
H4 and
2, 611 37 Brno (Czechoslovakia)
J. TURANEK Institute of LandscapeEcology, Czechoslovak Academy of Science, Na sctdkbch 7,370 05 ceskk Bucejovice (Czechoslovakia) and V. KAHLE Institute of Analytical Chemistry, ESAV, Leninova 82, 611 42 Brno (Czechoslovakia) (Received July 3rd, 1987)
Lactate dehydrogenase (LDH, E.C. 1.1.1.27) is a part of the glycolytic system and is ubiquitous in animal cellsl. Considerable evidence has been accumulated for the multiple forms of lactate dehydrogenase l. There are two major structural genes corresponding to the H and M subunits, the tetrameric molecule of the enzyme occurring in five isoenzymes (H4, H3M, H2M2, HM3, M4). The abundance of these isoenzyme forms is different in various tissues of vertebrate organisms, H4 and MH3 prevailing in the heart muscle Is2. Lactate dehydrogenase is used in analytical biochemistry in coupled optical tests for the determination of several enzyme activities, e.g., alanine aminotransferase, aspartate aminotransferase, phosphoglycerate kinase, myokinase, creatine kinase and pyruvate kinase 3. It is also used for the enzymatic determination of numerous metabolites, such as lactate, pyruvate, NADH and NAD, ADP and AMP, phosphoenolpyruvate, 2_phosphoglycerate, 3-phosphoglycerate and creatine3. Lactate dehydrogenase is found in the cytoplasm and is easily extracted into solution when cells are broken. The purification procedures mostly involve ammonium sulphate fractionation2p4 and ion-exchange chromatography2p5. Several isolation procedures using affinity resins have also been described6-8. The objective of the present paper is to describe a quick semi-preparative procedure for the isolation of H4 and MH3 isoenzymes of lactate dehydrogenase using ammonium sulphate fractionation and three high-performance chromatographic steps. Hydrophobic interaction chromatography on Spheron-Mikro was used in the first chromatographic separation since this method proved to be very useful in the purification of malate dehydrogenase from beef heartg. EXPERIMENTAL
Materials
Beef hearts were obtained from a slaughter house and were stored at - 60°C. 0021-9673/87/$03.50
%
1987 Elsevier Science Publishers B.V.
NOTES
373
NADH and sodium pyruvate were obtained from Reanal (Budapest, Hungary) and Lachema (Brno, Czechoslovakia), respectively. The materials used for the electrophoretic separations were mostly from Serva (Heidelberg, F.R.G.). Enzyme preparation
Beef heart tissue (150 g) was homogenized with 300 ml of 20 mA4 sodium phosphate buffer (pH 7). The supernatant obtained after centrifugation at 6000 g for 30 min was precipitated with ammonium sulphate, most of the LDH activity being salted out between 40 and 60% saturation. These initial procedures were carried out at 4°C the following chromatographic steps at room temperature (the eluted fraction being kept in an ice-bath). The sediment of crude LDH was dissolved in 0.1 M sodium phosphate buffer (pH 7) and separated in two steps on a glass column (120 mm x 12 mm I.D.) packed with Spheron-Micro (12 pm, Lachema). The column was attached to two pumps (P-500) and a liquid chromatography controller (LCC-500) from Pharmacia (Uppsala, Sweden). The starting and terminating buffers were 0.1 M sodium phosphate (pH 7) with ammonium sulphate to 30% saturation and 0.02 M sodium phosphate (pH 7) (flow-rate 5 ml/min). The samples were injected with a V-7 valve equipped with a lo-ml superloop (Pharmacia) and sample loops of various volumes. The separations were evaluated by an UV-1 monitor (J. = 280 nm), a FRAC-100 collector (Pharmacia) and an LCC-500 apparatus. The fractions containing LDH were concentrated to ca. 50 mg protein per ml with an ultrafiltration cell and an XM-30 membrane (Amicon, Danvers, MA, U.S.A.). The sample was divided into two portions and applied to an Ultropac TSK 3000 SWG column (600 mm x 21.5 mm I.D.; LKB, Bromma, Sweden) attached to the above-mentioned chromatographic system; 20 mM sodium phosphate buffer (pH 7) was used as the mobile phase (flow-rate 6 ml/min). The active fractions were pooled, their pH was brought to 8 and this solution was injected, in two portions, onto a Mono Q HR 5/5 column (50 mm x 5 mm I.D.) from Pharmacia, attached to the equipment described. The starting and terminating buffers were 20 mM sodium phosphate buffer (pH 8) and the same buffer with 0.8 M sodium chloride (flow-rate 2 ml/min). Enzyme analysis
