LOW RESISTANCE JUNCTIONS IN CRAYFISH : II. Structural Details and Further Evidence for Intercellular Channels by Freeze-Fracture and Negative Staining

Low resistance junctions between axons of crayfish ganglia are studied by freeze-fracture and negative staining. In freeze-fracture, fracture planes that go through a junctional membrane expose two faces, both internal, called face A and face B. Face A belongs to the internal membrane leaflet and faces the gap. Face B belongs to the external membrane leaflet and faces the axoplasm. Face A displays pits, 60–100 Å in diameter, arranged in a hexagonal array with a unit cell of ~200 Å. An ~25 Å bump is frequently seen at the center of each pit. Some pits are occupied by a globule ~125 Å in diameter, which displays a central depression ~25 Å in size. Face B contains globules also arranged in a fairly regular hexagonal pattern. The center-to-center distance between adjacent globules is most frequently ~200 Å; however, occasionally certain globules are seen separated by a distance as short as ~125 Å. The top surface of the globules occasionally displays a starlike profile and seems to contain a central depression ~25 Å in diameter. In negatively stained preparations of membranes from the nerve cord, two types of membranes are seen containing a fairly regular pattern. In one, globules ~95 Å in diameter form a hexagonal close packing with a unit cell of ~95 Å. In the other, globules of the same size are organized in a larger hexagonal array with a unit cell of ~155 Å (swollen arrangement). Some of the globules forming the swollen arrangement are seen containing six subunits. The six subunits form a hexagon which is skewed with respect to the main rows of hexagons in such a way that the subunits lie on rows which make an angle of ~37° with the main rows.     

EXCITABLE MEMBRANE ULTRASTRUCTURE : I. Freeze Fracture of Crayfish Axons

Cross-sectioned and cross-fractured crayfish axons display regions in which axon and Schwann cell surface membranes are regularly curved and project into the axoplasm. At these regions (projections) the two membranes run precisely parallel, separated by a gap of 130–140 Å. Longitudinal fractures through the axons expose the inner fractured surface of either the internal (face A) or the external (face B) leaflet of axon and adjacent Schwann cell surface membranes. On both membranes the projections appear as elongated structures oriented with the long axis parallel to the long axis of the nerve fiber. On face A of the axon surface membrane they are seen as elongated indentations 0.5–1.2-µm long, 0.12–0.15-µm wide. The indentations contain parallel chains of globules. The chains repeat every 120–125 Å and are oriented obliquely in such a way that if one looks at the axon surface from the extracellular space, the axis of the chains is skewed counterclockwise to the long axis of the indentations by an acute angle (most often 55–60°). The globules repeat along the chain every 80–85 Å. Globules of adjacent chains are in register in such a way that the axis on which globules of neighboring chains are aligned forms an angle of 75–85° with the axis of the chains. The complex structure can be defined as a globular array with a rhomboidal unit cell of 80–85 x 120–125 Å. On face B of the axon surface membrane the complementary image of these structures is seen. The projections of the Schwann cell surface membrane also contain groupings of globules; however, these differ from those in the axonal projections in size, pattern of aggregation, and fracture properties. Several possible interpretations of the meaning of these membrane specializations could be proposed. They could be: (a) structures involved in the mechanism of excitation, (b) regions of presumed metabolic couplings, and (c) areas of cell-to-cell adhesion.     

Myelin Organization in the Nodal,Paranodal, and Juxtaparanodal Regions Revealed by Scanning X-Ray Microdiffraction

