Many enzymes have been reported to exist in more than one form within the same species. By several methods a number of molecular forms of lactic dehydrogenase (LDH) have been found in differing amounts in the various organs of one individual (1-4). This article describes studies which were undertaken in an attempt to elucidate the molecular nature of these multiple enzymatic forms and to follow their development. Most of the developmental studies were made on the chicken.Two "pure" lactic dehydrogenases occur in the chicken. One of them (CM) is found principally in the breast muscle, and the other (CH) in the heart, of adult chickens. These two enzymes are separate entities as judged by physical, enzymatic, and immunochemical criteria (1, 2, 5). During embryonic development the LDH enzymes of the chicken breast muscle shift from the enzymes related to CH, through several intermediate enzyme types, and appear in the adult as pure CM. The characterization of these intermediate enzyme types by several independent methods has led to the development of the hypothesis, which we present in this article, that the intermediate enzyme types which appear during embryonic development are "hybrid" enzymes, consisting of both CM and CH components. Furthermore, these "hybrids" also occur in the adult tissues of the chicken and in other animals. Immunological ApproachIn order to use immunological methods for the study of the development and structure of specific proteins, it is necessary that the immune systems be 962 fully characterized. Two immune systems were used in this study: (i) CH- (6), immunoelectrophoresis (7), and quantitative precipitation analyses (8, 9). Anti-CH was shown to be heterogeneous when measured by double diffusion in agar. After absorption with CM, two bands remained when tested with crude chicken heart extracts, only one of which showed LDH activity (10, 11). This absorption did not decrease the complement (C') fixation titer of CH-anti-CH (Fig. 1), nor did it remove antibody capable of neutralizing CH enzymatic activity. Thus, the heterogeneity in anti-CH reflected antibodies to impurities in the CH immunizing antigen (probably CM) and not anti-CH cross-reacting with CM. Figure 1 shows the C' fixation curves of CM and CH with anti-CM and anti-CH. After absorption with CM, anti-CH showed no reduction in titer against CH. Moreover, the C' fixation curves were identical when either crystalline CH or crude heart extracts were used as antigen. At the dilutions of antisera used (1/5000 for anti-CH and 1/2200 for anti-CM) no cross reactions between CM and CH are detectable. Figure 2 demonstrates the ability of C' fixation to resolve a mixture of pure CM and CH. The heights of the curves at peak fixation are the same in the mixture as in the two pure systems. By the shift in the abscissa at peak fixation it is possible to calculate the percentage of total enzyme activity which reacts with either anti-CM or anti-CH (9, 12). In an artificial mixture of pure CM and CH (Fig. 2), the percentages calculated by C' fi...

EIGHTEEN FIGURESAn important contribution to our k ledge of limb development was made by Saunders in 1948. He demonstrated that apical growth of the wing in the chick embryo was suppressed when the thickened apical ectodermal cap or ridge of the limb bud was extirpated. Excision of this apical ectodermal ridge resulted in the development of partial limbs, lacking distal structures. The amount of distal deficiency depended on the stage subjected to the procedure. Early limb buds, deprived of the apical ectodermal ridge, developed only girdle elements and humeral stumps, etc. Wing buds of a "wingless" mutation (Waters and Bywater, '43) in chickens were shown (Zwilling, '49) to lack apical ectodermal ridges.The relative importance of ectodermal ridge and mesoderm in regard to a variety of developmental relations (determinaation of pattern, polarity, etc.) was left obscure. Extension of our knowledge of ectoderm -mesoderm interaction in the limb would shed light not only on normal developmental relations but might yield information about the mechanism responsible for a variety of genetically altered limb conditions (Duplicate, polydactyly, wingless, etc.) . With these objectives in view an attempt was made to transfer the ectoderm from This investigation was supported in part by a research grant [CI-l79O(R)]

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