Enzyme Polymorphism and Function During Embryonic Development*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIEE, Vol. 7, No. 3 Copyright 1977, Institute for Clinical Science Enzyme Polymorphism and Function During Embryonic Development* MICHAEL L. NETZLOFF, M.D., and OWEN M. RENNERT, M.D. Division of Genetics, Endocrinology and Metabolism., Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL ABSTRACT Alterations in multiple molecular forms of enzymes have been described during normal embryogenesis. Changes in electrophoretic patterns, which differ from the normal isozyme ontogeny, occur in embryos and their yolksacs during incipient maldevelopment secondary to teratogen exposure. One such isozyme change, in response to a teratogenic regimen using 9-methyl pteroylglutamic acid (), is persistence of lactate dehydrogenase-5 (LDH-5) beyond its time of normal involution in the rat yolk-sac. Since LDH-5 is an allosteric, regulatory enzyme which favors anaerobic metabolism, the cellular respiration of 9-methyl -treated embryos was investigated and found to be depressed. However, no changes were found in the oxidative metabolism of visceral yolk-sacs from similarly treated pregnancies. A possible explanation for the unchanged oxygen consumption is the observed simultaneous quantitative alterations in other LDH-yolk-sac isozymes following 9-methyl treatment. Other potential causes include known changes in isozymes other than LDH, limitation of enzyme function by its substrate or co-factor or the presence of a functionally inert LDH-5 isozyme. Changes in LDH and other isozyme patterns and their associated metabolic alterations may eventually prove useful in predicting chemical teratogenicity. Introduction Alterations in protein synthesis and content have been described during both normal and abnormal embryonic development.1,13 Similar changes occur in enzymatically active proteins which exist in multiple molecular forms.8,12,14 These * Supported in part by grants from the National Foundation/March of Dimes (Basil O Connor Research Grant) and The American Heart Association, Florida Affiliate (76-AG-303) isozymes, or isoenzymes, may be separated electrophoretically and their pattern of relative concentration is dependent upon their subcellular or cellular localization, tissue of origin, as well as the stage of differentiation of the organism.12 A remarkably constant ontogeny of isozymes occurs during embryonic development to produce the eventual adult pattern.14,16 This evolution is accomplished by sequential additions, de

2 ISOZYM E FU TION DU RIN G EM BRYOGENESIS 217 letions and changes in concentration of isozymes as maturation progresses.12 Differentiation of a single fertilized ovum into the multiplicity of cell types in the adult organism must involve alterations in the isozyme constitution of these cells, since their specific enzymic complement is the best biochemical correlate with their normal individuality.16 In addition, abnormal isozyme ontogeny characterizes embryonic maldevelopment at the molecular level.5,6,7,8 The constancy of isoenzyme patterns from individual tissues suggests that each isozyme has its own particular function both in adult cells and at each sequential level in the differentiation of embryonic cells.13 Distinctive biochemical properties and metabolic functions of individual isozymes are also indicated by their restriction to specific cellular or subcellular locations, but the actual functional purpose of such restricted placement remains mostly unknown.15 Few of the actual isozymes which probably exist have been recognized to allow functional studies. Isozymes are frequently identified by electrophoresis which depends upon differences in net charge. Since most amino acid substitutions do not alter the net charge on an enzyme, it is likely that most isoenzymes have thus far escaped detection.15,16 The ontogeny of isozyme patterns reflects the changing sequences of gene activation and repression during embryonic development. The isozymes of lactate dehydrogenase (LDH) serve as a good example of genetic control and its modification. LDH s are tetrameric enzymes hybridized most commonly between two different types of polypeptides, A- and B-types, each of which is controlled by its own structural gene. The possible combinations of isozymes include A4(LDH- 5), AsB(LDH-4), A2B2(LDH-3), AB3 (LDH-2) and B4(LDH-1). The properties of the different types of LDH isozymes differ depending on relative A or B polypeptide content with respect to physicalchemical characteristics. These observations, as well as that of the contrasting localization of A- or B-form isozymes in tissues with markedly different energy pathways and requirements, suggests that the B-type isozyme participates in aerobic and A-type in anaerobic metabolism. In the first detailed studies of LDH ontogeny in a mammalian species, Markert and Ursprung demonstrated that the embryonic form of the mouse enzyme was almost entirely A-type, while some tissues developed a predominately B-type pattern at specific rates during maturation.14 Since most of the organs which they studied had an increasingly heterogeneous cell population during development, the electrophoretic pattern may represent a combination of individual isozyme patterns from different types of cells. The resultant pattern may depend heavily on the relative numerical contribution of a cell type to the organ under study. Although Solomon et al have demonstrated the possibility of enzymatic differentiation in the absence of mitosis,20 Johnson and Spinuzzi minimized the problem of cellular heterogeneity by studying the yolk-sac of the rat.6,7 This organ is of embryonic origin and is composed of a limited number of cell types. The ontogeny of LDH isozyme patterns was studied during normal embryogenesis and compared to those found during abnormal embryonic development following exposure to a teratogenic regimen of folic acid deficiency and pteroylglutamic acid () antagonism with 9-methyl. Maternal treatment with this regimen on days 10 to 13 of gestation results in a greater than 80 percent malformation rate among embryos, a limitation which must be met to obtain reproducible findings.9 Electrophoresis in acrylamide gels fol-

