A plasma membrane proton ATPase in specialized cells of rat epididymis

Acidification of the luminal fluid in the epididymis is believed to play an important role in sperm maturation. Previous studies have shown that specialized cells in the epithelium lining the epididymis contain high levels of carbonic anhydrase and that these cells have rod-shaped intramembraneous particles when examined by freeze fracture. Both of these features are characteristic of proton-transporting intercalated cells in the kidney collecting duct. We now show that apical cells in the head of the epididymis and clear cells in the body and tail of the epididymis express high levels of a vacuolar proton-pumping adenosinetriphosphatase on their apical plasma membranes and on intracellular vesicles. By analogy with kidney intercalated cells, these cell types may be specialized for acid secretion in the epididymis.

Histochemical Demonstration of an ATP-Dependent Ca2+-Pump in Bullfrog Myocardial Cells

In the present investigation different finestructural and histochemical procedures were employed to demonstrate normal morphology and Ca2+-transport mechanism in bullfrog atrial myocardium. For normal morphology specimens were fixed in 1% OsO4, 2.5% glutaraldehyde or liquid propane at - 185 °C before they were prepared for conventional embedding and freeze-etching, respectively. Special interest was focussed on the caveolae system, which is composed of single, spherical membrane invaginations (diameter, 85 nm), randomly distributed at the entire cell periphery. The caveolae enlarge the cell surface by 24.5% and occupy 8.5% of the cellular volume. The caveolae membrane contains a few intramembraneous particles with a diameter of 8.4 nm comparable to the size of ATPases as found in other cells. For histochemistry the specimens were first stabilized by treatment with 50% glycerol, 0.025% glutaraldehyde or 0.15% formaldehyde and then incubated in a medium containing 4 mᴍ CaCl2, 4 mᴍ EGTA, 5 mᴍ MgCl2, 5 mᴍ K2C2O4, 5 mᴍ ATP and 20 mᴍ histidine at pH 7.0. This incubation always succeeded in the formation of electron-dense deposits with elliptical shape, measuring 100 -200 nm in length and 10 -30 nm in diameter. According to X-ray spectra they deliver a characteristic calcium-peak and can be found within two different cellular compartments: in small invaginations of the sarcolemma, i. e. the caveolae-system, and in the intrafibrillar sarcoplasmic reticulum. The elliptical deposits can be clearly distinguished from round electron-dense granules measuring 16 nm in diameter, which are located within randomly distributed small vesicles and composed of potassium and phosphate. Contrary to the elliptical deposits the round granules are also present in controls and seem to be identical with the so-called atrial granules. In comparison to observations obtained with the same method in other muscular systems and derived from various control experiments the results of this study favour the existence of an ATP-dependent Ca2+-pumping mechanism in frog atrial muscle bound to both, the sarcoplasmic reticulum and the caveolae system.

Enlarged Crystalline Patches on the Plasma Membrane of Yeast Protoplasts

The crystalline patches of intramembraneous particles that form in the yeast plasma membrane, under stationary state physiological conditions, represent a potentially interesting specimen for high resolution electron microscopy. Isolation of these crystalline membrane patches first requires removal of the cell wall and the formation of osmotically fragile yeast protoplasts. In developing a procedure for the isolation of these crystalline membrane patches, we have found that the intramembraneous particles form much larger crystalline patches in protoplasts than in intact yeast cells. We have performed deep etch experiments and have found that the crystalline array of particles is not expressed on the extracellular surface of the plasma membrane.

Lipidic intramembraneous particles

Freeze-fracturing splits membranes into 2 halves, thus allowing an examination of the membranes interior. The 5-10 nm particles visible on both monolayers are widely assumed to be proteinaceous in nature.Most membranes do not reveal impressions complementary to particles on the opposite face. Even when it is considered that shadowing, contamination or fracturing itself might obscure complementary pits, there is no satisfactory explanation why under similar physical circumstances matching halves of other membranes can be visualized. A prominent example of uncomplementarity is found in the erythrocyte membrane. It is well established that band III protein and possibly glycophorln represent these uncomplementary structures.On the other hand a number of experiments has made us aware that particles of the Eeoheriohia ooli outer membrane are basically constructed of lipid micels (i.e., lipo- polysaccharlde). These particles have complementary Impressions on the opposite face (Fig. 1).