X-ray diffraction has provided extensive information about the arrangement of lipids and proteins in multilamellar myelin. This information has been limited to the abundant inter-nodal regions of the sheath because these regions dominate the scattering when x-ray beams of 100 µm diameter or more are used. Here, we used a 1 µm beam, raster-scanned across a single nerve fiber, to obtain detailed information about the molecular architecture in the nodal, paranodal, and juxtaparanodal regions. Orientation of the lamellar membrane stacks and membrane periodicity varied spatially. In the juxtaparanode-internode, 198–202 Å-period membrane arrays oriented normal to the nerve fiber axis predominated, whereas in the paranode-node, 205–208 Å-period arrays oriented along the fiber direction predominated. In parts of the sheath distal to the node, multiple sets of lamellar reflections were observed at angles to one another, suggesting that the myelin multilayers are deformed at the Schmidt-Lanterman incisures. The calculated electron density of myelin in the different regions exhibited membrane bilayer profiles with varied electron densities at the polar head groups, likely due to different amounts of major myelin proteins (P0 glycoprotein and myelin basic protein). Scattering from the center of the nerve fibers, where the x-rays are incident en face (perpendicular) to the membrane planes, provided information about the lateral distribution of protein. By underscoring the heterogeneity of membrane packing, microdiffraction analysis suggests a powerful new strategy for understanding the underlying molecular foundation of a broad spectrum of myelinopathies dependent on local specializations of myelin structure in both the PNS and CNS.     

Chloroplast membrane structure. Intramembranous particles of different sizes make contact in stacked membrane regions.

The supramolecular architecture of stacked thylakoid membrane regions of class II spinach chloroplasts has been investigated by means of freeze-fracture electron microscopy. Such membranes contain two basic types of intramembranous particles: laarge particles, which are found on the fracture face of the lumenal membrane leaflet (Bs face), and smaller ones which are found on the fracture face of the external leaflet (Cs face). By analyzing thylakoid membranes containing geometrical arrangements of intramembranous particles it is shown (a) that within the plane of each membrane approximately two small particles are associated with each large particle, and (b) that normal thylakoid stacking involves the connection of large particles of one membrane to small particles of the other and vice versa. If the two types of particles are related to Photosystems I and II, as suggested by circumstantial evidence, then our observations provide support for the idea that maximum Photosystem I-photosystem II interaction is obtained by intermembrane subunit interaction in grana stacks. To this end, our results suggest that stacking should enhance the quantum yield at very low light intensities.     

STRUCTURE OF MEMBRANE HOLES IN OSMOTIC AND SAPONIN HEMOLYSIS

Serial section electron microscopy of hemolysing erythrocytes (fixed at 12 s after the onset of osmotic hemolysis) revealed long slits and holes in the membrane, extending to around 1 µm in length. Many but not all of the slits and holes (about 100–1000 Å wide) were confluent with one another. Ferritin and colloidal gold (added after fixation) only permeated those cells containing membrane defects. No such large holes or slits were seen in saponin-treated erythrocytes, and the membrane was highly invaginated, giving the ghost a scalloped outline. Freeze-etch electron microscopy of saponin-treated membranes revealed 40–50 Å-wide pits in the extracellular surface of the membrane. If these pits represent regions from which cholesterol was extracted, then cholesterol is uniformly distributed over the entire erythrocyte membrane.     

MEMBRANE FUSION IN A MODEL SYSTEM : Mucocyst Secretion in Tetrahymena

The freeze-fracture, freeze-etch technique can be employed to reveal new details of the process of fusion of two unit membranes For this study, mucocyst discharge in Tetrahymena pyriformis provides a model system with certain general implications The undischarged mature mucocyst is a saclike, membrane-bound, secretory vesicle containing crystalline material The organelle tip finds its way toward a special site, a rosette of 150 Å diameter particles within the plasma membrane. To match this site, the mucocyst membrane forms an annulus of 110 Å diameter particles, above whose inner edge the rosette particles sit. Discharge of some mucocysts is triggered by fixation. As discharge proceeds, the organelle becomes spherical and its content changes from crystalline to amorphous. The cytoplasm between the two matching membrane sites is squeezed away and the membranes fuse Steps in membrane reorganization can be reconstructed from changes in rosette appearance in the fracture faces. First, a depression in the rosette—the fusion pocket—forms. The rosette particles spread at the lip as the pocket deepens and enlarges from 60 to 200 nm. The annulus particles then become visible at the lip, indicating completed fusion of the A fracture faces of mucocyst and plasma membranes The remaining B faces of the two membranes have opposite polarities When the content of the mucocyst is released, the edges of these faces join so that the unit membrane runs uninterruptedly around the lip and into the pocket.     

