Size Of Intramembranous Particles Research Articles

Prestin forms oligomer with four mechanically independent subunits

Prestin is the motor protein of cochlear outer hair cells (OHCs) with the unique capability of performing direct, rapid, and reciprocal electromechanical conversion. Prestin consists of 744 amino acids with a molecular mass of ∼ 81.4 kDa. The predicted membrane topology and molecular mass of a single prestin molecule appear inadequate to account for the size of intramembrane particles (IMPs) expressed in the OHC membrane. Although recent biochemical evidence suggests that prestin forms homo-oligomers, most likely as a tetramer, the oligomeric structure of prestin in OHCs remains unclear. We obtained the charge density of prestin in the gerbil OHCs by measuring their nonlinear capacitance (NLC). The average charge density (22,608 μm − 2) measured was four times the average IMP density (5686 μm − 2) reported in the freeze-fracture study. This suggests that each IMP contains four prestin molecules, based on the general notion that each prestin transfers a single elementary charge. We subsequently compared the voltage dependency and the values of slope factor of NLC and somatic motility simultaneously measured from the same OHCs to determine whether NLC and motility are fully coupled and how prestin subunits function within the tetramer. We showed that the voltage dependency and slope factors of NLC and motility were not statistically different, suggesting that NLC and motility are fully coupled. The fact that the slope factor is the same between NLC and motility suggests that each prestin monomer in the tetramer is in parallel, each interacting independently with cytoplasmic or other partners to facilitate the mechanical response.

Connexins, pannexins, innexins: novel roles of “hemi-channels”

Connexins, pannexins, innexins: novel roles of “hemi-channels”

Membrane Particle Distribution in the Sternal Epithelia of the Terrestrial Isopod Porcellio scaber Latr. (Crustacea, Oniscidea) during CaCO3 Deposit Formation and Resorption, a Freeze-Etch Analysis

The anterior sternal epithelium of terrestrial isopods transports cuticular Ca2+ to and from large sternal CaCO3 deposits. We analyzed the anterior and posterior sternal epithelium by the means of the freeze-etch technique and measured the size distribution and density of intramembrane particles (IMPs) during three different molting stages. At least three IMP size classes around 4.5, 7.7, and 9.4 nm can be distinguished on the P-face of the apical and basolateral plasma membrane. An additional size class of around 12.8 nm is restricted to the apical compartment. In the anterior sternal epithelium, the density of these large particles changes by a factor of 1.9 during the molt cycle, suggesting a role in CaCO3 formation and/or resorption. The density of the smaller IMPs rises transiently by a factor of 1.3 in the posterior sternal epithelium only. The IMP density of the basolateral plasma membrane increases significantly by a factor of 1.4 and 1.3 in the anterior and posterior sternal epithelia, respectively. The results indicate that increases in the IMP density contribute to the differentiation to an increased transport activity during the cyclic enlargements of the plasma membrane surface area in the anterior sternal epithelium.

Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice.

It has been controversial whether high water permeability in the thin descending limb of Henle (TDLH) is required for formation of a concentrated urine by the kidney. Freeze-fracture electron microscopy (FFEM) of rat TDLH has shown an exceptionally high density of intramembrane particles (IMPs), which were proposed to consist of tetramers of aquaporin-1 (AQP1) water channels. In this study, transepithelial osmotic water permeability (Pf) was measured in isolated perfused segments (0.5-1 mm) of TDLH in wild-type (+/+), AQP1 heterozygous (+/-), and AQP1 null (-/-) mice. Pf was measured at 37 degrees C using a 100 mM bath-to-lumen osmotic gradient of raffinose, and fluorescein isothiocyanate (FITC)-dextran as the luminal volume marker. Pf was (in cm/s): 0.26 +/- 0.02 ([+/+]; SE, n = 9 tubules), 0.21 +/- 0.01 ([+/-]; n = 12), and 0.031 +/- 0.007 ([-/-]; n = 6) (P < 0.02, [+/+] vs. [+/-]; P < 0.0001, [+/+] vs. [-/-]). FFEM of kidney medulla showed remarkably fewer IMPs in TDLH from (-/-) vs. (+/+) and (+/-) mice. IMP densities were (in microm-2, SD, 5-12 micrographs): 5,880 +/- 238 (+/+); 5,780 +/- 450 (+/-); and 877 +/- 420 (-/-). IMP size distribution analysis revealed mean IMP diameters of 8.4 nm ([+/+] and [+/-]) and 5.2 nm ([-/-]). These results demonstrate that AQP1 is the principal water channel in TDLH and support the view that osmotic equilibration along TDLH by water transport plays a key role in the renal countercurrent concentrating mechanism. The similar Pf and AQP1 expression in TDLH of (+/+) and (+/-) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/-) mice.