The activity of LDH was assayed spectrophotometrically in the presence of NADH and pyruvate at 25°C (ref. 3); the protein concentration was calculated from the absorbance at 280 nm. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected by means of the Monotests (Boehringer, Mannheim, F.R.G.). The spectrophotometric measurements were performed on a Cary 118 instrument (Varian, Palo Alto, CA, U.S.A.). Electrophoretic analysis of LDH was carried out in 7% polyacrylamide gels as describedlO. The gels were stained for protein with Coomassie Brilliant Blue R 250 and for the LDH activity according to ref. 11. RESULTS AND DISCUSSION
The commonly used ammonium sulphate fractionation was carried out as the first purification step in the isolation procedure described. It brought about a four-
NOTES
374 TABLE I PURIFICATION OF THE H4 AND MH3 ISOENZYMES FROM BEEF HEART
OF LACTATE DEHYDROGENASE
Details of the procedure as in the text. Fraction
Crude supernatant Ammonium sulphate fractionation (40-60%) Hydrophobic chromatography (Spheron-Micro)* Gel permeation chromatography (Ultropac TSK 3000 SWG)** Anion-exchange chromatography (Mono Q) H4 MH3 M2H2
l
Puri$cation fold
Recovery
Protein
Total activity
Specific activity
(WI
iv)
i Ulmll
6100
12 600
2.1
(1)
(IGO)
840
7000
8.3
4
55
105
4800
45.7
22
38
17
3610
212
101
29
5.4 3.5 2.8
1510 1440 210
280 410 75
W)
25
* The separations were carried out in three steps. * The separations were carried out in two steps.
fold puridication of LDH and a reduction of the sample volume (Table I). Moreover, this step was compatible with the subsequent hydrophobic interaction chromatography; the salted-out homogenate could be used directly without removal of salts. The chromatography on a Spheron-Micro column proved to be a convenient method for further purification of LDH (Fig. 1). The recovery of LDH activity was good (cu. 70%), however the purity of the final preparation was not as high as in the case ofmalate dehydrogenaseg (see Table I). The main reason for this was that LDH was eluted in a large protein peak at the higher salt concentration owing to its lower hydrophabicity in comparison with malate dehydrogenase. The electrophoretic analysis showed that all the LDH isoenzymes were present in the peak with LDH activity (Fig. l), H4 and MH3 being abundant, M2H2 with moderate activity and M3H and M4 being present in negligible amounts. The subsequent step, i.e., gel permeation chromatography, brought about a ca. five-fold purification of LDH, the separation af low-molecular-weight protein being especially good (Fig. 2, Table’ I). The recovery of LDH in this step was nearly 90%. Moreover, the chromatography yielded nearly pure myoglobin as a by-product; it was eluted as the last peak in the chromatagram shown in Fig. 2 and was identified spectrophotometrically and electropharetically. The material passed through un Ultropac TSK 3000 SWG column was suitable for the last purification step, i.e., ion exchange-chromatography, since the ionic strength of the mobile phase used was low (see Experimental). The conditions for the chromatography on the Mono Q column were similar to those used in ref. 12. The MH3 and H4 isaenzymes of LDH were obtained as homogeneous proteins with high LDH activities (Fig. 3), H4 constituting the highest peak, MH3 the peak preceding it. The homogeneity of these isoenzymes was determined by electraphoresis followed
NOTES
2.