LOW RESISTANCE JUNCTIONS IN CRAYFISH : I. Two Arrays of Globules in Junctional Membranes

The ultrastructure of low resistance junctions between segments of lateral giant fibers in crayfish is studied in sections from specimens fixed either by conventional methods or by glutaraldehyde-H₂O₂ or by glutaraldehyde-lanthanum. Cross sections through junctions fixed by conventional glutaraldehyde display the usual trilaminar profile of two parallel membranes separated by a narrow gap. Most of the junctional regions appear covered by 500–800 Å vesicles which lie on both sides of the junction in rows adjacent to the membranes. Gross sections through junctions fixed by glutaraldehyde-H₂O₂ display, in regions containing vesicles, membranes with a beaded profile. The beads correspond to globules ~125 Å in width and ~170 Å in height arranged in a hexagonal pattern with a unit cell of ~200 Å. The globules of one membrane match precisely with those of the adjacent membrane, and opposite globules seem to come in contact with each other at the center of the junction. The membrane of the vesicles also contains globules. Occasionally the globules of the vesicles seem to join with those of the junctional membranes, apparently forming intracellular junctions. Injunctions negatively stained by lanthanum the globules are seen organized into two arrangements. Areas containing globules in a hexagonal array with a unit cell of ~200 Å (swollen pattern) are seen adjacent to areas in which the globules are more closely and disorderly packed (close packing), the minimum center-to-center distance between adjacent globules being ~125 Å. At higher magnification each globule appears composed of six subunits arranged in a circle around a central region occupied by lanthanum (possibly a pit).     

THE VISUALIZATION OF THE PHOTOSYNTHETIC COUPLING FACTOR IN EMBEDDED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were stained with aqueous uranyl acetate immediately after glutaraldehyde-osmium fixation but before dehydration and embedding. Under these conditions, the lamellae are shown in thin sections to have 95-Å x 115-Å coupling factor particles on their surfaces. The particles can be seen only on the matrix side of nonopposed thylakoids, and are shown to occur on both stromal and granal lamellae, regardless of the organization of the lamellae into stacks. It is estimated that, in native, fully coupled chloroplast lamellae, there is on the average one coupling factor for every 500 chlorophyll molecules. The morphological appearance of the particles is not affected by a variety of buffers, by changes in illumination or temperature, or by alterations in the energy state of the membranes during preparation. The particles can be removed from the membranes with low concentrations of Na₂EDTA, and the photophosphorylating activity of the membranes is concomitantly lost. Both the activity and the appearance of the particles can be restored to the membranes by rebinding EDTA-extracted coupling factors to the uncoupled membranes.     

PRODUCTION AND RELEASE OF A LOCUS-SPECIFIC RIBONUCLEOPROTEIN PRODUCT IN POLYTENE NUCLEI OF DROSOPHILA HYDEI

A specific, 0.1–0.3-µm large ribonucleoprotein complex consisting of a central core with stalklike extensions on top of which 280–320-Å ribonucleoprotein particles are situated is found in an experimentally activated chromosome region, 2–48C, of the polytene chromosomes of Drosophila hydei. Alkaline hydrolysis, RNAse digestion, and uranyl-EDTA-lead staining indicated the ribonucleoprotein character of the 280–320-Å particles, whereas the central core seems to be devoid of RNA. The characteristic complexes are present in the nucleoplasm and at the nuclear membrane, but absent from the cytoplasm. It is suggested that the large RNP complexes are the specific products of the puff at 2–48C. Complexes similar to the ones described have not been observed in any other region of the polytene salivary gland chromosomes of this species.     

STRUCTURAL DIFFERENTIATION OF STACKED AND UNSTACKED CHLOROPLAST MEMBRANES : Freeze-Etch Electron Microscopy of Wild-Type and Mutant Strains of Chlamydomonas

Wild-type chloroplast membranes from Chlamydomonas reinhardi exhibit four faces in freeze-etchreplicas: the complementary Bs and Cs faces are found where the membranes are stacked together; the complementary Bu and Cu faces are found in unstacked membranes. The Bs face carries a dense population of regularly spaced particles containing the large, 160 ± 10 A particles that appear to be unique to chloroplast membranes. Under certain growth conditions, membrane stacking does not occur in the ac-5 strain. When isolated, these membranes remain unstacked, exhibit only Bu and Cu faces, and retain the ability to carry out normal photosynthesis. Membrane stacking is also absent in the ac-31 strain, and, when isolated in a low-salt medium, these membranes remain unstacked and exhibit only Bu and Cu faces. When isolated in a high-salt medium, however, they stack normally, and Bs and Cs faces are produced by this in vitro stacking process. We conclude that certain particle distributions in the chloroplast membrane are created as a consequence of the stacking process, and that the ability of membranes to stack can be modified both by gene mutation and by the ionic environment in which the membranes are found.     

