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Multiple Molecular Forms of Enzymes
Multiple Molecular Forms of Enzymes ELLIOT S. VESELL, M.D.*
During my years as a medical student, it was my good fortune to work with Dr. Edward D. Frank in the Surgical Laboratory of Dr. Jacob Fine. The experience was rewarding because the experiments were interesting and because the environment was unique. The atmosphere that Dr. Fine created in his laboratory was indeed special. Ideas became animated because they were treated with rare reverence and were invested with unusual significance. This made working in Dr. Fine's laboratory an exciting, rich, and rewarding experience. Every experiment followed logically the preceding one and led inexorably to the next. At each stage interest and suspense grew; there was genuine enthusiasm to perform these experiments that were designed to test the validity of exciting ideas. If the hypothesis proved to be correct, the next experiment was clear; if the idea had to be rejected, alternatives arose and, accordingly, new experimental models were devised. Thus, pursuit of the mechanisms that caused shock and subsequently death assumed the nature of an absorbing chess game or of a battle waged against the hostile forces of nature. The laboratory was composed of congenial, skilled, and devoted scientists. These included Howard and Edward Frank, Alexander and Selma Rutenburg, Fritz Schweinburg, Herbert Ravin, Carlo Palmerio, Edward Friedman, and Chester Rosoff. Many of them helped me. We all contributed to one another's projects. Experiments conducted by these dedicated individuals often continued late into the night. The Friday afternoon research luncheons run by Dr. Fine served as a focus for the exchange of ideas and for realigning forces to attack new aspects of the problem. Individuals interested in the comments and suggestions of their associates presented their projects. The sessions were lively and stimulating. They frequently led to new ideas and approaches. My study concerned alterations of serum enzymes in hemorrhagic and endotoxic shock. After the first year of medical school I had worked with Dr. Alexander G. Beam in Dr. Henry G. Kunkel's laboratory at the Rockefeller Institute. We had discovered that the activities of the en-
*Professor and Chairman, Department of Pharmacology, and Professor of Genetics, Pennsylvania State University College of Medicine, Hershey, Pennsylvania Surgical Clinics of North America- Vol. 49, No.3, June, 1969
571
572
ELLIOT Peak 5
Peak 4 -
Peak 3
Peak 2
S.
VESELL
Peak I
160 Myocardial Infarction serum
120 80 40 0 120 80 40 0 ~
.~
U 0
:I: 0
120 80
-' !!.
40
::>
0
C
.
280
,, ,,
Leukemic serum
240
,f
.
200
1
1.2 1.0 ~
,
160
1.4
0.8
,,
120
06
80
04
40
02
0 5
9L!
..~
"0
§ Q. 0
0 13
15
17
19
21
23
25
27
29
31
33
35
37
Segment numoer
Figure 1. Distribution of lactate dehydrogenase (LDH) activity in normal human serum and sera from patients with myocardial infarction, chronic granulocytic leukemia, and infectious hepatitis. These four sera were separated simultaneously on a starch block. Note that in each disease there are alterations in individual LDH activity peaks from the normal pattern. The curve with the broken line for the leukemic serum represents the protein concentration. The origin is indicated by the arrow.