A280
l-
37.5
s
Fig. 1, Chromatography of crude lactate dehydrogenase on a Spheron-Micro column.Buffers:A, 0.1 M sodium phosphate (PH 7) with ammonium sulphate to 30% saturation; B, 0.02 M sodium phosphate (pH absorbance at 280 nm (Azso); - - - ~ -, gradient; 1, LDH activity. 7). V, = Elution volume; -, Approximately 200 mg of protein were applied to the column (flow-rate 5 ml/min). Fig. 2. Chromatography of partially purified lactate dehydrogenase on an Ultropac TSK 3000 SWG column 20 mM sodium phosphate (pH 7) was used as the mobile phase (flow-rate 6 ml/min); other details as in Fig. 1. Approximately 50 mg of protein were injected.
by parallel staining for LDH activity and protein. No protein fractions except the H4 and MH3 isoenzymes of LDH were detected in these two main fractions. On the other hand, the M2H2 isoenzyme was not homogeneous, its activity being less than 7% of the total LDH activity (see Fig. 3 and Table I), and the other two isoenzymes (M3H and M4) were completely missing. The results obtained by electrophoresis were confirmed by the values of the specific activities of the isoenzymes (see Table I), which are comparable with those given by the supplier of LDH13 in the case of the H4 and MH3 isoenzymes. When a column of identical dimensions packed with DEAE-Spheron-Micro was used instead of the Mono Q column, similar results were obtained, but the efficiency of separation was slightly worse. The rapid chromatographic procedure described here is suitable for the preparation of several milligrams of the homogeneous H4 and MH3 isoenzymes of beef heart LDH. The chromatographic steps (see Table I) can be carried out in cc1. 5 h, includ ng the concentration with the ultrafiltration cell. Therefore the whole procedure s1arting from the heart tissue can be performed in one working day. The purified isoenz’ mes of LDH can be used for enzymological studies as well as for analytical purpo, I es. The H4 isoenzyme is more suitable in the former case since it is comprised of only one type of subunits, whereas the MH3 isoenzyme is more convenient in the latter case due to its higher specific activity (see Table I; cj, ref. 13). The purified isoenzymes are devoid of any contaminating enzyme activity, i.e., aminotransferase and their amounts (Table I) are sufficient for more than 2000 assays of alanine ami-
376
NOTES
2-
A280
0
20
40 V, (ml1
Fig. 3. Separation of isoenzymes of lactate dehydrogenase on a Mono Q column. Buffers: A, 20 mM sodium phosphate (pH 8); B, same as A but with 0.8 M sodium chloride. Full and broken arrows correspond to high and low LDH activities, respectively; other details as in Fig. 1. Approximately 7 mg of protein were injected (flow-rate 2 ml/min). The isoenzymes of LDH identified by electrophoresis were: M2H2 (peak indicated with a broken arrow); MH3 (full arrow, Azso = 1.15) and H4 (fuI1 arrow, Azso = 2.03).
notransferase or aspartate aminotransferase; the LDH activity required for these assays is 1.2 U/ml, cJ, refs. 14 and 15. It is interesting that even the partially purified LDH (after hydrophobic interaction chromatography) can be used for the determination of alanine aminotransferase activity since this sdample is devoid of any detectable activity from this aminotransferase. On the other hand, this preparation contains a measurable activity due to aspartate aminotransferase, ca. 0.2% of the LDH activity. If these preparations were to be used for the assay of aspartate aminotransferase in human blood sera, the LDH activity would enhance the blank values of aspartate aminotransferase by ca. 20%, cJ, ref. 14. ACKNOWLEDGEMENT
The authors thank Dr. M. Smri for his gift of Spherons and for valuable suggestions. REFERENCES 1 G. W. Schwert and A. D. Winer, in P. D. Boyer (Editor), The Enzymes, Vol. 7, Academic Press, New York, 2nd ed., 1963, p. 127. 2 J. J. Holbrook, A. Liljas, A. Steindel and M. G. Rossmann, in P. D. Boyer (Editor), The Enzymes, Vol. 11, Academic Press, New York, 3rd ed., 1975, p. 191. 3 Biochemica Information, Boehringer, Mannheim, 1973. 4 G. J&csai, Acta Phvsiol. Acad. Sri. Hunf.. 20 (1962) 339.