3 218 N E T ZLO FF AN D RENNERT BB IB D AY 10 DA Y 11 var B H H DAY 12 var H DAY 13 DAY 14 KC DAY 2 0 Fig u r e 1. Diagram of electrophoretic analyses of lactate dehydrogenase on days and 20 of gesta tion for normal control rat embryo yolk-sacs () and embryonic yolk-sacs from maternal animals deficient beginning on day 10 and ending on day 13 (). Zones of mobility are numbered from anode (+) to cathode ( ). Var is a zone of enzymic activity not present in every electrophoresis and is at the limit of resolution; < is a region of staining which will appear later in gestation in normal tissues, but which has appeared earlier than normal in -deficient yolk-sacs; X is a zone present only in the control tissue on day 14. The intensity of staining is indicated by the relative shading of the bands. (Modified from Johnson, E. M. and Spinuzzi, R.: J. Embryol. Exp. Morph. 26: , 1966; figure 5). lowed by specific LDH enzyme staining showed changing patterns throughout normal development, represented in fig ure 1. For example, LDH-3 was absent on days 10 and 11, but appeared by day 12. LDH-5 was present in large amounts on day 10, but decreased to the limit of reso lution on days 11, 12, and 13 to disappear altogether by day 14. Teratogenic treat ment with 9-methyl caused early appearance of LDH-3 on day 11 and per sistence of LDH-5 in markedly increased amounts on days 11, 12 and 13 in the rat yolk-sac. LDH-5 has been shown by Fritz to be an allosteric, regulatory enzyme which catalyzes reactions interconverting aerobic and anaerobic metabolisms.4 The purpose of this study is to assess the cel lular respiration of yolk-sacs from normal control and 9-methyl -treated em bryos and thereby seek functional differ ences in oxidative metabolism corre sponding to the teratogen-induced LDH-5 isozyme changes, such as were seen in the embryos themselves.18 Materials and Methods The teratogenic regimen and its admin istration have been described in detail previously.7,17,18 In brief, it consisted of breeding female rats and calling day 0 of gestation as the first day on which sperm was found in the vaginal smear. The sub sequent days of gestation were numbered sequentially, and deficiency was

4 ISOZYM E FU TION DU RIN G EM BRYOGEN ESIS 219 begun on day 10 by administering 9-methyl by gastric intubation and replacing stock diet with a folatedeficient one containing 10 mg of 9-methyl per 100 g diet. Pregnant rats were sacrificed by cervical dislocation on day 12 of gestation, and the visceral yolk-sac was dissected free from Reichert s membrane, amnion, vitelline vessels and any residual chorioallantoic placenta. The yolk-sacs were then pooled in lots of three to four and placed in phosphate buffered Ringer s solution containing 0.2 percent D-glucose.10 Oxygen uptake was measured by the Warburg direct technique,22 and protein determination after the method of Lowry et al was used.11 Average respiratory rates were expressed ± their standard errors in microliters of oxygen per hour per miligram of embryonic protein (Qo2). Comparison was made for significant differences using the Student s t test.3 Results As can be seen from table I, determinations of oxygen consumption by embryonic yolk-sacs from maternal animals treated with 9-methyl and by control yolk-sacs show no significant differences. The protein content per individual yolk-sac is 0.65 mg for experimental and 0.78 mg for control embryonic yolk-sacs. Discussion A predictable ontogeny of isozymes accompanies normal embryonic development. Likewise, a reproducibly altered evolution of isozymes is associated with maldevelopment of embryos from mothers treated with chemical teratogens. These changed patterns of functional proteins are among the first signs of teratogenesis, and precede, often by days, any evidence of dysmorphology. It is not unreasonable that these alterations of specific enzymes during the period of T A B L E I Oxygen Consumption by Yolk-Sacs from Normal Control and 9-Methyl Pteroylglutamic Acid-Treated Embryos Normal Control +9-Methyl Number of maternal animals Number of determinations Qq 2 (Ul 02/hr/mg 15.7 ± ± 0.9 protein) ± S.E. Mg protein per ± ± yolk-sac ± S.E. embryogenesis should be instrumental in causing structural maliformations. The persistence of LDH-5 in the 9-methyl -treated system suggested that anaerobic metabolism might be abnormally prolonged during incipient malformation, without the normal conversion to aerobic pathways. Such retardation of molecular development could result in deficient elaboration of high energy compounds necessary for normal differentiation. Reduced amounts of ATP and ADP,2 as well as decreased rates of oxygen consumption18 are reported for abnormally developing embryos from mothers treated with the identical 9-methyl regimen. The rat yolk-sac is an ideal organ in which to study oxygen consumption since its membrane-like thickness allows adequate gaseous diffusion for accurate Qo2 determinations.18 Using the visceral yolk-sac, no differences were found between experimental and control Qo2 values, despite the observed LDH-5 changes. One conclusion which may be true is that the presence of an enzyme does not assure function of that enzyme.17 However, alternate explanations may pertain. Changes in other LDH isozymes in addition to LDH-5 also occur in the yolk-sac in response to the 9-methyl teratogen treatment. These include quantitative differences in the amount of staining activity in LDH-1, -3, and -4. In addition,