zymes lactate and malate dehydrogenase (LDH and MDH) were present in multiple forms in human serum and tissues. 4 As shown in Figure 1, LDH activity exists in three major forms in normal human serum. The distribution of total activity among these forms was fairly constant in normal serum, but the proportions were altered dramatically in various disease states, such as myocardial infarction, hepatitis, and leukemia. 4. 6 Normally the LDH peak (peak 1) which migrated electrophoretically on a starch block at pH 8.6 with a mobility between alpha-1 globulin and albumin contained 31 to 37 per cent of the total serum LDH activity. The alpha-2-globulin peak had 43 to 55 per cent of the total LDH activity and the beta globulin peak exhibited 12 to 23 per cent.4 In sera from patients with myocardial infarction, peak 1 was differentially elevated; early in viral hepatitis, peaks 5 and 4 became prominent, whereas normally they were present only in trace amounts. In sera from patients with leukemia peak 2 was raised. These observations led to the introduction into clinical diagnosis of electrophoretic separation of plasma and other body fluids and tissues to
573
MULTIPLE MOLECULAR FORMS OF ENZYMES
determine how much of the total enzyme activity was present in each of the multiple forms. Analysis of these individual molecular forms of LDH provided a diagnostic specificity lacking when only total serum enzyme activity was obtained. Electrophoretic separations of serum enzymes are therefore utilized routinely as a convenient, economical, and rapid diagnostic technique in many hospitals in this country and abroad. The pathophysiologic basis of altered serum LDH patterns is suggested in Figure 2, which shows the LDH patterns of various human tissues obtained at autopsy. The supernatants of the tissue homogenates were separated by starch gel electrophoresis in borate buffer pH 8.3. Then the gels were sliced and stained in a buffered solution containing a tetrazolium salt, lactate, and nicotinamide-adenine dinucleotide. 6 Figure 2 shows that each tissue contains five forms of LDH. However, the distribution of total activity among the five forms differs in various tissues. Skeletal muscle, liver and skin have mainly LDH 5, whereas heart muscle and kidney contain predominantly LDH 1 and 2. Thus, in myocardial infarction LDH 1 is released from heart muscle into the circulation, and plasma LDH 1 becomes elevated. Conversely, in hepatitis, LDH 5 and LDH 4, the main LDH forms in liver, are liberated into the blood. Other conditions in which altered serum LDH patterns contribute diagnostic information include pancreatitis, various malignant tumors, pulmonary embolus and infarction, muscular dystrophy, and megaloblastic anemias. In each of these disease states, single organs or tissues are affected and only one or two serum LDH peaks are altered. In Dr. Fine's laboratory, Dr. Edward Frank and I investigated plasma LDH patterns during experimental hemorrhagic shock in the dog and endotoxic shock in the rabbit. We discovered that during hemorrhagic shock in the dog all of the plasma peaks were raised. 5 By studying arterial-venous differences in total LDH activity, we concluded that elevations of plasma LDH activity
•
•
•
t
Lung . Heart , .•... Kidney
•• Figure 2. Starch gel showing lactate dehydrogenase CLDH) isozyme patterns of various human tissues obtained at autopsy. Note that each tissue has five bands of LDH activity, but that the distribution of total activity among the five bands differs in various tissues.
574
ELLIOT
S.
VESELL
resulted from enzyme released from at least three separate sources - the tissues of an extremity, the kidney, and the liver.5 These experiments suggested that even after a diagnosis had been established, LDH fractionations might prove useful. They could provide both qualitative and quantitative information in following the clinical progress of a patient to determine whether an abnormal serum pattern tended to return toward normal with treatment or whether it became more distorted, that is, whether there was extension of the disease process to other tissues, as in shock. In the past decade much attention has been focused on the fact that many, if not most, enzymes exist in multiple molecular forms, either within a single cell or within different tissues of an organism. The word isozyme was coined in 1959 by Markert and M9illeil to call attention to this situation. With the definition of isozymes as multiple molecular forms of an enzyme exhibiting similar substrate specificity, enzyme heterogeneity assumed the position of a general biological principle. The chemical basis of several isozymic systems has been elucidated. In the case of LDH, Appella and Markert! showed that each of the five commonly encountered LDH isozymes is a tetramer composed of A or B type monomers or both, assorted in all possible combinations of four. Thus, LDH 1 = BBBB; LDH 2 = BBBA; LDH 3 = BBAA; LDH 4 = BAAA; LDH 5 = AAAA. Various biochemical, genetic, and immunologic types of proof of this hypothesis have been provided. 2 The five isozymes of catalase in maize and aldolase in mammalian tissues probably have a similar structural basis. Three isozymes of alkaline phosphatase in certain species probably result from two subunits A and B combined to form various dimers; the three alkaline phosphatase isozymes would therefore have the structures AA, AB, and BE. A system composed of two subunits assembled to form trimers would be expected to yield four isozymes of structure AAA, AAB, ABB, and BBB. In addition to this mechanism involving the combination of two or more dissimilar subunits, isozymes can arise from different polymeric states of a single subunit, as in glycogen phosphorylase, or from a single polymeric form to which varying amounts of a prosthetic group are attached, as in different amounts of nicotinamide-adenine dinucleotide bound to some alcohol dehydrogenases, of sialic acid to several alkaline and acid phosphatases, and of manganese to certain arginases and glutamine synthetases. Enzymatic cleavage of proteins, including removal of whole residues or of amino, carboxyl, or hydroxyl groups, is part of the large category of catabolic processes that theoretically could produce multiple forms of enzymes. Multiple forms of certain enzymes such as MDH have been said to represent configurational isomers or "conformers," signifying that they are identical in primary structure but vary in tertiary or quaternary structure. Finally, multiple forms of enzymes may be created artifactually during preparative procedures. These artifactually created forms, though technically important, are without biological significance. Of the numerous biologic applications of enzymatic heterogeneity, determination of serum LDH isozymes in the diagnosis and management
MULTIPLE MOLECULAR FORMS OF ENZYMES
575
of various disease states was historically the first and remains the most popular. Subsequently, few areas of biology have escaped investigation by this powerful analytic technique. For the geneticist, isozymes have provided a sensitive technique for discovering new polymorphic systems, for marking chromosomes, and for following changing gene action. For the biochemist, they have offered a fresh approach to the control and regulation of intermediary metabolism. For the student of growth and development, they are precise markers along the stages of ontogeny and phylogeny. The future promises additional applications.
