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Lactate dehydrogenase isozymes in human and rat fetal liver and lung
Life Sciences Vol. 8, Part II, pp . 33-37, 1969 . Printed in Great Britain.
Pergamon Press
LACTATE DEHYDROGENASE ISOZYMES IN HUMAN AND RAT FETAL LIVER AND LUNG Ralph P. Francesconi and Claude A . Villee Department of Biological Chemistry, Harvard Medical School, and Laboratory of Reproductive Biology, Boston Hospital for Women. (Received 30 September 1968 ; in final form 30 October 1968) Introduction In many species of vert6brates, lactate dehydrogenase (ED 1.1 .1.27, L-lactate: NAD oxidoreductase) exists in at least five electrophoretically separable forms or isozymes (1).
The presence of five forms has been explained
by postulating that there are two types of subunits which associate in the five possible combinations to form tetramers (2, 3) .
Quantitative and qualitative
variations in the distribution of isozymes have been reported during development (4), in malignant tissues (5), during liver regeneration (6), and among the various tissues of a given individual (7) . Such alterations have been attributed to changes in the relative rates of synthesis of the two subunits . LDH-1 and LDH-5 were separated by DEAE column chromatography and electrophoresis, mixed with 1 M NaCl, and frozen overnight (8).
On sub-
sequent starch gel electrophoresis, all five isozymes appeared in approximately the expected proportions of 1:4 :6 :4 :1 .
However, the discovery of a
sixth isozyme of LDH (9) in testis and sperm required that a third kind of subunit be present in these tissues . Further evidence of a third polypeptide subunit came from the demonstration of as many as nine forms of LDH in the muscle, testes or sperm, heart, and ovary of several species of fish (10) .
33
34
LDH ISOZYMES
Vol. 8, No. 2
Methods In the present investigation multiple molecular forms of LDH were demonstrated by polyacrylamide gel electrophoresis according to the methods of Ornstein (11) and Davis (12) .
Human fetuses, 13 and 17 weeks of gestational
age, were obtained immediately at therapeutic abortion and fetal rats, 19 days gestational age, were obtained by Cesarean section.
Ten percent homogen-
ates of liver and lung were prepared and centrifuged at 20, 000 x g. One tenth ml of the supernatant solution was subjected to disc gel electrophoresis, and isozyme patterns were developed with the standard techniques utilizing nitrobluetetrazolium, phenazine methosulfate, nicotinamide adenine dinucleotide, and lactate in Tris HCl buffer, 0 .1 M pH 7. 6 (5) . Results In the lungs and liver from both human fetal specimens, LDH isozymes 1, 2, and 3 appeared to be identical with regard to electrophoretic mobility and subbanding patterns (Fig . 1) .
However, LDH-4, while clearly pre
sent in the fetal liver preparation, was absent from the zymograms of lung tissue of 13 and 17 weeks fetuses.
The isozyme patterns of liver and lung
from 19 day fetal rats did not demonstrate this degree of similarity with regard to electrophoretic mobility (Fig . 2) . The absence of LDH-4 from human fetal lung is of interest as an example of tissue specificity of isozyme patterns, and for its bearing on the theory of the random association of subunits to form a tetrameric enzyme molecule . Intensely staining isozymes corresponding to LDH-1, LDH-2, and LDH-3 were present, and one would expect LDH-4 and 5 also to be present, especially since isozymes 1, 2 and 3 of liver and lung were identical and liver
Vol . a, No. 2
LDH MOZYMES
35
FIG . 1
Human Fetal
s
ÌiI' Liver
ii Lung 13 Week
+
*11
Ii :, Liver laag 17 Week
Electrophoregram of LDH isozymes from human fetal liver and lung . time of electrophoresis was approximately 50 minutes in all cases . FIG. 2
Rat Fetal
Liver
Lung
19 Day
Electrophoregram of LDH isozymes from rat fetal liver and lung .
The
36
LDH ISOZYMES
Vol . 8, No. 2
contained LDH-4. It is possible that in human fetal lung the independent synthesis of the A polypeptide is much less than that of subunit B and the fraction of LDH activity present in LDH-4 is so small that it is not detected by this staining method .
