Clement L. Markert

LACTATE DEHYDROGENASE IN TESTIS: DISSOCIATION AND RECOMBINATION OF SUBUNITS.

Lactate dehydrogenase from beef tissues may be resolved electrophoretically into five isozymes each of which is a tetramer. These tetramers can be dissociated into monomers by freezing in 1M sodium chloride. On thawing, reassociation into functional tetramers occurs. On the basis of charge and amino acid composition there are two kinds of monomers. Lactate dehydrogenase-1 contains one kind of monomer and lactate dehydrogenase-5 the other kind. A mixture of equal quantities of these two isozymes, after dissociation and reassociation, leads to the production of all five isozymes in the expected proportions of 1:4:6:4:1.

Dissociation of lactate dehydrogenase into subunits with guanidine hydrochloride.

Abstract Analysis of proteins commonly reveals that the functional molecule is a polymer or aggregate of smaller polypeptide chains. Furthermore, it is increasingly evident that many proteins with enzymatic activity exist in multiple molecular forms, or as isozymes, even within the cells of a single organism. Among these is lactate dehydrogenase (LDH), which has been extensively investigated and shown to be resolvable into numerous isozymes (Markert and Moller, 1959) . In searching for an explanation of this molecular heterogeneity we have subjected crystalline LDH to a variety of physico-chemical analyses. This report presents the results of treating the enzyme with guanidine hydrochloride — a hydrogen bonding reagent which effectively ruptures the hydrogen bonds responsible for the secondary structure of many proteins. With the destruction of their secondary structure the polypeptide chains unfold and aggregates of such polypeptide chains then commonly dissociate. LDH behaves in this way and appears to be dissociated into four inactive subunits by treatment with guanidine hydrochloride.

The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse

Abstract The enzyme lactate dehydrogenase (LDH) was shown to exist as five electrophoretically distinct molecular varieties, or isozymes, within the tissues of the mouse. Nearly every tissue or organ of the adult mouse contains all five isozymes of LDH, but in proportions that are highly specific for the tissue. The pattern of isozyme distribution in embryonic tissues differs from the adult pattern. Most embryonic tissues initially contain principally LDH-5; as development proceeds LDH activity is gradually transposed toward the LDH-1 end of the spectrum. The LDH isozymes differ in kinetic properties, e.g., substrate concentration optima; these differences apparently enable the isozymes to fulfill distinct metabolic roles in cellular metabolism. LDH-5 is more abundant in tissues subject to relative anaerobiosis, and the isozymes at the other end of the spectrum are more abundant in highly oxygenated tissues. The isozyme patterns in the mouse are satisfactorily explained by the previously elaborated subunit hypothesis ( Markert, 1962 ), which pictures the LDH molecule as composed of four polypeptide subunits that may be separated into two distinct varieties, A and B, each presumably under the control of a separate gene. Assortment of these subunits in all possible combinations of four generates the five isozymes of LDH. The relative proportions of A and B synthesized by a cell would determine the tissue-specific patterns of LDH isozymes either in the adult or during the course of embryonic or neonatal development.

THE MOLECULAR BASIS FOR ISOZYMES

Proteins are always large molecules. Almost none have molecular weights of less than 10,000, and a few are polymerized aggregates with molecular weights of approximately one million. The number of amino acid residues required for the synthesis of such proteins varies from a low of about 100 to as many as 10,000. Not more than a few hundred amino acid residues are joined through peptide links to form a single chain, bu t several chains may associate to form large polymers. At one time it was believed tha t molecules of such large size might be subject to numerous mistakes in synthesis to yield a kind of microheterogeneity (Colvin et al., 1954). On this view a purified preparation of a protein would be an average mixture of many slightly different molecules. Our present understanding of protein synthesis renders this view implausible. Although mistakes in synthesis do occur, they appear to be insignificant in number. However, the primary polypeptide chain, once formed, is subject to many chemical modifications, particularly through the reactive amino, carboxyl, or hydroxyl groups of several of its amino acid residues. Disulfidebridging, hydrogen-bonding, and various other types of interaction between polypeptide chains all contribute to a secondary and tertiary threedimensional structure that may exist in several distinctive alternate conformations. In addition, most of the larger proteins are polymers of two or more polypeptide chains which may be identical or different. All these possibilities for affecting protein structure raise questions concerning the molecular homogeneity or heterogeneity of any particular protein preparation. How much variability does in fact exist, and what kind of variation can occur without changing the essential biological identity or physiological role of a protein a re questions tha t have been asked repeatedly and answered in accord with the knowledge and insights available a t the time. Moreover, the preparation of proteins for examination, their extraction and purification, have always led to the possibility of denaturation or changes of a purely artifactual nature. Biochemists have been very sensitive to such possibilities and have been reluctant t o conclude tha t heterogeneity in their finished preparations reflected a corresponding heterogeneity in the native state within the cell. This reserve has been reinforced by the strength of the one-gene-one-enzyme concept and by a recent appreciation of the precision of the molecular mechanisms of protein synthesis, all of which led to the expectation that each enzyme would generally be present in an organism in a single molecular form. Nevertheless, occasional claims were made for heterogeneity of single enzymes, but these had little impact until simple, easy methods for assessing