Intramembrane structure of the sensory axon terminals in bullfrog muscle spindles

Much physiologic and morphologic research has been done into the sensory mechanism of the frog muscle spindle. However, no freeze-fracture study has described in detail the shape and intramembrane structure of the nonmyelinated sensory axon terminals of the frog muscle spindle. In this study, muscle spindles were isolated from the red part of bullfrog semitendinous muscles. Chemically fixed spindles were subjected to freeze fracturing. The sensory axon endings were reconstructed, and the size and density of intramembrane particles (IMPs) were measured along the sensory nerve endings. The axon terminals had four distinctive parts: parent trunks (>0.5 microm in diameter), primary branches (0.15-0.5 microm), terminal branches (<0.1 pm), and varicosities (0.02-0.5 microm). IMPs ranged from 5 nm to 21 nm in diameter and were present in the intramembrane space of the plasma membrane all throughout the nonmyelinated sensory nerve endings. Mean IMP sizes in the protoplasmic face (PF) and the external face (EF), respectively, were 8.1 nm and 8.4 nm in the parent trunks, 8.8 nm and 8.8 nm in the primary branches, 9.4 nm and 9.0 nm in the varicosities, and 8.7 nm and 8.7 nm in the terminal branches. Mean IMP size in the PF was smallest in the parent trunk and largest in the varicosity. Mean IMP densities (numbers of IMPs per microm2) in the PF and the EF, respectively, were 2,500 and 700 in the parent trunks, 2,200 and 500 in the primary branches, 1,700 and 400 in the varicosities, and 1,000 and 300 in the terminal branches. Density decreased with the tapering of the axon terminal, with IMPs distributed evenly in the PF and the EF. The characteristic intramembrane structure of sensory nerve endings is discussed.

Dynamics of activity and structure of the tonoplast vacuolar-type H+-ATPase in plants with differing CAM expression and in a C3 plant under salt stress

Differences in the activity and structure of the vacuolar H+-ATPase (V-ATPase, EC 3.6.1.3) were investigated in the C3/CAM intermediate plantKalanchoe blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, andHordeum vulgare cv. Carina, grown with or without 150 mM NaCl. InK. blossfeldiana ATP-hydrolysis and H+-transport activity were higher with higher expression of CAM than in plants with very weak CAM. This was mainly due to a larger amount of V-ATPase. Statistical analysis of the diameter of intramembrane particles (IMPs) on freeze-fractures of tonoplast vesicles showed that IMPs were larger in tonoplast vesicle preparations ofK. blossfeldiana with strong CAM expression (9.1 nm) than in preparations ofK. blossfeldiana with low CAM expression (7.3 nm). As there is evidence that the majority of IMPs on freeze-fractures of tonoplast vesicles corresponds to the V0 domain of V-ATPase, the higher activity of V-ATPase inK. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase inK. blossfeldiana with stronger CAM is discussed in relation to the requirement for a higher proton pumping capacity for nocturnal malate accumulation in the vacuole. The ATP-dependent H+-pumping activity inH. vulgare was higher under salt stress than in control plants, while the rates of ATP-hydrolysis and the size of IMPs were not affected by the salt treatment. The data presented here indicate that different mechanisms might increase the transport capacity of V-ATPase to meet the higher requirements of secondary active transport related to CAM expression and adaptation to salt stress.