ULTRASTRUCTURE OF BARNACLE GIANT MUSCLE FIBERS

Increasing use of barnacle giant muscle fibers for physiological research has prompted this investigation of their fine structure. The fibers are invaginated by a multibranched system of clefts connecting to the exterior and filled with material similar to that of the basement material of the sarcolemmal complex. Tubules originate from the surface plasma membrane at irregular sites, and also from the clefts They run transversely, spirally, and longitudinally, making many diadic and some triadic contacts with cisternal sacs of the longitudinal sarcoplasmic reticulum. The contacts are not confined to any particular region of the sarcomere. The tubules are wider and their walls are thicker at points of contact with Z material. Some linking of the Z regions occurs across spaces within the fiber which contain large numbers of glycogen particles. A-band lengths are extremely variable, in the range 2.2 µm–20.3 µm (average 5.2 µm) Individual thick filaments have thin (110 Å) hollow regions alternating with thick (340 Å) solid ones. Bridges between thick filaments occur at random points and are not concentrated into an M band The thin:thick filament ratio is variable in different parts of a fiber, from 3:1 to 6:1. Z bands are basically perforated, but the number of perforations may increase during contraction.     

Freeze-etched membrane faces and photosynthetic activity in normal and mutantTradescantia chloroplasts

Summary Membrane structure and photosynthetic activity was investigated in normal and mutant plastids ofTradescantia albiflora cv.aureo-vittata. In the stacked membrane regions (the macrograna) of mutant plastids, the B fracture faces lack both 170 Å particles and photosystem II (PS II) activity. The C face has the normal 110 Å particles, and photosystem I (PS I) activity is also similar to that in normal chloroplasts. In dilated macrograna the particle size on the C face significantly decreases, and as progressive plastid destruction occurs so PS I activity also disappears. It has been concluded that the integrity of B face particles is related to PS II activity, rather than for membrane stacking. A similar correlation seems to be valid for C face particles and PS I activity.     

Freeze fracture studies of reaction centers from Rhodospirillum rubrum in chromatophores and liposomes

In freeze-fractures of chromatophores of Rhodospirillum rubrum the reaction centers are seen as hexagonal arranged particles of 13 nm diameter with a density of around 5,500 particles per m². Similar regions on the cytoplasmic membrane suggest that these parts are the prospective invagination sites.Isolated reaction centers are easily incorporated into liposomes. In freeze fractures of liposomes particles similar in shape and size, although less dense as in chromatophores are observed. In negative staining much smaller units of only 5 nm in diameter are found indicating that reaction centers occur in the membrane as tri- or tetramers. There is a strong correlation between particle density in chromatophores and titratable reaction centers remaining in these membranes after extraction of reaction centers by detergents; both values are in good agreement with the yield of reaction centers at a given detergent concentration.Abbreviations LDAO Lauryldimethylamine oxide - PF protoplasmic fracture face - EF exoplasmic fracture face     

ELECTRON MICROSCOPY OF ORAL CELLS IN VITRO : II. Subsurface and Intracytoplasmic Confronting Cisternae in Strain KB Cells

Two special areas involving membranous components in strain KB cells were studied by electron microscopy. The first area described is that of the subsurface regions of two apposing cells in which flattened cisternae (one cisternae in each subsurface region) with membranes spaced 110–230 A apart were found in a confrontation alignment. The long dimension of the profiles of these cisternae ranges from 0.5 to 2 µ. At these intercellular contact areas, each cisterna is closely applied to the adjacent plasma membrane; the intervening space is 60–100 A. We have named the cisternae in these roughly symmetrical areas of cell contact the subsurface confronting cisternae. Communications between these cisternae and those of the rough-surfaced endoplasmic reticulum also were observed. The second area described is that of the intracytoplasmic confronting cisternae. These cisternae were observed as oval or round images about 0.3–1.4 µ in diameter, each image being composed of a pair of concentrically arranged confronting cisternae with membranes spaced 200–400 A apart. The apposing membranes of the two confronting cisternae are electron opaque, smooth, and free of ribosomes, whereas the unapposed membranes are less dense, scalloped, and associated with ribosomes. The spacing between the two intracytoplasmic confronting cisternae is 70–110 A.     

