Journal of Ultrastructure Research
Morphologie du glycogène: Etude au microscope électronique de colorations négatives du glycogène particulaire
La morphologie fine du glycogène particulaire isolé d'un homogénat de foie de rat à pH 5,2 par centrifugation différentielle, a été étudiée au microscope électronique par la méthode de coloration négative. Parallèlement, pour chaque condition expérimentale, le glycogène a été également fixé au permanganate, inclus au méthacrylate et examiné en coupes. Cette étude morphologique a été guidée et complétée par l'examen des opalescences relatives des suspensions de glycogène.
Le glycogène isolé du foie et examiné par coloration négative et par coloration positive après fixation au permanganate, se présente sous forme de particules de 40–200 mμ de diamètre qui montrent une organisation complexe en plusieurs étages. Pour des raisons pratiques, les structures qui correspondent à ces différents degrés d'organisation ont été désignées sous les noms de particules α, β, et γ. La particule entière, sous sa forme complexe, appelée particule α, apparaît constituée d'éléments de petit diamètre, de 30 mμ environ, les particules β. Dans la constitution de ces dernières entrent des éléments d'un troisième ordre de grandeur, les particules γ en forme de filaments de 3 mμ de diamètre, disposés régulièrement suivant des plans perpendiculaires. Ces deux structures β et γ qui représentent deux degrés distincts dans l'organisation de la particule entière α, correspondent aussi à des degrés différents de dissociation du glycogène particulaire sous l'influence de milieux acides.
Examinés au microscope électronique, des spécimens recueillis à différents pH, montrent deux étapes dans leur dissociation : (a) la dissociation des particules α, qui débute à pH 4,5 et qui s'accentue aux pH plus bas pour être compléte à pH 3,0, (b) la dissociation des particules β à des pH inférieurs à pH 3,0 qui aboutit à la libération des éléments γ.
Summary
Particulate glycogen has been isolated from rat liver by a rapid method of differential centrifugation which consists in acidifying the liver homogenate at pH 5.2, so that a single low speed centrifugation eliminates the agglutinated mitochondria and microsomes. The negative staining method has been used for the electron microscope study of the isolated glycogen and the results have been compared with those obtained, on sections, by the positive staining produced by fixing the glycogen with permanganate. The structure of the particulate glycogen has been studied : (1) on the glycogen isolated by differential centrifugation and kept in conditions which seem to preserve its structure, (2) on the glycogen treated with a series of solutions at different pH, ranging from pH 7.0 to 1.7.
The normal glycogen particle, as isolated from the liver, appears as a complex unit 60–200 mμ in diameter which shows an organization in two levels, each level corresponding to a well defined structure: (a) the complex particle which represents the highest level of organization found in the liver glycogen has been designated by the name α particle; within this particle, smaller units, 20–30 mμ in diameter, may be recognized and have been called β particles; (b) the β particle results from the regular disposition in two perpendicular planes of filaments or rods, 3 mμ in diameter and 20 mμ in length.
The particulate glycogen added to acid solutions at different pH, prepared with two different acids, acetic acid and phosphotungstic acid, shows between pH 7.0 and 1.7 a characteristic diminution of their opalescence. The curve obtained by measuring the relative opalescence of the glycogen suspensions at various pH values in the acidic range, may be divided in four parts corresponding to different slopes and thus to different degrees of degradation of the glycogen.
Samples taken at different pH, corresponding to successive points on the opalescence curve, have been examined by electron microscopy. The glycogen particles show a dissociation of their structure in such a way that the two levels of organization observed in the complex α particle are dislocated successively at definite pH values and that the resulting particles are released in a pure form. The most representative stages in the dissociation process have been illustrated in the present work, namely: the partial dissociation of the α particles, the complete dissociation of the α particles in a uniform population of β particles, and the dissociation of the β particles with the release of small γ elements.
The degree of opalescence of the glycogen suspension at different pH values related to the morphological findings emphasizes the following observations: (1) no significant changes in the opalescence and in the structure of the glycogen particles are observed between pH 7.0 and 5.0; (2) the complete dissociation of the α particles occurs at about pH 3.0 and corresponds to the rapid drop in the opalescence; (3) the dissociation of the β particles and the release of the γ elements constitute the last step of degradation observed in the pH range studied and corresponds to a stabilization of the opalescence of the corresponding glycogen suspension.
The glycogen particle, as it is observed in its complete form, may be conceived as a two-stage structure: the macromolecular organization of the γ elements in a regular pattern constitutes a first unit with definite size and shape, the β particle. Beyond this level of organization, the β particle may be reproduced and then aggregated in a larger unit, the α particle.
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