NOTES
371
5 G. von Beisenherz, H. J. Boltze, T. Biicher, R. Czok, K. H. Garbade, E. Meyer-Arendt and G. Fileiderer, Z. Naturforsch.. Teil 3, 8 (1953) 555. 6 K. Mosbach, H. Guilford, P.-O. Larsson, R. Ohlsson and M. Scott, Biochem. J., 125 (1971) 20. 7 K. Mosbach, H. Guilford, R. Ohlason and M. Scott, Biochem. J., 127 (1972) 625. 8 C. R. Lowe and P. D. G. Dean, Biochem. J., 133 (1973) 515. 9 Z. SkabrahovB, J. Turinek, J. Kov&i and Z. Glatz, J. Chromatogr., 369 {1986) 426. 10 B. J. Davis, Ann. NY Acad. Sci., 121 (1961) 404. 11 I. H. Fine and L. A. Costello, Methods Enzymol., 4 (1963) 958. 12 L. A. Haff, L. G. Flgerstam and A. R. Barry, J. Chromatogr., 266 (1983) 409. 13 Biochemical Catalogue, Boehringer, Mannheim, 1986. 14 COT opt. Monotest, Boehringer, Mannheim, 1980. 15 GPT opt. Monotest, Boehringer, Mannheim, 1980.
Note
High-performance MH3 isoenzymes
liquid chromatographic of lactate dehydrogenase
Z. SKABRAHOVA*, J. KOVkR and Z. GLATZ Department of Biochemistry, Purkynje University, Kotlihkh
purification of the from beef heart
H4 and
2, 611 37 Brno (Czechoslovakia)
J. TURANEK Institute of LandscapeEcology, Czechoslovak Academy of Science, Na sctdkbch 7,370 05 ceskk Bucejovice (Czechoslovakia) and V. KAHLE Institute of Analytical Chemistry, ESAV, Leninova 82, 611 42 Brno (Czechoslovakia) (Received July 3rd, 1987)
Lactate dehydrogenase (LDH, E.C. 1.1.1.27) is a part of the glycolytic system and is ubiquitous in animal cellsl. Considerable evidence has been accumulated for the multiple forms of lactate dehydrogenase l. There are two major structural genes corresponding to the H and M subunits, the tetrameric molecule of the enzyme occurring in five isoenzymes (H4, H3M, H2M2, HM3, M4). The abundance of these isoenzyme forms is different in various tissues of vertebrate organisms, H4 and MH3 prevailing in the heart muscle Is2. Lactate dehydrogenase is used in analytical biochemistry in coupled optical tests for the determination of several enzyme activities, e.g., alanine aminotransferase, aspartate aminotransferase, phosphoglycerate kinase, myokinase, creatine kinase and pyruvate kinase 3. It is also used for the enzymatic determination of numerous metabolites, such as lactate, pyruvate, NADH and NAD, ADP and AMP, phosphoenolpyruvate, 2_phosphoglycerate, 3-phosphoglycerate and creatine3. Lactate dehydrogenase is found in the cytoplasm and is easily extracted into solution when cells are broken. The purification procedures mostly involve ammonium sulphate fractionation2p4 and ion-exchange chromatography2p5. Several isolation procedures using affinity resins have also been described6-8. The objective of the present paper is to describe a quick semi-preparative procedure for the isolation of H4 and MH3 isoenzymes of lactate dehydrogenase using ammonium sulphate fractionation and three high-performance chromatographic steps. Hydrophobic interaction chromatography on Spheron-Mikro was used in the first chromatographic separation since this method proved to be very useful in the purification of malate dehydrogenase from beef heartg. EXPERIMENTAL
Materials
Beef hearts were obtained from a slaughter house and were stored at - 60°C. 0021-9673/87/$03.50