Distribution of intramembranous particle size in the muscle plasma membrane of the mdx mouse

We investigated whether the reduced intramembranous particles (IMP) in the muscle plasma membrane in mdx mice reflects a preferential depletion of a particular size of the IMP. The experiments were performed using the freeze-fracture method to analyze the frequency distribution of the size of IMP, the density of orthogonal array and caveolae in the extensor digitorum longus (EDL) and soleus (SOL) muscles obtained from mdx and control mice. We detected a reduced density of IMP and orthogonal array, and the increased density of caveolae in EDL muscle but not in SOL in the mdx mouse compared with those of the same muscles in control animals. The reduction of IMP was, however, not limited to any specific size of IMP. Our results suggest that the dystrophin associated glycoprotein present in the membrane does not reflect a specific size of IMP. Therefore, our findings indicate that the mechanism of reduced IMP in dystrophinopathy may be different from that of diminished dystrophin binding glycoprotein associated with dystrophin deficiency in Duchenne muscular dystrophy.

Membrane modifications in chick osteoclasts revealed by freeze‐fracture replicas

Remarkable differences among various membranes of bone cells became evident by examination of freeze-fracture replicas. In osteoclasts, three types of intramembranous particles (IMPs) were identified based on their size and shape: two sizes of isolated globular particles (8 and 12 nm in diameter) and rod-shaped, linear aggregates (8 x 30 nm in dimension). Furthermore, the density and distribution pattern of these IMPs enabled us to distinguish three different domains of membranes of osteoclasts including ruffled border, clear zone, and basolateral regions, as were also observed in thin sections. The highest density of IMPs was 3,500-4,000/microns2 in the ruffled border membrane, and these IMPs included linear aggregates among the usual globular particles. Linear aggregated particles were also observed in the membrane of cytoplasmic vesicles in the vicinity of the ruffled border region, but not in this membrane in other bone cells. In attached osteoclasts, the distribution patterns and densities of IMPs in each ruffled-finger and -plate were extremely variable, from closely to the loosely packed membrane particles. Focal aggregates of membrane particles were also frequently encountered. An important outcome of the present study was the finding that the presence of linear aggregated particles proved to be an additional criterion for distinguishing membrane domains in freeze-replicas of osteoclasts. The surface of the clear zone membrane was not smooth in profile, but revealed a number of eminences that were almost free of particles. Basolateral membranes exhibited a particle density of 2,400/microns2. Globular particles were homogeneously scattered in random fashion on their exposed fracture faces. In some cases, aggregates of IMPs on the basolateral membranes were encountered. In comparison with the ruffled fingers, microprojections from the basolateral surface showed a lesser density of IMPs and were devoid of rod-shaped or linear aggregated particles. Differences between osteoblasts and osteocytes were apparent in the density and the size of IMPs. The membranes of osteoblasts and osteocytes contained the same types of globular particles as seen in osteoclasts. Various sizes of gap junctions were located only on basolateral membranes of the osteoblasts. In contrast, no cellular junctions were observed between osteoclasts and any other type of cells.

Freeze-fracture electron microscopic observations on the effects of sulphydryl group reagents on human erythrocyte membranes

Freeze-fracture electron microscopy of human red blood cells at pH 7.4 and 5.5 reveals the presence of membrane elevations (50–100 nm diameter). These are also observed after incubation of the erythrocytes with N-ethylmaleimide but not after incubation with p-chloromercuribenzene sulphonate. Neither of the sulphydryl-group reagents affects the distribution or size of intramembrane particles. The findings are discussed in the light of the effects of mercurials on erythrocyte membrane proteins.

Transient inactivation of the thylakoid photosystem II light-harvesting protein kinase system and concominant changes in intramembrane particle size during photoinhibition of [formula omitted

Transient inactivation of the thylakoid photosystem II light-harvesting protein kinase system and concominant changes in intramembrane particle size during photoinhibition of [formula omitted

Transient inactivation of the thylakoid photosystem II light-harvesting protein kinase system and concomitant changes in intramembrane particle size during photoinhibition of Chlamydomonas reinhardtii.