STRUCTURAL AND FUNCTIONAL HETEROGENEITY OF THE SURFACE OF RAT LEUKEMIA CELLS

Rat leukemia cells IRC 741 in suspension culture form single cytoplasmic protrusions by which the cells preferentially adhere to one another. The induction and/or maintenance of these protrusions is sensitive to changes in intercellular contact, pH, temperature, and nutritional conditions. The protrusions are stable, rigid structures which take part in intercellular adhesion but not in adhesion to substrata. Movement on substrata occurs by means of ruffling membranes formed on the main cell body. This asymmetry in cellular form and function is associated with specialized cell surface regions. Ultrastructurally, the cell surface over the protrusions lacks microvilli, and is covered with a 3,000–4,000-Å thick cell coat consisting of 200–500-Å electron-dense particles in an amorphous matrix. In contrast, the surface over the main cell body has microvilli and a 200-Å wide cell coat which lacks particles. The extracellular particles overlying the protrusions have electron-lucent cores, are protease- and pepsin-resistant, and do not stain with colloidal iron, while the matrix in which they are embedded is sensitive to proteolytic enzymes and contains acidic moieties. The negative surface charge density over the protrusions is higher than that over the main cell body, as shown by the orientation of the cells in an electric field. The unexpected observation that a region of higher charge density is one of increased intercellular adhesiveness might be explained, in part, by the rigidity of the protrusions and by the very small radius of curvature of the overlying extracellular particles. The protrusions permit the observation of discrete regions, differing in charge density, on the surface of living leukemia cells.     

MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.     

Alteration of human erythrocyte plasma membranes by perfringolysin O as revealed by freeze-fracture electron microscopy. Studies on Clostridium perfringens exotoxins V

When human erythrocyte membranes were treated with perfringolysin O (Clostridium perfringens θ-toxin) and examined by electron microscopy after freeze-fracture, two ultrastructural alterations were observed in fracture faces of membrane. (1) A random aggregation of intramembranous particles was seen in the fracture face of the protoplasmic half (PF face) of all membranes treated with the toxin, even if at a low concentration (40 hemolytic units/ml). On the other hand, the aggregation in the fracture face of the exoplasmic half (EF face) was observed only in membranes treated with a high concentration (3300 hemolytic units/ml) for 2 h. (2) Round protrusions and ‘cavities’ with 30 nm in diameter were visible in EF and PF faces of membranes treated with a high concentration, respectively. These structures were always protruded toward cytoplasmic side, but did not appear to form holes through the membrane.Ring and are shaped structures with a dark center of 26 nm and a distinct border of 5 nm in width were observed when the toxin alone was negatively stained at a very high concentration (170 000 hemolytic units/ml). These structures were also produced in the presence of cholesterol even if the toxin concentration was low.     

SUBSTRUCTURE OF AMPHIBIAN MOTOR END PLATE : Evidence for a Granular Component Projecting from the Outer Surface of the Receptive Membrane

End-plate membrane has been examined at amphibian myoneural junctions by means of transmission electron microscopy of thin tissue sections. The postjunctional membrane exhibits morphologically specialized dense, convex patches which are located superficially facing the axon terminal but do not extend into the depths of the junctional folds. In the specialized regions the plasma membrane is ~ 120 Å thick and trilaminar. The outer dense lamina is thickened by the presence in it of granular elements ~60–120 Å in diameter which are spaced semiregularly at ~100–150-Å intervals and which border the junctional cleft directly. In these regions the concentration of the granules is of the order of ~ 10⁴/µm², which is in the same range as the estimated concentration of receptor sites at other vertebrate cholinergic junctions. Filamentous projections can sometimes be seen extending from the granules to the overlying basement membrane, and in oblique views a reticular pattern may appear both in these patches and in the basement membrane. The cytoplasmic surface of the specialized membrane is covered with an amorphous and filamentous dense material whose distribution coincides with that of the granules visible in the outer layer and which may be connected to them across the membrane. In unosmicated specimens stained with permanganate and uranyl acetate the specialized regions exhibit the same morphological features but stand out sharply in contrast to adjacent areas of unspecialized membrane which appear only faintly. Such preparations are particularly useful in assessing the extent of the specialized membrane. It is proposed that the granules visible at the outer surface of the end-plate membrane represent acetylcholine receptors and that in amphibians, as in annelids, the receptors at myoneural junctions are concentrated into patches which occupy less than the total postjunctional membrane surface area.     