REFERENCES 1. Appella, E., and Markert, C. L.: Dissociation of lactate dehydrogenase into subnunits with guanidine hydrochloride. Biochem. Biophys. Res. Commun., 6:171,1961. 2. Kaplan, N. 0.: Lactate dehydrogenase - Structure and function. Brookhaven Sympos. BioI., 17:131, 1964. 3. Markert, C. L., and Ml!lller, F.: Multiple forms of enzymes: Tissue, ontogenetic, and species specific patterns. Proc. Nat. Acad. Sci., 45:753,1959. 4. Vesell, E. S., and Beam, A. G.: Localization oflactic dehydrogenase activity. Proc. Soc. Exp. BioI. Med., 94:96, 1957. 5. Vesell, E. S., Feldman, M. P., and Frank, E. D.: Plasma lactic dehydrogenase activity in experimental hemorrhagic shock. Proc. Soc. Exp. BioI. Med., 101 :644, 1959. 6. Vesell, E. S., Osterland, K. C., Beam, A. G., and Kunkel, H. G.: Isozymes oflactic dehydrogenase; their alterations in arthritic synovial fluid and sera. J. Clin. Invest., 41 :2012, 1962. Department of Pharmacology Pennsylvania State University College of Medicine Hershey, Pennsylvania 17033
During my years as a medical student, it was my good fortune to work with Dr. Edward D. Frank in the Surgical Laboratory of Dr. Jacob Fine. The experience was rewarding because the experiments were interesting and because the environment was unique. The atmosphere that Dr. Fine created in his laboratory was indeed special. Ideas became animated because they were treated with rare reverence and were invested with unusual significance. This made working in Dr. Fine's laboratory an exciting, rich, and rewarding experience. Every experiment followed logically the preceding one and led inexorably to the next. At each stage interest and suspense grew; there was genuine enthusiasm to perform these experiments that were designed to test the validity of exciting ideas. If the hypothesis proved to be correct, the next experiment was clear; if the idea had to be rejected, alternatives arose and, accordingly, new experimental models were devised. Thus, pursuit of the mechanisms that caused shock and subsequently death assumed the nature of an absorbing chess game or of a battle waged against the hostile forces of nature. The laboratory was composed of congenial, skilled, and devoted scientists. These included Howard and Edward Frank, Alexander and Selma Rutenburg, Fritz Schweinburg, Herbert Ravin, Carlo Palmerio, Edward Friedman, and Chester Rosoff. Many of them helped me. We all contributed to one another's projects. Experiments conducted by these dedicated individuals often continued late into the night. The Friday afternoon research luncheons run by Dr. Fine served as a focus for the exchange of ideas and for realigning forces to attack new aspects of the problem. Individuals interested in the comments and suggestions of their associates presented their projects. The sessions were lively and stimulating. They frequently led to new ideas and approaches. My study concerned alterations of serum enzymes in hemorrhagic and endotoxic shock. After the first year of medical school I had worked with Dr. Alexander G. Beam in Dr. Henry G. Kunkel's laboratory at the Rockefeller Institute. We had discovered that the activities of the en-
*Professor and Chairman, Department of Pharmacology, and Professor of Genetics, Pennsylvania State University College of Medicine, Hershey, Pennsylvania Surgical Clinics of North America- Vol. 49, No.3, June, 1969
571
572
ELLIOT Peak 5
Peak 4 -
Peak 3
Peak 2
S.
