Endocytosis via galactose receptors in vivo : Ligand size directs uptake by hepatocytes and/or liver macrophages

We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction.     

Localization of uroplakin Ia, the urothelial receptor for bacterial adhesin FimH, on the six inner domains of the 16 nm urothelial plaque particle

The binding of uropathogenic Escherichia coli to the urothelial surface is a critical initial event for establishing urinary tract infection, because it prevents the bacteria from being removed by micturition and it triggers bacterial invasion as well as host cell defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium and its urothelial receptor, uroplakin Ia (UPIa). To localize the UPIa receptor on the 16 nm particles that form two-dimensional crystals of asymmetric unit membrane (AUM) covering >90 % of the apical urothelial surface, we constructed a 15 A resolution 3-D model of the mouse 16 nm AUM particle by negative staining and electron crystallography. Similar to previous lower-resolution models of bovine and pig AUM particles, the mouse 16 nm AUM particle consists of six inner and six outer domains that are interconnected to form a twisted ribbon-like structure. Treatment of urothelial plaques with 0.02-0.1 % (v/v) Triton X-100 allowed the stain to penetrate into the membrane, revealing parts of the uroplakin transmembrane moiety with an overall diameter of 14 nm, which was much bigger than the 11 nm value determined earlier by quick-freeze deep-etch. Atomic force microscopy of native, unfixed mouse and bovine urothelial plaques confirmed the overall structure of the luminal 16 nm AUM particle that was raised by 6.5 nm above the luminal membrane surface and, in addition, revealed a circular, 0.5 nm high, cytoplasmic protrusion of approximately 14 nm diameter. Finally, a difference map calculated from the mouse urothelial plaque images collected in the presence and absence of recombinant bacterial FimH/FimC complex revealed the selective binding of FimH to the six inner domains of the 16 nm AUM particle. These results indicate that the 16 nm AUM particle is anchored by a approximately 14 nm diameter transmembrane stalk, and suggest that bacterial binding to UPIa that resides within the six inner domains of the 16 nm AUM particle may preferentially trigger transmembrane signaling involved in bacterial invasion and host cell defense.     

Growth and fragmentation of silver nanoparticles in their synthesis with a fs laser and CW light by photo-sensitization with benzophenone.

The photo-sensitization synthetic technique of making silver nanoparticles using benzophenone is studied using both a laser and a mercury lamp as light sources. The power and irradiation time dependence of the synthesized nanoparticle absorption spectra and their size distribution [as determined by transmission electron microscopy (TEM)] are studied in each method and compared. In the laser synthesis, as either the laser power or the irradiation time increases, the intensity of the surface plasmon resonance absorption at 400 nm is found to increase linearly first, followed by a reduction of the red edge of the plasmon resonance absorption band. The TEM results showed that in the laser synthesis low powers and short irradiation times produce nanoparticles around 20 nm in diameter. Increasing the power or irradiation time produces a second population of nanoparticles with average size of 5 nm in diameter. These small particles are believed to be formed from the surface ablation of the large particles. The surface plasmon absorption band is found to be narrower when the nanoparticles are produced with laser irradiation. Throughout the exposure time with the CW lamp, the plasmon resonance absorption band of the particles formed first grows in intensity, then blue shifts and narrows, and finally red shifts while decreasing in intensity. The TEM results for lamp samples showed particle formation and growth, followed by small nanoparticle formation. The above results are discussed in terms of a mechanism in which, the excited benzophenone forms the ketal radical, which reduces Ag+ in solution and on the Ag nanoparticle surface. As the time of irradiation or the light energy increases the benzophenone is consumed, which is found to be the limiting reagent. This stops the formation of the normal large nanoparticles while their photo-ablation continues to make the small particles.     

Surface structure of Uukuniemi virus.