Light-dependent reduction of the plastoquinone pool regulates the activity of the thylakoid-bound protein kinase which phosphorylates the light harvesting chlorophyll a,b-protein complex (LHC II) and regulates energy distribution between photosystems II (PS II) and I (Staehelin, L. A., and C. J. Arntzen, 1983, J. Cell Biol., 97:1327-1337). Since reduction of plastoquinone by PS II is abolished in photoinhibited thylakoids due to loss of the secondary electron acceptor QB protein (Kyle, D. J., I. Ohad, and C. J. Arntzen, 1984, Proc. Natl. Acad. Sci. USA, 81:4070-4074), it was of interest to examine the activity of the LHC II protein kinase system during photoinhibition and recovery of PS II activity. The kinase activity was assessed both in vivo and in vitro in Chlamydomonas cells exposed to high light intensity (photoinhibition) and recovery at low light intensity. The kinase activity was progressively reduced during photoinhibition and became undetectable after 90 min. The inactive LHC II-kinase system could not be reactivated in vitro either by light or by reduction of the plastoquinone pool following addition of reduced duroquinone (TMQH2). The LHC II polypeptides were dephosphorylated in vivo when cells, prelabeled with [32P]orthophosphate before exposure to high light intensity, were transferred to photoinhibiting light in the presence of [32P]orthophosphate. In vivo recovery of the LHC II-kinase activity, elicited by the addition of TMQH2 to the assay system, did not require restoration of QB-dependent electron flow or de novo protein synthesis, either in the cytoplasm or in the chloroplast. Mild sonication of thylakoids isolated from photoinhibited cells restored the ability of the LHC II protein kinase system to be activated in vitro by addition to TMQH2. Restoration of the light-activated LHC-II kinase required recovery of QB-dependent electron flow. At the structural level, photoinhibition did not affect the ratio of grana/stroma thylakoids. A reduction of approximately 20% of the 11-17-nm intramembrane particles and an equivalent increase in the number of 6-10.5-nm particles was observed on the E-fracture faces of stacked thylakoid membranes. Similar but smaller changes were observed also on the E-fracture faces of unstacked thylakoid membranes (more 10-14-nm and less 6-9-nm particles) and P-fracture faces of stacked thylakoid membranes (more 6-8- and less 9.5-13-nm particles). All these structural changes were reversed to normal values during recovery of PS II activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Hyperthermia on the Plasma Membrane Structure of Chinese Hamster V79 Fibroblasts: A Quantitative Freeze-Fracture Study

Hyperthermia in the range 41-45 degrees C can induce wide biochemical, physiological, and morphological changes in mammalian cells both in vivo and in vitro. In general, its effects on membranes, particularly on the plasma membrane, are still poorly understood. To investigate the effects of heat on this cell structure, Chinese hamster V79 fibroblasts were exposed to 43 degrees C hyperthermia for 1 h, immediately fixed with glutaraldehyde after treatment, and freeze-fractured for electron microscopic examination. Particular attention was given to the density and size of intramembranous particles (IMPs) on both protoplasmic (PF) and external (EF) fracture faces of the plasma membrane. The quantitative study performed by an interactive image analyzer on the IMPs, generally reported as plasma membrane proteins, showed in heat-treated cells a statistically significant increase in their density and size on both fracture faces. The differences observed demonstrate that in our experimental conditions, hyperthermia in plasma membranes produces structural changes whose biological significance has to be clarified. Moreover, our findings seem to support recent data indicating an involvement of membrane proteins in the cell response to hyperthermia.

Protein secretion in Tetrahymena thermophila: Characterization of the secretory mutant strain sb281

The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion. Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210. In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described. SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling. Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts. In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells. A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane. Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type.