Fine structure of the chloroplast membranes ofEuglena gracilis as revealed by freeze-cleaving and deep-etching techniques

Summary The chloroplasts ofEuglena gracilis have been examined by freeze-cleaving and deep-etching techniques.The two chloroplast envelope membranes exhibit distinct fracture faces which do not resemble any of the thylakoid fracture faces.Freeze-cleaved thylakoid membranes reveal four split inner faces. Two of these faces correspond to stacked membrane regions, and two to unstacked regions. Analysis of particle sizes on the exposed faces has revealed certain differences from other chloroplast systems, which are discussed. Thylakoid membranes inEuglena are shown to reveal a constant number of particles per unit area (based on the total particle number for both complementary faces) whether they are stacked or unstacked.Deep-etchedEuglena thylakoid membranes show two additional faces, which correspond to true inner and outer thylakoid surfaces. Both of these surfaces carry very uniform populations of particles. Those on the external surface (the A surface) are round and possess a diameter of approximately 9.5 nm. Those on the inner surface (the D surface) appear rectangular (as paired subunits) and measure approximately 10 nm in width and 18 nm in length. Distribution counts of particles show that the number of particles per unit area revealed by freeze-cleaving within the thylakoid membrane approximates closely the number of particles exposed on the external thylakoid surface (the A surface) by deep-etching. The possible significance of this correlation is discussed. The distribution of rectangular particles on the inner surface of the thylakoid sac (D surface) seems to be the same in both stacked and unstacked membrane regions. We have found no correlation between the D surface particles and any clearly defined population of particles on internal, freeze-cleaved membrane faces. These and other observations suggest that stacked and unstacked membranes are similar, if not identical in internal structure.     

ROLE OF THE SARCOPLASMIC RETICULUM IN GLYCOGEN METABOLISM : Binding of Phosphorylase, Phosphorylase Kinase, and Primer Complexes to the Sarcovesicles of Rabbit Skeletal Muscle

Sarcoplasmic vesicles and β-glycogen particles 30–40 mµ in diameter were isolated from perfused rabbit skeletal muscle by the differential precipitation-centrifugation method. This microsomal fraction was subjected to zonal centrifugation on buffered sucrose gradients, in a B XIV Anderson type rotor, for 15 hr at 45,000 rpm in order to separate the two cytoplasmic organelles. Zonal profiles of absorbance at 280 mµ, proteins, glycogen, and enzymatic activities (phosphorylase b kinase, phosphorylase b, and glycogen synthetase) were performed. Whereas the entire synthetase activity was found combined with the glycogen particles, 39% of phosphorylase and 53% of phosphorylase b kinase activities, present in the microsomal fraction, were recovered in the purified vesicular fraction (d = 1.175). This latter fraction consists of vesicles, derived from the sarcoplasmic reticulum, and of small particles 10–20 mµ in diameter attached to the outer surface of the membranes. These particles disappear after α-amylase treatment. Incubation of the sarcovesicular fraction with ¹⁴C-labeled glucose-1-phosphate confirms the localization of a polysaccharide synthesis at the level of the membranes. "Flash activation" of phosphorylase b, i.e. Ca "activation" of phosphorylase kinase followed by a conversion of phosphorylase b into a, was demonstrated in the purified sarcovesicular fraction. Moreover, the active enzymatic sites were detected on the membranes by electron microscopy. The presence of binding sites between the membranes of the sarcoplasmic vesicles and a glycogen-enzyme complex suggests that this association plays a role in the glycogenolysis during muscle contraction.