Uukuniemi virus, grown in chicken embryo fibroblasts, has been studied by electron microscopy using negative staining, thin sectioning, and freeze-etching techniques. The spherical virus particle measures about 95 nm in diameter. Its envelope consists of a 5-nm thick membrane covered by 8- to 10-nm long surface projections. These are composed of two polypeptides species of about the same size. Both of them can be removed by digestion with the proteolytic enzyme thermolysin except for a small fragment. The enzyme-treated particles are smooth surfaced and extremely deformable. The glycopolypeptides are clustered to form hollow cylindrical morphological units, 10 to 12 nm in diameter, with a 5-nm central cavity. Both negative staining and freeze-etching suggest that these units are penton-hexon clusters arranged in a T = 12, P = 3, icosahedral surface lattice. The membrane to which the surface subunits are attached is probably a lipid bilayer as evidenced by its double-track appearance in thin sections and the tendency of the freeze fracturing to occur within it. The strand-like nucleoprotein appears from thin-sectioning results to be to a large part located in a zone underneath the membrane.     

Size and lipid composition of chylomicrons of different Svedberg units of flotation

Chylomicrons from thoracic duct lymph of rabbits which were fed corn oil were separated in a preparative ultracentrifuge into subfractions of different S(f) values in order to compare their size, as determined by electron microscopy, with that expected from ultracentrifugation data. The lipid composition of the chylomicrons of different S(f) values was also correlated with their morphology in order to elucidate more about their structure. Although the diameter distribution of chylomicrons from subfractions of lower S(f) ranges corresponded approximately to the expected size distribution, that of the higher S(f) ranges contained many small particles. The TG:PL ratio showed a highly significant correlation with the V:SA ratio of chylomicrons from all subfractions. The findings were consistent with the hypothesis that, irrespective of the S(f) range of chylomicrons, the core is comprised of TG, while PL is spread as a monomolecular layer on the surface of the particles.     

Core-shell Au nanoparticle formation with DNA-polymer hybrid coatings using aqueous ATRP

We report here a direct surface-grafting approach to forming DNA-containing polymer shells outside of Au nanoparticles using aqueous atom transfer radical polymerization (ATRP). In this approach, DNA molecules were immobilized on Au particles to introduce ATRP initiators on the surface. The same DNA molecules also acted as particle stabilizers through electrostatic repulsion and allowed particles to stay suspended in water. The immobilized ATRP initiators prompted polymer chain growth under certain conditions to form thick polymer shells outside of the particles. The formation of DNA-polymer hybrids outside of Au nanoparticles was characterized using absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and gel electrophoresis. The presence of thick polymer shells improved particle stability in high ionic strength media, whereas particles with the DNA coating only aggregated. A visible color difference between these two particle solutions was clearly observed, providing the basis for DNA sensing in homogeneous solutions.     

Some viscoelastic properties of human erythrocyte spectrin networks end-linked in vitro

We have succeeded in making macroscopic networks of end-linked human erythrocyte spectrin. The network junctions were made using erythrocyte protein 4.1 irreversibly attached to 5 nm (diameter) colloidal gold particles. Rotary shadowing electron microscopy verifies that the protein 4.1-labelled colloidal gold particles bind only to the tail end of the spectrin molecules. Electron micrographs of protein 4.1-labelled colloidal gold particles incubated at 4 degrees C with spectrin dimers reveal that 1-5 spectrin dimers attach to each protein 4.1-labelled colloidal gold particle yielding a spider-like appearance of these complexes. Incubation with a low concentration of spectrin tetramers instead of dimers leads to extensive formation of spectrin microaggregates whereas use of spectrin concentrations higher than 3 mg/ml and a molar ratio between spectrin tetramers and protein 4.1/Au of 4 leads to formation of macroscopic spectrin networks. We have quantitated the viscoelastic properties of such end-linked macroscopic spectrin networks using a gravitational pendulum viscoelastometer. We find that in vitro end-linked spectrin networks can be described by linear viscoelastic theory. The dynamic storage modulus increases almost linearly with the spectrin-protein 4.1/gold particle concentration when the spectrin concentration exceeds about 3 mg/ml and the molar ratio between spectrin tetramers and protein 4.1/Au is 4. At a spectrin concentration of 6 mg/ml and the same ratio between spectrin and protein 4.1/Au, we find a dynamic storage modulus at low frequency of about 80 dyn/cm2. This is in adequate agreement with what is predicted by simple elastomer theory.     

Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry

Effect of particle size on labeling intensity in protein A-gold immunocytochemistry was studied. Catalase labeling of rat liver peroxisomes was used as a labeling model. Ultra-thin sections of Lowicryl K4M-embedded rat liver were stained for catalase with protein A-gold (pAg) probes. Five different sizes of colloidal gold probes, from 5 nm to 38 nm in diameter, were prepared. Labeling intensity decreased as the particle size of the pAg probes increased. The highest labeling was obtained by the 5-nm pAg probe and the lowest by the 38-nm pAg probe. Quantitative analysis also showed that labeling density was inversely proportional to the size of gold particles. The results suggest that the pAg probe with small gold particles has high sensitivity.     

Phospholipid-stabilized nanoparticles of cyclosporine a by rapid expansion from supercritical to aqueous solution

The purpose of this research was to form stable suspensions of submicron particles of cyclosporine A, a water-insoluble drug, by rapid expansion from supercritical to aqueous solution (RESAS). A solution of cyclosporine A in CO₂ was expanded into an aqueous solution containing phospholipid vesicles mixed with nonionic surfactants to provide stabilization against particle growth resulting from collisions in the expanding jet. The products were evaluated by measuring drug loading with high performance liquid chromatography (HPLC), particle sizing by dynamic light scattering (DLS), and particle morphology by transmission electron microscopy (TEM) and x-ray diffraction. The ability of the surfactant molecules to orient at the surface of the particles and provide steric stabilization could be manipulated by changing process variables including temperature and suspension concentration. Suspensions with high payloads (up to 54 mg/mL) could be achieved with a mean diameter of 500 nm and particle size distribution ranging from 40 to 920 nm. This size range is several hundred nanometers smaller than that produced by RESAS for particles stabilized by Tween 80 alone. The high drug payloads (≈10 times greater than the equilibrium solubility), the small particle sizes, and the long-term stability make this process attractive for development.     

Enhanced viscoelasticity of human cystic fibrotic sputum correlates with increasing microheterogeneity in particle transport

Current biochemical characterizations of cystic fibrosis (CF) sputum do not address the high degree of microheterogeneity in the rheological properties of the mucosal matrix and only provide bulk-average particle diffusion coefficients. The viscoelasticity of CF sputum greatly reduces the diffusion rates of colloidal particles, limiting the effectiveness of gene delivery to underlying lung cells. We determine diffusion coefficients of hundreds of individual amine-modified and carboxylated polystyrene particles (diameter 100-500 nm) embedded in human CF sputum with 5 nm and 33 ms of spatiotemporal resolution. High resolution multiple particle tracking is used to calculate the effective viscoelastic properties of CF sputum at the micron scale, which we relate to its macroscopic viscoelasticity. CF sputum microviscosity, as probed by 100- and 200-nm particles, is an order of magnitude lower than its macroviscosity, suggesting that nanoparticles dispersed in CF sputum are transported primarily through lower viscosity pores within a highly elastic matrix. Multiple particle tracking provides a non-destructive, highly sensitive method to quantify the high heterogeneity of the mucus pore network. The mean diffusion coefficient becomes dominated by relatively few but fast-moving particles as particle size is reduced from 500 to 100 nm. Neutrally charged particles with a diameter <200 nm undergo more rapid transport in CF sputum than charged particles. Treatment with recombinant human DNase (Pulmozyme) reduces macroviscoelastic properties of CF sputum by up to 50% and dramatically narrows the distribution of individual particle diffusion rates but surprisingly does not significantly alter the ensemble-average particle diffusion rate.     

Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro

Both phagocytosis (of particles) and pinocytosis (of solutes) occur in macrophages. It is not known, however, whether particles, if they are small enough, can enter by pinocytosis, nor whether there is a minimum size of particle capable of triggering phagocytic uptake. These questions have been investigated by studying, in vitro, the uptake by rat peritoneal macrophages of particles ranging in diameter from 30 nm to 1100 nm. Percoll (30 nm diameter) and polystyrene beads (100, 300, 600, 800 or 1100 nm diameter) were 125I-iodinated and their uptake by macrophages was measured in the absence or presence of metabolic and cytoskeletal inhibitors. Since uptake, expressed as an Endocytic Index (microliter/10(6) cells per h), increased steadily with the duration of incubation and was inhibited by low temperature or metabolic inhibitors, it was concluded that true endocytosis, and not a superficial cell-association, was being measured. Rates of clearance increased with increasing particle diameter. The rate of uptake of Percoll was 10-times, and of 100 nm polystyrene beads 100-times, the rate of fluid-phase pinocytosis, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. Polystyrene beads of 1100 nm diameter were captured at 700-times this rate. The differential effects of colchicine and cytochalasin B on the uptake of 125I-labelled polyvinylpyrrolidone and of 1100 nm polystyrene beads were taken as indicators of their effects on pinocytosis and phagocytosis respectively. It is concluded that Percoll, although particulate, is captured by pinocytosis. The pattern of inhibition of uptake of polystyrene particles suggests that there is no radical discontinuity between pinocytic and phagocytic uptake, but that the contribution of phagocytosis steadily increases with increasing particle diameter. The results are discussed.     

Qualitative and quantitative changes in hepatic lipoprotein particles following acute injury of the rat liver induced by thioacetamide

Acute intoxication of the rat liver with a single dose of 100 mg thioacetamide (TAA)/kg body weight causes within 48 h a fatty liver and a heterogeneous reaction in the hepatocytes. This affects principally the centrilobular liver parenchymal cells (zone 3) and to a lesser extent the periportal ones (zone 1). Ultrastructural analysis was performed to determine to what extent the formation of lipid-carrying particles of the very low density type (VLDL) is changed in affected hepatocytes in zones 1 and 3. Being morphologically the most conspicuous site of VLDL processing, the Golgi complex was chosen for quantitation by measuring its volume, VLDL content and particle size. The concentration and composition of the liver lipids were determined, biochemically. After TAA treatment of the liver the number of Golgi-VLDL particles is significantly reduced to about 50% in both the lobular zones examined. In addition, distinct classes of size-modified Golgi-VLDL particles appear which show an abnormally wide size distribution pattern. In periportal hepatocytes the size distribution of Golgi-VLDL particles shows a clear shift towards smaller particles homogeneous in size (mean diameter 39 nm). In contrast, centrilobular hepatocytes contain particles of very heterogeneous size, the mean diameter of which is nearly doubled (77 nm). The decrease in VLDL particle number and their size modification induced by TAA is not accompanied by significant changes in the volume of the Golgi complex. Biochemical analysis showed that the accumulation of lipids in the TAA-treated liver, mainly evident morphologically as drop-like deposits in the central area of the liver lobules, is due to an increase in triglycerides (TG) by 23 mumol/g liver wet weight, which represents nearly 95% of the accumulated lipids. Despite the striking elevation of the absolute cholesterol ester (CHOL-E) content (2 mumol/g liver wet weight), this corresponds to only 5% of the newly accumulated lipids. Our electron optical and biochemical results support the suggestion that, in spite of the markedly different intralobular reaction of TAA-intoxicated hepatocytes, the formation of triglyceride-carrying particles is altered significantly in both lobular zones examined.     

Sizing of protein A-colloidal gold probes for immunoelectron microscopy

Gold particles in colloidal solutions often vary considerably in size. The finest sols (diameter less than 15 nm), especially, are very heterogeneous, as is indicated by coefficients of variance (CV) of 25- 35%. We have complexed staphylococcal protein A with gold particles (PA/Au) and then fractionated the preparations by glycerol or sucrose gradient centrifugation into very homogeneous subfractions. In this way, PA/Au probes of almost any size between 4.5 and 15 nm could be prepared. The variation of the gold particles in these fractions resulted in CV's between 9 and 16%. The reactivity of the PA/Au complex was not affected by the gradient procedure, as was shown by single- and double-labeling immunocytochemistry of ultrathin cryosections of rat pancreatic tissue.     

Plasmonic Properties of β-Sn Nanoparticles in Ordered and Disordered Arrangements

This work investigates the localized surface plasmon resonance (LSPR) of β-Sn also known as white tin. Recently, studies on arrays of β-Sn nanoparticles have shown that these arrays possess strong optical features caused by diffractive effects in the particle grating (Johansen et al., Phys Rev B 84:113405–113408, 2011). In the presence of the grating, the LSPR could not clearly be distinguished in the spectra. To get a better understanding of the plasmonic properties of the particles, we have now eliminated the diffractive effects by placing the particles in a random distribution. The particles were fabricated by electron beam lithography on a fused silica substrate and investigated by optical transmission measurements. In the random configuration, a clear LSPR is observed at 530 nm for particles with a diameter of 155 nm and a height of 50 nm.     