If, for example, nine B subunits were made for every one A sub-
unit and these underwent random association to form tetramers, the fraction of .LDH activity in LDtl-1 to 5 would be 0. 66, 0.29, 0. 05, 0.0036 and 0. 0001 respectively .
In this circumstance, the more than twelve fold difference in
staining between LDH-1 and LDH-3 should be detectable, yet in fetal lung LDH-3 was, if anything, more darkly stained than LDH-1 or LDH-2 . Alternatively, one might suppose that in certain tissues - such as the lung - the association of subunits does not occur at random and that the formation of AlB 3 is prevented.
A third possibility is that the AlB3 tetramer
is formed but is enzymically inactive . Acknowledgement This work was supported by Grant #HD06 from the National Institute of Child Health and Human Development. References 1.
B.O . WIGGERT and C.A . VILLEE, J. Biol . Chem . , 239 (2), 444 (1964) ; C. L. MARKERT and F. MOLLER, Proc . Nat. Acad . Sci. , 45, 753 (1959) .
2.
E. APPELLA and C. L. MARKERT, Biochem. Biophys . Res. Comm . , 6 (3), 171 (1961) .
3.
A. C . WILSON, R. D. CAHN, and N. O. KAPLAN, Nature , 197, 331 (1963) .
4.
R. D. CAHN, Dev. Biol . , 9, 327 (1964) .
Vol . 8, No. 2
LDH ISOZYMES
37
5.
D. KADLECOVA, V. KOLAR, and Z. MECHI, Neoplasma, 14 (5), 473 (1967) .
6.
M. M. THALER, R. P. FRANCESCONI, and C .A . VILLES, _Life Sciences, 7 (4), 175 (1968) .
7.
A.E . EMERY, Biochem. J . , 105, 599 (1967) .
8.
C . L . MARKERT, Science, 140,
9.
A . BLANCO and W. H. ZINKHAM, Science, 139, 601 (1963) .
329 (1963) .
10 .
E . GOLDBERG, Science, 148, 391 (1965) .
11 .
L. ORNSTEIN, Ann. N.Y .
12 .
B . J. DAVIS, Ann. N.Y . Acad . Sci., 121, 404 (1964) .
ad . Sci, 121, 321 (1964) .
Pergamon Press
LACTATE DEHYDROGENASE ISOZYMES IN HUMAN AND RAT FETAL LIVER AND LUNG Ralph P. Francesconi and Claude A . Villee Department of Biological Chemistry, Harvard Medical School, and Laboratory of Reproductive Biology, Boston Hospital for Women. (Received 30 September 1968 ; in final form 30 October 1968) Introduction In many species of vert6brates, lactate dehydrogenase (ED 1.1 .1.27, L-lactate: NAD oxidoreductase) exists in at least five electrophoretically separable forms or isozymes (1).
The presence of five forms has been explained
by postulating that there are two types of subunits which associate in the five possible combinations to form tetramers (2, 3) .
Quantitative and qualitative
variations in the distribution of isozymes have been reported during development (4), in malignant tissues (5), during liver regeneration (6), and among the various tissues of a given individual (7) . Such alterations have been attributed to changes in the relative rates of synthesis of the two subunits . LDH-1 and LDH-5 were separated by DEAE column chromatography and electrophoresis, mixed with 1 M NaCl, and frozen overnight (8).
On sub-
sequent starch gel electrophoresis, all five isozymes appeared in approximately the expected proportions of 1:4 :6 :4 :1 .
However, the discovery of a
sixth isozyme of LDH (9) in testis and sperm required that a third kind of subunit be present in these tissues . Further evidence of a third polypeptide subunit came from the demonstration of as many as nine forms of LDH in the muscle, testes or sperm, heart, and ovary of several species of fish (10) .
33
34
LDH ISOZYMES
Vol. 8, No. 2
Methods In the present investigation multiple molecular forms of LDH were demonstrated by polyacrylamide gel electrophoresis according to the methods of Ornstein (11) and Davis (12) .
Human fetuses, 13 and 17 weeks of gestational
age, were obtained immediately at therapeutic abortion and fetal rats, 19 days gestational age, were obtained by Cesarean section.