A miniaturized system for electrophoresis on polyacrylamide gels

The design, construction, and use of a simple economical system for gel electrophoresis are described, along with effective procedures and the required materials. Examples of successful use of the system are presented.

Malate dehydrogenase isozymes of the marine snail, Ilyanassa obsoleta

Abstract Malate dehydrogenase (MDH) exists in several isozymic forms in the marine snail, Ilyanassa obsoleta. The isozymes may be classified in two distinct groups, mitochondrial and supernatant. The kinetic properties of these two groups of MDHs resemble those reported for mammalian and avian MDHs except that the relative electrophoretic mobilities of the two groups of isozymes are reversed. The organs of I. obsoleta all contain the same MDH isozymes as revealed by starch gel electrophoresis of organ homogenates. However, quantitative differences in relative abundance were commonly observed, but these differences may be artifacts of preparation since they were not repeatable. All of the supernatant MDH isozymes were apparently of the same molecular weight and were all convertible to a single form by prolonged exposure to 2-mercaptoethanol. This conversion was reversible by removal of the mercaptoethanol. However, the mitochondrial MDH isozymes were not affected by mercaptoethanol. Comparisons of Ilyanassa and pig heart mitochondrial and supernatant MDH isozymes showed parallel responses to mercaptoethanol by the homologous preparations from the two species.

PHYSICOCHEMICAL NATURE OF ISOZYMES

The properties of living cells are in large part a reflection of the enzymes that they contain, and these enzymes are in turn a manifestation of the activity of the nucleic acids of the cells. We have all become familiar with the prevailing hypothesis that relates the primary structure of enzymes (the linear sequence of amino acids) to a corresponding sequence of nucleotides in DNA and/or RNA. This relationship was long ago aptly summed up as the one gene-oneenzyme hypothesis. One reasonable prediction of this hypothesis is that a homozygous organism should synthesize identical molecular replicas of each of its various protein molecules, and this should be true for all the cells of the organism. We were surprised, therefore, to discover from our own work and by a perusal of the literature that single enzymes commonly existed in multiple molecular forms, or isozpes, within the tissues or cells of a single organism. These isozymes are not artifacts of analysis but exhibit characteristic patterns of distribution in each tissue (FIGURE 1). Moreover the tissue patterns are species specific (FIGURE 2). The characteristically different isozyme patterns of adult tissues must have arisen during the course of embryonic development, and direct analysis shows this to be true. The adult pattern is the end product of a long sequence of gradual changes during ontogeny. The remarkable specificity in isozyme pattern that characterizes each tissue implies a significant physiological role for isozymes even though they are essentially alike in enzymatic activity. The existence of isozymes poses important biological problems as well as problems in the physical chemistry of proteins. I t is of great importance to know whether the distinctions among isozymes lie in the primary, secondary, or tertiary structure of the molecule, for a knowledge of such differences may provide an insight into the biological significance of multiple molecular forms of enzymes and, perhaps, also into the mechanisms of protein synthesis. Accordingly we present the results of a physicochemical study on the recrystallized, separated isozymes of lactate dehydrogenase obtained from beef hearts.