Crystalloid Inclusions in Species of Leucocytozoon,Parahaemoproteus, and Plasmodium*†

Cytoplasmic vacuoles seen in methanol-fixed, Giemsa's-stained ookinetes of Leucocytozoon simondi, Parahaemoproteus fringillae and Plasmodium gallinaceum, when studied with the electron microscope, were found to correspond with crystalloid inclusions of similar structure, particle size, and arrangement. Cytochemical examination of these “crystalloids” revealed their lipid-protein nature. Morphologically similar inclusions were found also in ookinetes of Leucocytozoon ziemanni and Parahaemoproteus velans. In L. simondi, crystalloid is formed rapidly after fertilization, from amorphous electron dense material seen in mature macrogametocytes. The arrangement and distribution of crystalloids in the zygote, ookinete, oocyst, and sporozoite are described. On the basis of differences in structure and particle size, it is proposed that the crystalloid inclusions in Haemosporina be divided into 2 types. Type I—lipid-protein in nature, characterized by electron dense irregularly spherical particles, 25–40 nm in diameter, with individual particles not invested by membrane. Type II—probably virus, characterized by electron dense, irregularly spherical, membrane-bounded particles, with a diameter usually greater than 40 nm.     

Formation of phospholipid-rich HDL: a model for square-packing lipoprotein particles found in interstitial fluid and in abetalipoproteinemic plasma

The major bovine HDL subfraction, fraction I-HDL, was incubated with increasing amounts of dimyristoylphosphatidylcholine (DMPC). HDL size, as determined by gradient gel electrophoresis and electron microscopy, increased with increasing HDL-phospholipid to DMPC mole ratios. Control fraction I-HDL were spherical, hexagonally-packing particles with a peak on gradient gel electrophoresis at 12.3 +/- 0.1 nm; at a ratio of 1:0.5, larger, mainly spherical particles with a peak at 12.9 +/- 0.08 nm were formed. At a ratio of 1:1, occasional square-shaped particles were seen by electron microscopy; by gradient gel analysis, the mean diameter of the HDL-product increased to 13.7 +/- 0.1 nm. At the 1:2 ratio, extensive domains of square-packing particles were noted; the major size peak of this product was 14.6 +/- 0.08 nm. In all incubations with DMPC, a small 9.4 +/- 0.08 nm product was formed; it was most pronounced at the 1:2 ratio. The large, less dense particles generated by incubation contained apolipoprotein A-I and small molecular weight proteins. The 9.4 nm product contained only apolipoprotein A-I. The less dense product formed during incubation at the 1:2 ratio had a decreased protein-to-lipid ratio relative to control HDL and a 2-fold increase in percent phospholipid. At a 1:2 ratio, incorporation of DMPC into fraction I-HDL results in the loss of one molecule of apolipoprotein A-I; the resultant particle is a stable phospholipid-rich and protein-poor HDL which has a square-packing geometry. These phospholipid-laden HDL are morphologically similar to lipoproteins isolated from interstitial fluid or from plasma of abetalipoproteinemic patients. Our data suggest that the unusual morphological properties of the latter biologically formed particles may be due to increases in the polar lipid contents, and concomitant decreases in surface protein.     

Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene

Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.     

Atomic force microscopy investigation of isolated virions of murine leukemia virus

Virions of mouse leukemia virus spread on glass substrates were visualized by atomic force microscopy. The size distribution mode was 145 nm, significantly larger than that for human immunodeficiency virus particles. The distribution of particle sizes is broad, indicating that no two particles are likely identical in content or surface features. Virions possess knoblike protrusions, which may represent vestiges of budding from cell membranes. Particles which split open allowed imaging of intact cores with diameters of 65 nm. They also permitted estimation of viral shell thickness (35 to 40 nm) and showed the presence of a distinct trough between the shell and the core surface.     

Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria.

The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.