5 22 0 N ET ZLO FF AND RENNERT alterations occur in other isozyme patterns such as those of malate and glucose-6-phosphate dehydrogenases in the same 9-methyl -treated system.6,7 The net oxygen consumption may be influenced by the resultant combination of all the enzyme alterations, including LDH-5 activity. The individual isozyme changes may balance and cancel out any net effect on oxidative metabolism. Another important explanation involves the dependence of many different reactions in a cell on the NADH/NAD+ ratio. The interconversion reaction of pyruvate and lactate catalyzed by LDH is accompanied by NADH < > NAD+ oxidoreduction. The principal function of the isozymes of LDH may be to regulate this ratio in various metabolic pathways,14 rather than, for instance, simply to provide more lactate as the hydrogen storage reservior needed during anaerobiosis or to interconnect glycolysis with the aerobic Krebs cycle. Thus, metabolic requirements other than oxygen may determine the predominant LDH isozyme type and function within a cell, and these reactions may be the ones primarily affected by a teratogen. The indirect action of oxygen on the biosynthesis of LDH is indicated by the lack of correlation between the degree of aerobic-anaerobic metabolism in a particular species of embryo and the expected proportion of A- vs. B-type subunits.16 Many examples exist of embryo species which are substantially less well oxygenated possessing less A-type activity than a more aerobic species, the reverse of what would be expected extrapolating from data on adult tissues. Thus, exposure to the directive effects of concentration of metabolites other than oxygen may determine the pattern of early LDH ontogeny. The role of the maternal organism in providing some of the metabolites should be recognized, since it may profoundly effect embryonic LDH isoenzyme differentiation. The presence of LDH-5 on day 10 and its involution during normal embryogenesis between days 10 and 11 in the rat may explain the decreasing aerobic glycolysis, decreased lactate formation and decreased glucose utilization observed by Neubert et al between days 11 and 12 of gestation in the rat.19 The normally diminishing LDH-5 activity would both limit the conversion of pyruvate to lactate and prevent the regeneration of NAD+, which sustains operation of glycolysis under anaerobic conditions.21 Prolongation of this isoenzyme in the teratogen-treated system may limit the normal conversion to Krebs cycle metabolism and thus restrict the energy necessary for differentiation. This seems to be the case for rat embryos treated with 9-methyl, but similar findings were not obtained for their yolk-sacs. Changes occur during incipient malformation in the isozymes of alkaline and acid phosphatases, lactate, malate and glucose-6-phosphate dehydrogenases as well as esterases. The reproducible correlation between treatment with a chemical teratogen and specific alterations in electrophoretic isozyme patterns suggests the latter may be useful in predicting chemical teratogenicity. Before this possibility can be realized, the myriad other types of isozymes and their ontogeny during normal embryonic development must be studied. Acknowledgments Thanks are extended to Dr. J. M. Smith, Jr., D i rector, Process and Analytical Research Section of Lederle Laboratories, for the generous provision of the 9-methyl pteroylglutamic acid, and to Ms. Maria Neves and Mr. Steve Salzman for their technical assistance.

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