VESELL
Peak I
160 Myocardial Infarction serum
120 80 40 0 120 80 40 0 ~
.~
U 0
:I: 0
120 80
-' !!.
40
::>
0
C
.
280
,, ,,
Leukemic serum
240
,f
.
200
1
1.2 1.0 ~
,
160
1.4
0.8
,,
120
06
80
04
40
02
0 5
9L!
..~
"0
§ Q. 0
0 13
15
17
19
21
23
25
27
29
31
33
35
37
Segment numoer
Figure 1. Distribution of lactate dehydrogenase (LDH) activity in normal human serum and sera from patients with myocardial infarction, chronic granulocytic leukemia, and infectious hepatitis. These four sera were separated simultaneously on a starch block. Note that in each disease there are alterations in individual LDH activity peaks from the normal pattern. The curve with the broken line for the leukemic serum represents the protein concentration. The origin is indicated by the arrow.
zymes lactate and malate dehydrogenase (LDH and MDH) were present in multiple forms in human serum and tissues. 4 As shown in Figure 1, LDH activity exists in three major forms in normal human serum. The distribution of total activity among these forms was fairly constant in normal serum, but the proportions were altered dramatically in various disease states, such as myocardial infarction, hepatitis, and leukemia. 4. 6 Normally the LDH peak (peak 1) which migrated electrophoretically on a starch block at pH 8.6 with a mobility between alpha-1 globulin and albumin contained 31 to 37 per cent of the total serum LDH activity. The alpha-2-globulin peak had 43 to 55 per cent of the total LDH activity and the beta globulin peak exhibited 12 to 23 per cent.4 In sera from patients with myocardial infarction, peak 1 was differentially elevated; early in viral hepatitis, peaks 5 and 4 became prominent, whereas normally they were present only in trace amounts. In sera from patients with leukemia peak 2 was raised. These observations led to the introduction into clinical diagnosis of electrophoretic separation of plasma and other body fluids and tissues to
573
MULTIPLE MOLECULAR FORMS OF ENZYMES
determine how much of the total enzyme activity was present in each of the multiple forms. Analysis of these individual molecular forms of LDH provided a diagnostic specificity lacking when only total serum enzyme activity was obtained. Electrophoretic separations of serum enzymes are therefore utilized routinely as a convenient, economical, and rapid diagnostic technique in many hospitals in this country and abroad. The pathophysiologic basis of altered serum LDH patterns is suggested in Figure 2, which shows the LDH patterns of various human tissues obtained at autopsy. The supernatants of the tissue homogenates were separated by starch gel electrophoresis in borate buffer pH 8.3. Then the gels were sliced and stained in a buffered solution containing a tetrazolium salt, lactate, and nicotinamide-adenine dinucleotide. 6 Figure 2 shows that each tissue contains five forms of LDH. However, the distribution of total activity among the five forms differs in various tissues. Skeletal muscle, liver and skin have mainly LDH 5, whereas heart muscle and kidney contain predominantly LDH 1 and 2. Thus, in myocardial infarction LDH 1 is released from heart muscle into the circulation, and plasma LDH 1 becomes elevated. Conversely, in hepatitis, LDH 5 and LDH 4, the main LDH forms in liver, are liberated into the blood. Other conditions in which altered serum LDH patterns contribute diagnostic information include pancreatitis, various malignant tumors, pulmonary embolus and infarction, muscular dystrophy, and megaloblastic anemias. In each of these disease states, single organs or tissues are affected and only one or two serum LDH peaks are altered. In Dr. Fine's laboratory, Dr. Edward Frank and I investigated plasma LDH patterns during experimental hemorrhagic shock in the dog and endotoxic shock in the rabbit. We discovered that during hemorrhagic shock in the dog all of the plasma peaks were raised. 5 By studying arterial-venous differences in total LDH activity, we concluded that elevations of plasma LDH activity
•
•
•
t
Lung . Heart , .•... Kidney
•• Figure 2. Starch gel showing lactate dehydrogenase CLDH) isozyme patterns of various human tissues obtained at autopsy. Note that each tissue has five bands of LDH activity, but that the distribution of total activity among the five bands differs in various tissues.
574
ELLIOT
S.