Animal-vegetal polarity in the plasma membrane of a molluscan egg: A quantitative freeze-fracture study

Using freeze-fracture electron microscopy, the numerical particle distribution in the fertilized Nassarius egg plasma membrane has been analyzed in four areas at different positions along the animal-vegetal axis of the egg. These areas can be distinguished by distinct microvilli patterns and differences in microvilli densities. In all areas, more IMPs (intramembrane particles) are present on the P face than on the corresponding E face. The ratio of the number of IMPs present on E and P face is similar in all areas (0.48–0.55) except for the most animal part of the vegetal hemisphere, where relatively more IMPs remain attached to the exterior half of the fractured membrane ( E P ratio = 0.88). The IMP density at the vegetal pole of the egg is considerably higher than in the animal hemisphere and in the animal part of the vegetal hemisphere. This difference is due to an increased number of IMPs in all size classes (4–18 nm). In the area adjacent to the vegetal pole the density of particles is also higher than in the two more animal areas, but here the difference is exclusively due to the smaller IMP size classes (4–8 nm). Statistical analysis of our data reveals that the area adjacent to the vegetal pole patch is significantly different from the other areas with respect to the distribution of the IMPs over the different IMP size classes. These results demonstrate the polar organization of the Nassarius egg plasma membrane. The possible role of this surface heterogeneity in the spatial organization of the egg cell and the later embryo is discussed.

Membrane specializations of neuritic growth cones in vivo: A quantitative IMP analysis

The internal structure of the membranes of axonal and Schwann cell growth processes was examined by freeze-fracture in the growing olfactory nerve, a simple in vivo system consisting of a homogeneous neuronal population. Excision of the mature nerve of adult bullfrogs provides well-synchronized primary neuritic outgrowth that is highly enriched in growth cones at its distalmost segment. The extreme uniformity of olfactory axons in terms of their diameter and their intramembrane particle (IMP) composition permits clear identification of the cellular origin of the growth cone structures observed in replicas. In vivo, growth cones of the olfactory nerve appear as irregularly shaped enlargements of the distal tip; filopodia are only infrequently exposed by the fracture plane. Axonal and Schwann cell growth cones are distinguished by 1) the larger size of the Schwann cell growth cone and the smaller diameter of its attached processes, and 2) the distinct differences in IMP composition of Schwann cell and axonal growth cones and cell processes. Schwann cell growth cones display a uniformly high IMP density on their P-face leaflet, with the exception of circumscribed moundlike protrusions that are relatively free of IMPs. In contrast, axonal growth cones display sharp regional variation in IMP density on their P-face: broad regions almost devoid of IMPs are interspersed with zones of high IMP density. Cytotic profiles occur within high IMP density zones located, most often, at the base of the axonal growth cone. A comparison of IMP size histograms of both high and low-density regions of axonal growth cones and that of the neighboring distal shaft of the axon indicates a strict partitioning of membrane components between these two regions. The IMP profile of the axonal growth cone, notable for its relative enrichment in large-diameter particles, suggests that IMP components of the growth cone are delivered to the distal tip by a mechanism that is distinct from the lateral diffusion process described for particles of the growing axon's shaft [cf. Small and Pfenninger, 1984]. The IMP profile of the concave P-face leaflet of the internal vesicles found clustered at the base of the growth cone is more similar in composition to the profile of the neuronal shaft than that of the growth cone.

Asymmetric mass distribution of [formula omitted] in membranes studied by freeze-fracture-etch electron microscopy

SDS-purified porcine kidney (Na + + K +)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: F A , which shows a rather smooth background with many intramembranous particles, and F B , which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that F A faces are covered with many intramembranous particles, while the etched external faces (E A) are either irregularly granulated or reveal many particles half the size of intramembranous particles. F B faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (E B) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated F A convex fracture faces and irregularly granulated or smooth etched E A surfaces, indicating that the F A face is the protoplasmic fracture face and that the majority of the protein mass of the (Na + + K +)-ATPase is located on the cytoplasmic half of the membrane.

Components of the plasma membrane of growing axons. II. Diffusion of membrane protein complexes.

