Alex B. Novikoff

NUCLEOSIDEPHOSPHATASE ACTIVITIES OF CYTOMEMBRANES

Publisher Summary This chapter discusses the nucleosidephosphatase activities of cytomembranes. Light and electron microscopic studies are based on (1) nucleosidephosphatase activities in the plasma membranes of many cells, (2) nucleosidediphosphatase activity, of narrow substrate range, in the endoplasmic reticulum and nuclear membrane of some cells, (3) nucleosidediphosphatase activity, with wider substrate range, in the Golgi apparatus, and (4) acid phosphatase activity in the lysosomes. They illustrate the extent to which enzyme cytochemistry can be brought to the level of ultrastructure by incubation of simply prepared frozen sections of formalin-fixed tissues. The endoplasmic reticulum enzyme has been isolated from acetone powders of rat liver microsomes and beef adrenal, and its properties have been compared with those of the enzyme within frozen sections of fixed tissue. These observations lead to speculations regarding (1) the functional roles of the nucleosidephosphatases of different cytomembranes; (2) the intimate topographical relation that exists between Golgi lamellae and lysosomes; (3) the lysosomal nature of early secretory granules, and some mature ones; and (4) dynamic interrelations that may exist among the membranes of endoplasmic reticulum, Golgi apparatus, and lysosomes.

MICROPEROXISOMES AND PEROXISOMES IN RELATION TO LIPID METABOLISM

We will begin with the last topic in the conference program abstract of this paper: what we call constellations. These constellations include: (a) “cytosolic” lipid spheres (in the sense that the spheres are not enclosed by membranes visible by conventional electron microscopy-whatever their relation might be to the “cytoskeleton” of Keith Porter’ or the “cytosol” of Sheldon Penman’); (b) endoplasmic reticulum or ER; (c ) peroxisomes (in rat hepatocytes, in proximal renal tubules, and in a few other cell types) or microperoxisomes (in all animal cell types studied and in some plant cells as well); and (d) mitochondria. These constellations show such intimate cytological association that they must have functional significance. But electron microscopy, even when supplemented with cytochemically valid reactions for enzyme localizations [for a list of those we use in our laboratory, see ref. 3) can only be qualitative. Such methods could describe the presence and distributions of peroxisomes [also of lysosomes) among the various cell types and tissues. With cytochemistry, we were able to describe particles hitherto not known to exist. And so, Christian de Duve’s brilliant PANEL DISCUSSION in the last New York Academy of Sciences Conference held in 196g4 needs emendation regarding microperoxisomes. In the present Conference‘ we have lost the word “Microbodies.“ and have gained the word “International.“ A good bargain, yes? Yet, cytochernistry cannot yield quantitative data such as obtained by biochemists and molecular biologists. Nor can it establish the concept of peroxisome or the concept of lysosome. Each concept involves a collection of enzymes, quantitatively assayed, within a distinctive cytoplasmic particle. On the other hand, in 1979, P. Lazarow et al.,& from their extensive gel analyses. should not have drawn the conclusion that the peroxisome membrane and ER are not continuous. Paul was unable to attend the meeting and thus Ann Hubbard had to refute our comment to this effect, which we read at the meeting. During our comment we projected FIGURES 10 and 11 of the present publication. The most recent review of the peroxisome-glyoxysome field is that of N. E. T01bert.~ The constellations are shown diagrammatically in FIGURE 1, an unpublished diagram. and in two micrographs, one from a rat hepatocyte’ (FIG. 2) and another

Unusual lysosomes in hamster hepatocytes

Abstract Residual bodies (lysosomes) in hepatocytes of the Syrian golden hamster possess a relatively large electron-lucent lipid inclusion. In males on normal chow the lipid inclusions are large enough, in periportal hepatocytes, to be visible by light microscopy when frozen sections are stained by Oil red O. Thus, the pericanalicular arrangement of the lysosomes may be revealed either by Oil red O staining or by incubation for acid phosphatase activity. Demonstrable acid phosphatase activity is restricted to the area surrounding the lipid inclusion and, usually, to a region at one pole of the lysosome that contains electron-opaque materials characteristic of residual bodies generally. Feeding either female or male hamsters cholesterol-supplemented chow results in a dramatic increase in the number of lipid-containing lysosomes visible by light microscopy. Chemical determinations demonstrate a rapid and sustained rise in liver cholesterol levels, and the presence of cholesterol in lysosomes is shown by electron microscopy of tissue fixed with digitonin-containing aldehydes. Polarizing microscopy reveals the pericanalicular arrangement of anisotropic lipid. Electron microscopic images suggest that the lipid inclusion forms in GERL or neighboring ER. Discontinuing the cholesterol diet leads to a gradual disappearance of the electron-lucent lipid; residual bodies now resemble lipofuscin granules. It is suggested that hamster liver may serve as a model for the study of inherited disorders of lipid metabolism such as Wolmans disease and cholesteryl ester storage diseases, and other diseases in which lysosomes are filled with electron-lucent lipid.

Human chorionic trophoblasts, decidual cells, and macrophages: A histochemical and electron microscopic study☆

Human chorion, amnion, and adherent decidua were studied in term placentas obtained at spontaneous normal delivery and at cesarean section from women not in labor. There were no differences in morphologic features or in distribution of acid phosphatase reaction product in the two delivery groups--in trophoblasts, decidual cells, or macrophages of the decidual layer. No diffusion of acid phosphatase reaction product from lysosomes was evident. The significance of these findings is discussed in the context of current hypotheses concerning cellular event associated with labor.

Cytochemical and electron microscopic demonstration of organelle translocation and the inhibition of such translocation by colchicine during the mitosis of rat hepatocytes

Abstract Hepatocyte lysosomes, mitochondria, and peroxisomes show a dramatic translocation during mitosis induced by partial hepatectomy. During prophase, all three organelles move to the perinuclear cytoplasm. In metaphase, they become concentrated in the polar regions. During telophase, these organelles form clusters in the juxtanuclear regions. This organelle translocation is inhibited by the administration of a low concentration of colchicine, suggesting an involvement of microtubules in their movement.

Cytochemical Staining Reactions for Enzymes in Cytoplasmic Organelles

Recent reports on the use of lead precipitating procedures for localizing intracellular phosphatases and criticism of these procedures (see (1) and (2) for the most recent interchange of views) have left undiminished the value of using the following phosphatases as cytochemical “markers” for light and electron microscopy (3, 4): plasma membrane — nucleoside phosphatases (e.g., Mn++ — and Mg++-stimulated “ATPase”, 5’-nucleotidase, Co++-stimulated CMPase, etc.); endoplasmic reticulum (ER) of liver, kidney, endocrine-secreting cells, etc. -- a nucleoside diphosphatase (NDPase) that hydrolyzes IDP, UDP, GDP (also TPP) but not CDP or ADP (5, 6); Golgi apparatus -- NDPase, thiamine pyrophosphatase (TTPase) (see 6)); Golgi-endoplasmic reticulumlysosome (GERL) -- acid phosphatase; and lysosomes -- acid phosphatase. Optimal oxidation at high pH and relatively high H2O2 concentration of diaminobenzidine may be used to “mark” peroxisomes because of their high catalase content (7, 8). Mitochondria oxidize diaminobenzidine optimally at low pH and low H2O2 concentration (7).

The Induction of Sulfatide, Ganglioside and Cerebroside Storage in Organized Nervous System Cultures

Although a number of animal models exist for the sphingolipidoses, tissue culture offers advantages in studying the development of the inclusion bodies characteristic of these diseases.