Ten percent homogen-
ates of liver and lung were prepared and centrifuged at 20, 000 x g. One tenth ml of the supernatant solution was subjected to disc gel electrophoresis, and isozyme patterns were developed with the standard techniques utilizing nitrobluetetrazolium, phenazine methosulfate, nicotinamide adenine dinucleotide, and lactate in Tris HCl buffer, 0 .1 M pH 7. 6 (5) . Results In the lungs and liver from both human fetal specimens, LDH isozymes 1, 2, and 3 appeared to be identical with regard to electrophoretic mobility and subbanding patterns (Fig . 1) .
However, LDH-4, while clearly pre
sent in the fetal liver preparation, was absent from the zymograms of lung tissue of 13 and 17 weeks fetuses.
The isozyme patterns of liver and lung
from 19 day fetal rats did not demonstrate this degree of similarity with regard to electrophoretic mobility (Fig . 2) . The absence of LDH-4 from human fetal lung is of interest as an example of tissue specificity of isozyme patterns, and for its bearing on the theory of the random association of subunits to form a tetrameric enzyme molecule . Intensely staining isozymes corresponding to LDH-1, LDH-2, and LDH-3 were present, and one would expect LDH-4 and 5 also to be present, especially since isozymes 1, 2 and 3 of liver and lung were identical and liver
Vol . a, No. 2
LDH MOZYMES
35
FIG . 1
Human Fetal
s
ÌiI' Liver
ii Lung 13 Week
+
*11
Ii :, Liver laag 17 Week
Electrophoregram of LDH isozymes from human fetal liver and lung . time of electrophoresis was approximately 50 minutes in all cases . FIG. 2
Rat Fetal
Liver
Lung
19 Day
Electrophoregram of LDH isozymes from rat fetal liver and lung .
The
36
LDH ISOZYMES
Vol . 8, No. 2
contained LDH-4. It is possible that in human fetal lung the independent synthesis of the A polypeptide is much less than that of subunit B and the fraction of LDH activity present in LDH-4 is so small that it is not detected by this staining method .
If, for example, nine B subunits were made for every one A sub-
unit and these underwent random association to form tetramers, the fraction of .LDH activity in LDtl-1 to 5 would be 0. 66, 0.29, 0. 05, 0.0036 and 0. 0001 respectively .
In this circumstance, the more than twelve fold difference in
staining between LDH-1 and LDH-3 should be detectable, yet in fetal lung LDH-3 was, if anything, more darkly stained than LDH-1 or LDH-2 . Alternatively, one might suppose that in certain tissues - such as the lung - the association of subunits does not occur at random and that the formation of AlB 3 is prevented.
A third possibility is that the AlB3 tetramer
is formed but is enzymically inactive . Acknowledgement This work was supported by Grant #HD06 from the National Institute of Child Health and Human Development. References 1.
B.O . WIGGERT and C.A . VILLEE, J. Biol . Chem . , 239 (2), 444 (1964) ; C. L. MARKERT and F. MOLLER, Proc . Nat. Acad . Sci. , 45, 753 (1959) .
2.
E. APPELLA and C. L. MARKERT, Biochem. Biophys . Res. Comm . , 6 (3), 171 (1961) .
3.
A. C . WILSON, R. D. CAHN, and N. O. KAPLAN, Nature , 197, 331 (1963) .
4.
R. D. CAHN, Dev. Biol . , 9, 327 (1964) .
Vol . 8, No. 2
LDH ISOZYMES
37
5.
D. KADLECOVA, V. KOLAR, and Z. MECHI, Neoplasma, 14 (5), 473 (1967) .
6.
M. M. THALER, R. P. FRANCESCONI, and C .A . VILLES, _Life Sciences, 7 (4), 175 (1968) .
7.
A.E . EMERY, Biochem. J . , 105, 599 (1967) .
8.
C . L . MARKERT, Science, 140,
9.
A . BLANCO and W. H. ZINKHAM, Science, 139, 601 (1963) .
329 (1963) .
10 .
E . GOLDBERG, Science, 148, 391 (1965) .
11 .
L. ORNSTEIN, Ann. N.Y .
12 .
B . J. DAVIS, Ann. N.Y . Acad . Sci., 121, 404 (1964) .
ad . Sci, 121, 321 (1964) .