Intramembrane particles (IMPs) of the plasmalemma of mature, synapsing neurons are evenly distributed along the axon shaft. In contrast, IMPs of growing olfactory axons form density gradients: IMP density decreases with increasing distance from the perikarya, with a slope that depends upon IMP size (Small, R., and K. H. Pfenninger, 1984, J. Cell Biol., 98: 1422-1433). These IMP density gradients resemble Gaussian tails, but they are much more accurately described by the equations formulated for diffusion in a system with a moving boundary (a Stefan Problem), using constants that are dependent upon IMP size. The resulting model predicts a shallow, nearly linear IMP density profile at early stages of growth. Later, this profile becomes gradually transformed into a steep nonlinear gradient as axon elongation proceeds. This prediction is borne out by the experimental evidence. The diffusion coefficients calculated from this model range from 0.5 to 1.8 X 10(-7) cm2/s for IMPs between 14.8 and 3.6 nm, respectively. These diffusion coefficients are linearly dependent upon the inverse IMP diameter in accordance with the Stokes-Einstein relationship. The measured viscosity is approximately 7 centipoise. Our findings indicate (a) that most IMPs in growing axons reach distal locations by lateral diffusion in the plasma membrane, (b) that IMPs--or complexes of integral membrane proteins--can diffuse at considerably higher rates than previously reported for iso-concentration systems, and (c) that the laws of diffusion determined for macroscopic systems are applicable to the submicroscopic membrane system.

Quantitative analysis of modulations in intramembrane particles during maturation of starfish oocytes

The cell membrane of oocytes of the starfish Asterina pectinifera was investigated during maturation by freeze-fracture electron microscopy. On the micrographs of freeze-fractured oolemmas at various stages of maturation induced by 1-methyladenine, the intramembrane particles (IMPs) were counted and the size of each IMP was measured using an image analyzer. A 5-min treatment with the hormone lead to a decrease in IMP density on the E face and an increase on the P face. The most remarkable change in the density, an increase of 87% on the E face and a decrease of 42% on the P face, occurred within the following 5 min, the late “hormone-dependent period.” The mean value of IMP size first decreased on both faces, then gradually increased until values similar to those of the immature oocyte were reached. The results revealed that the hormonal signal to the surface receptors induces reorganization of the membrane components at the time of its action on the ooplasm, and that the oolemma also changes as a result of maturation of the ooplasm.

Tannic acid improves the visualization of the human erythrocyte membrane skeleton by freeze-etching

The effect of fixatives on the membrane skeleton underlying the human erythrocyte membrane was examined by freeze-etching. An anastomosing fibrillar network was readily observed on the protoplasmic surface of the erythrocyte membrane treated with tannic acid. Such structure was much less defined in unfixed membrane or membrane fixed with glutaraldehyde or glutaraldehyde followed by osmium tetraoxide. Tannic acid caused a marked increase in diameter of the fibrillar components of the membrane skeleton and of the protoplasmic surface particles of inside-out vesicles prepared by alkali treatment but did not affect the size of intramembranous particles seen on fracture faces nor the appearance of exoplasmic surfaces. The improved visualization of the membrane skeleton after treatment with tannic acid resulted from interactions between tannic acid and exposed membrane proteins.

Random distribution of NADPH-specific flavoprotein and cytochrome P-450 in liver microsomes

The dilution of rabbit liver microsomes by soy-bean phospholipids was used as methodical approach to investigate the molecular organization of NADPH-dependent microsomal redox chain. The ultrastructural analysis of control and phospholipid diluted microsomes revealed that the incorporation of exogenous phospholipids into microsome membranes increased their surface area, as well as decreased the lateral density distribution and size of intramembrane particles. The dilution of microsome membranes by phospholipids slowed down the initial rate of cytochrome P-450 reduction by NADPH. The apparent second order rate constant of cytochrome P-450 reduction by NADPH: cytochrome P-450-reductase did not change in phospholipid-enriched microsomes. The results obtained provide strong evidence for the random distribution of NADPH-specific flavoprotein and cytochrome P-450 in liver microsome membranes.