%
1987 Elsevier Science Publishers B.V.
NOTES
373
NADH and sodium pyruvate were obtained from Reanal (Budapest, Hungary) and Lachema (Brno, Czechoslovakia), respectively. The materials used for the electrophoretic separations were mostly from Serva (Heidelberg, F.R.G.). Enzyme preparation
Beef heart tissue (150 g) was homogenized with 300 ml of 20 mA4 sodium phosphate buffer (pH 7). The supernatant obtained after centrifugation at 6000 g for 30 min was precipitated with ammonium sulphate, most of the LDH activity being salted out between 40 and 60% saturation. These initial procedures were carried out at 4°C the following chromatographic steps at room temperature (the eluted fraction being kept in an ice-bath). The sediment of crude LDH was dissolved in 0.1 M sodium phosphate buffer (pH 7) and separated in two steps on a glass column (120 mm x 12 mm I.D.) packed with Spheron-Micro (12 pm, Lachema). The column was attached to two pumps (P-500) and a liquid chromatography controller (LCC-500) from Pharmacia (Uppsala, Sweden). The starting and terminating buffers were 0.1 M sodium phosphate (pH 7) with ammonium sulphate to 30% saturation and 0.02 M sodium phosphate (pH 7) (flow-rate 5 ml/min). The samples were injected with a V-7 valve equipped with a lo-ml superloop (Pharmacia) and sample loops of various volumes. The separations were evaluated by an UV-1 monitor (J. = 280 nm), a FRAC-100 collector (Pharmacia) and an LCC-500 apparatus. The fractions containing LDH were concentrated to ca. 50 mg protein per ml with an ultrafiltration cell and an XM-30 membrane (Amicon, Danvers, MA, U.S.A.). The sample was divided into two portions and applied to an Ultropac TSK 3000 SWG column (600 mm x 21.5 mm I.D.; LKB, Bromma, Sweden) attached to the above-mentioned chromatographic system; 20 mM sodium phosphate buffer (pH 7) was used as the mobile phase (flow-rate 6 ml/min). The active fractions were pooled, their pH was brought to 8 and this solution was injected, in two portions, onto a Mono Q HR 5/5 column (50 mm x 5 mm I.D.) from Pharmacia, attached to the equipment described. The starting and terminating buffers were 20 mM sodium phosphate buffer (pH 8) and the same buffer with 0.8 M sodium chloride (flow-rate 2 ml/min). Enzyme analysis
The activity of LDH was assayed spectrophotometrically in the presence of NADH and pyruvate at 25°C (ref. 3); the protein concentration was calculated from the absorbance at 280 nm. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected by means of the Monotests (Boehringer, Mannheim, F.R.G.). The spectrophotometric measurements were performed on a Cary 118 instrument (Varian, Palo Alto, CA, U.S.A.). Electrophoretic analysis of LDH was carried out in 7% polyacrylamide gels as describedlO. The gels were stained for protein with Coomassie Brilliant Blue R 250 and for the LDH activity according to ref. 11. RESULTS AND DISCUSSION
The commonly used ammonium sulphate fractionation was carried out as the first purification step in the isolation procedure described. It brought about a four-
NOTES
374 TABLE I PURIFICATION OF THE H4 AND MH3 ISOENZYMES FROM BEEF HEART
OF LACTATE DEHYDROGENASE
Details of the procedure as in the text. Fraction
Crude supernatant Ammonium sulphate fractionation (40-60%) Hydrophobic chromatography (Spheron-Micro)* Gel permeation chromatography (Ultropac TSK 3000 SWG)** Anion-exchange chromatography (Mono Q) H4 MH3 M2H2
l
Puri$cation fold
Recovery
Protein
Total activity
Specific activity
(WI
iv)
i Ulmll
6100
12 600
2.1
(1)
(IGO)
840
7000
8.3
4
55
105
4800
45.7
22
38
17
3610
212
101
29
5.4 3.5 2.8
1510 1440 210
280 410 75
W)
25
* The separations were carried out in three steps. * The separations were carried out in two steps.