VESELL
resulted from enzyme released from at least three separate sources - the tissues of an extremity, the kidney, and the liver.5 These experiments suggested that even after a diagnosis had been established, LDH fractionations might prove useful. They could provide both qualitative and quantitative information in following the clinical progress of a patient to determine whether an abnormal serum pattern tended to return toward normal with treatment or whether it became more distorted, that is, whether there was extension of the disease process to other tissues, as in shock. In the past decade much attention has been focused on the fact that many, if not most, enzymes exist in multiple molecular forms, either within a single cell or within different tissues of an organism. The word isozyme was coined in 1959 by Markert and M9illeil to call attention to this situation. With the definition of isozymes as multiple molecular forms of an enzyme exhibiting similar substrate specificity, enzyme heterogeneity assumed the position of a general biological principle. The chemical basis of several isozymic systems has been elucidated. In the case of LDH, Appella and Markert! showed that each of the five commonly encountered LDH isozymes is a tetramer composed of A or B type monomers or both, assorted in all possible combinations of four. Thus, LDH 1 = BBBB; LDH 2 = BBBA; LDH 3 = BBAA; LDH 4 = BAAA; LDH 5 = AAAA. Various biochemical, genetic, and immunologic types of proof of this hypothesis have been provided. 2 The five isozymes of catalase in maize and aldolase in mammalian tissues probably have a similar structural basis. Three isozymes of alkaline phosphatase in certain species probably result from two subunits A and B combined to form various dimers; the three alkaline phosphatase isozymes would therefore have the structures AA, AB, and BE. A system composed of two subunits assembled to form trimers would be expected to yield four isozymes of structure AAA, AAB, ABB, and BBB. In addition to this mechanism involving the combination of two or more dissimilar subunits, isozymes can arise from different polymeric states of a single subunit, as in glycogen phosphorylase, or from a single polymeric form to which varying amounts of a prosthetic group are attached, as in different amounts of nicotinamide-adenine dinucleotide bound to some alcohol dehydrogenases, of sialic acid to several alkaline and acid phosphatases, and of manganese to certain arginases and glutamine synthetases. Enzymatic cleavage of proteins, including removal of whole residues or of amino, carboxyl, or hydroxyl groups, is part of the large category of catabolic processes that theoretically could produce multiple forms of enzymes. Multiple forms of certain enzymes such as MDH have been said to represent configurational isomers or "conformers," signifying that they are identical in primary structure but vary in tertiary or quaternary structure. Finally, multiple forms of enzymes may be created artifactually during preparative procedures. These artifactually created forms, though technically important, are without biological significance. Of the numerous biologic applications of enzymatic heterogeneity, determination of serum LDH isozymes in the diagnosis and management
MULTIPLE MOLECULAR FORMS OF ENZYMES
575
of various disease states was historically the first and remains the most popular. Subsequently, few areas of biology have escaped investigation by this powerful analytic technique. For the geneticist, isozymes have provided a sensitive technique for discovering new polymorphic systems, for marking chromosomes, and for following changing gene action. For the biochemist, they have offered a fresh approach to the control and regulation of intermediary metabolism. For the student of growth and development, they are precise markers along the stages of ontogeny and phylogeny. The future promises additional applications.
REFERENCES 1. Appella, E., and Markert, C. L.: Dissociation of lactate dehydrogenase into subnunits with guanidine hydrochloride. Biochem. Biophys. Res. Commun., 6:171,1961. 2. Kaplan, N. 0.: Lactate dehydrogenase - Structure and function. Brookhaven Sympos. BioI., 17:131, 1964. 3. Markert, C. L., and Ml!lller, F.: Multiple forms of enzymes: Tissue, ontogenetic, and species specific patterns. Proc. Nat. Acad. Sci., 45:753,1959. 4. Vesell, E. S., and Beam, A. G.: Localization oflactic dehydrogenase activity. Proc. Soc. Exp. BioI. Med., 94:96, 1957. 5. Vesell, E. S., Feldman, M. P., and Frank, E. D.: Plasma lactic dehydrogenase activity in experimental hemorrhagic shock. Proc. Soc. Exp. BioI. Med., 101 :644, 1959. 6. Vesell, E. S., Osterland, K. C., Beam, A. G., and Kunkel, H. G.: Isozymes oflactic dehydrogenase; their alterations in arthritic synovial fluid and sera. J. Clin. Invest., 41 :2012, 1962. Department of Pharmacology Pennsylvania State University College of Medicine Hershey, Pennsylvania 17033