IMMUNOCHEMICAL PROPERTIES OF LACTATE DEHYDROGENASE ISOZYMES

The enzyme lactate dehydrogenase (LDH) has been shown to exist in five distinct isozymic forms within the tissues of each mammal that has so far been examined. These isozymes of LDH may be separated from crystalline preparations of the enzyme or from tissue homogenates by electrophoresis; hence each bears a characteristic charge. However, they are all of equal molecular weight as shown by sedimentation studies with the ultracentrifuge ( Markert and Appella, 1961 ) . Physicochemical analysis, including the use of reagents such as guanidine hydrochloride or urea that rupture hydrogen bonds, demonstrates that the LDH molecule may be dissociated into four polypeptide subunits of equal molecular weight. Low pH will also bring about a dissociation of the LDH molecule into various subunits. Electrophoretic analysis of these subunits shows that they are of two different types with respect to the net charge on the polypeptide ( Appella and Markert, 1961 ). These observations, and others, led to the hypothesis that the five isozymes of LDH are generated in mammalian cells by the random assortment of two kinds of polypeptides, designated A and B, into all possible combinations of four (Markert, 1962; Appella and Markert, 1961). The formulae of these tetramers may be written as follows to correspond with the five isozymes: LDH-1 (A0B4), LDH-2 ( A1B3), LDH-3 ( A2B2), LDH-4 ( A3B1), and LDH-5 (A4BO). Each polypeptide is presumably encoded by a separate gene (Markert, 1963; Markert and Ursprung, 1962). Some of the properties arising from the tertiary and quaternary structure of these tetramers may be investigated through immunochemical techniques (FIGURE 1). In particular we wished to examine the species specificity of LDH, the properties of electrophoretically homologous isozymes from different tissues of the same animal, the degree of correspondence of antigenic properties among the five isozymes, and the relationship between antigenic and enzymatic surfaces of the LDH molecule.

EVOLUTION OF THE LACTATE DEHYDROGENASE ISOZYMES OF FISHES

ABSTRACT . The evolution of the L-lactate dehydrogenase (LDH) structural genes as well as the evolution of their regulation has been elucidated by investigating the number, electrophoretic properties, tissue specific expression, and immunochemical relatedness of the LDH isozymes in fishes. The most primitive vertebrates probably possessed a single LDH locus similar to the LDH A locus of today. This single locus genotype is reflected in the LDH phenotype of some contemporary Agnatha. A duplication of this ancestral locus, via polyploidization, has apparently given rise to the LDH A and B loci, each of which exhibits extensive homology in structure, function, and regulation throughout the vertebrates today. All of the Chondrichthyes examined possess both LDH subunits. A third LDH locus, LDH C, which arose from a duplication of the LDH B locus, is first observed in the primitive bony fishes, the chondrosteans. Many of the more primitive bony fishes (including the chondrosteans, holosteans, and some primitive teleost orders) exhibit a generalized function of the C gene, with the C 4 isozyme being synthesized in many tissues and possessing different relative electrophoretic mobilities from species to species. In the more advanced orders of teleosts the C gene is restricted sharply in function and in net charge. In most orders of advanced teleosts the C gene encodes a retinal specific isozyme with a large net negative charge, whereas in some families in the orders Cypriniformes and Gadiformes the C gene codes for a liver predominant isozyme with a net positive charge. The LDH C gene in the teleosts may be ancestral to the third LDH gene of birds and mammals (characteristically active in the primary spermatocyte). These studies demonstrate that the lactate dehydrogenase isozymes provide an excellent model system for studying the origin and evolution of the structure and regulation of homologous genes.

Production of replicable persistent changes in zygote chromosomes of Rana pipiens by injected proteins from adult liver nuclei

Abstract An investigation was carried out to test the hypothesis that cellular differentiation is based upon the acquisition of specific macromolecules by the chromosomes. Very small amounts of various protein fractions from adult Rana pipiens liver cells were injected into zygotes of the same species. The albumin fraction, and to a lesser extent the histones, when injected cause a highly reproducible cessation of cell division and arrested development in the late blastula stage. Nuclei from arrested blastulae were serially transplanted for seven generations without showing any recovery. It is concluded that the effect is primarily on the chromosomes and is replicated during cell division. Cytological examination of blastulae obtained from such nuclear transplantations shows the presence of normal and abnormal chromosome complements in the same individual. This indicates that at the time of transplantation the nuclei were normal, and that the abnormalities occurred later as a consequence of the arrest. The original chromosomal changes induced by the injected macromolecules must therefore be below the level of microscopic detection.