fold puridication of LDH and a reduction of the sample volume (Table I). Moreover, this step was compatible with the subsequent hydrophobic interaction chromatography; the salted-out homogenate could be used directly without removal of salts. The chromatography on a Spheron-Micro column proved to be a convenient method for further purification of LDH (Fig. 1). The recovery of LDH activity was good (cu. 70%), however the purity of the final preparation was not as high as in the case ofmalate dehydrogenaseg (see Table I). The main reason for this was that LDH was eluted in a large protein peak at the higher salt concentration owing to its lower hydrophabicity in comparison with malate dehydrogenase. The electrophoretic analysis showed that all the LDH isoenzymes were present in the peak with LDH activity (Fig. l), H4 and MH3 being abundant, M2H2 with moderate activity and M3H and M4 being present in negligible amounts. The subsequent step, i.e., gel permeation chromatography, brought about a ca. five-fold purification of LDH, the separation af low-molecular-weight protein being especially good (Fig. 2, Table’ I). The recovery of LDH in this step was nearly 90%. Moreover, the chromatography yielded nearly pure myoglobin as a by-product; it was eluted as the last peak in the chromatagram shown in Fig. 2 and was identified spectrophotometrically and electropharetically. The material passed through un Ultropac TSK 3000 SWG column was suitable for the last purification step, i.e., ion exchange-chromatography, since the ionic strength of the mobile phase used was low (see Experimental). The conditions for the chromatography on the Mono Q column were similar to those used in ref. 12. The MH3 and H4 isaenzymes of LDH were obtained as homogeneous proteins with high LDH activities (Fig. 3), H4 constituting the highest peak, MH3 the peak preceding it. The homogeneity of these isoenzymes was determined by electraphoresis followed
NOTES
2.
A280
l-
37.5
s
Fig. 1, Chromatography of crude lactate dehydrogenase on a Spheron-Micro column.Buffers:A, 0.1 M sodium phosphate (PH 7) with ammonium sulphate to 30% saturation; B, 0.02 M sodium phosphate (pH absorbance at 280 nm (Azso); - - - ~ -, gradient; 1, LDH activity. 7). V, = Elution volume; -, Approximately 200 mg of protein were applied to the column (flow-rate 5 ml/min). Fig. 2. Chromatography of partially purified lactate dehydrogenase on an Ultropac TSK 3000 SWG column 20 mM sodium phosphate (pH 7) was used as the mobile phase (flow-rate 6 ml/min); other details as in Fig. 1. Approximately 50 mg of protein were injected.
by parallel staining for LDH activity and protein. No protein fractions except the H4 and MH3 isoenzymes of LDH were detected in these two main fractions. On the other hand, the M2H2 isoenzyme was not homogeneous, its activity being less than 7% of the total LDH activity (see Fig. 3 and Table I), and the other two isoenzymes (M3H and M4) were completely missing. The results obtained by electrophoresis were confirmed by the values of the specific activities of the isoenzymes (see Table I), which are comparable with those given by the supplier of LDH13 in the case of the H4 and MH3 isoenzymes. When a column of identical dimensions packed with DEAE-Spheron-Micro was used instead of the Mono Q column, similar results were obtained, but the efficiency of separation was slightly worse. The rapid chromatographic procedure described here is suitable for the preparation of several milligrams of the homogeneous H4 and MH3 isoenzymes of beef heart LDH. The chromatographic steps (see Table I) can be carried out in cc1. 5 h, includ ng the concentration with the ultrafiltration cell. Therefore the whole procedure s1arting from the heart tissue can be performed in one working day. The purified isoenz’ mes of LDH can be used for enzymological studies as well as for analytical purpo, I es. The H4 isoenzyme is more suitable in the former case since it is comprised of only one type of subunits, whereas the MH3 isoenzyme is more convenient in the latter case due to its higher specific activity (see Table I; cj, ref. 13). The purified isoenzymes are devoid of any contaminating enzyme activity, i.e., aminotransferase and their amounts (Table I) are sufficient for more than 2000 assays of alanine ami-
376
NOTES
2-
A280
0
20
40 V, (ml1
Fig. 3. Separation of isoenzymes of lactate dehydrogenase on a Mono Q column. Buffers: A, 20 mM sodium phosphate (pH 8); B, same as A but with 0.8 M sodium chloride. Full and broken arrows correspond to high and low LDH activities, respectively; other details as in Fig. 1. Approximately 7 mg of protein were injected (flow-rate 2 ml/min). The isoenzymes of LDH identified by electrophoresis were: M2H2 (peak indicated with a broken arrow); MH3 (full arrow, Azso = 1.15) and H4 (fuI1 arrow, Azso = 2.03).
notransferase or aspartate aminotransferase; the LDH activity required for these assays is 1.2 U/ml, cJ, refs. 14 and 15. It is interesting that even the partially purified LDH (after hydrophobic interaction chromatography) can be used for the determination of alanine aminotransferase activity since this sdample is devoid of any detectable activity from this aminotransferase. On the other hand, this preparation contains a measurable activity due to aspartate aminotransferase, ca. 0.2% of the LDH activity. If these preparations were to be used for the assay of aspartate aminotransferase in human blood sera, the LDH activity would enhance the blank values of aspartate aminotransferase by ca. 20%, cJ, ref. 14. ACKNOWLEDGEMENT
The authors thank Dr. M. Smri for his gift of Spherons and for valuable suggestions. REFERENCES 1 G. W. Schwert and A. D. Winer, in P. D. Boyer (Editor), The Enzymes, Vol. 7, Academic Press, New York, 2nd ed., 1963, p. 127. 2 J. J. Holbrook, A. Liljas, A. Steindel and M. G. Rossmann, in P. D. Boyer (Editor), The Enzymes, Vol. 11, Academic Press, New York, 3rd ed., 1975, p. 191. 3 Biochemica Information, Boehringer, Mannheim, 1973. 4 G. J&csai, Acta Phvsiol. Acad. Sri. Hunf.. 20 (1962) 339.
NOTES
371
5 G. von Beisenherz, H. J. Boltze, T. Biicher, R. Czok, K. H. Garbade, E. Meyer-Arendt and G. Fileiderer, Z. Naturforsch.. Teil 3, 8 (1953) 555. 6 K. Mosbach, H. Guilford, P.-O. Larsson, R. Ohlsson and M. Scott, Biochem. J., 125 (1971) 20. 7 K. Mosbach, H. Guilford, R. Ohlason and M. Scott, Biochem. J., 127 (1972) 625. 8 C. R. Lowe and P. D. G. Dean, Biochem. J., 133 (1973) 515. 9 Z. SkabrahovB, J. Turinek, J. Kov&i and Z. Glatz, J. Chromatogr., 369 {1986) 426. 10 B. J. Davis, Ann. NY Acad. Sci., 121 (1961) 404. 11 I. H. Fine and L. A. Costello, Methods Enzymol., 4 (1963) 958. 12 L. A. Haff, L. G. Flgerstam and A. R. Barry, J. Chromatogr., 266 (1983) 409. 13 Biochemical Catalogue, Boehringer, Mannheim, 1986. 14 COT opt. Monotest, Boehringer, Mannheim, 1980. 15 GPT opt. Monotest, Boehringer, Mannheim, 1980.