Anomalies in sedimentation. I. Stability theory of sedimenting entanglements in the "tight-bending" limit

A simple model is introduced to investigate the stability of a sedimenting entanglement. The sedimenting entanglement is represented by a sedimenting sieve. Solvent can pass through it, but single-chain molecules that flow into it become entangled and their flow decreases or, if permanent entanglements form, ceases entirely. With this model we are able to find the conditions under which the mass of a sedimenting entanglement remains constant, grows or decays to a stable value, grows beyond limit, or decays to the mass of a single chain. The theory is applied to the sedimentation of small concentrations of large chain molecules in solutions of small chain molecules in solutions of small chain molecules for the case in which the entanglements are long-lived. Equations are derived which, (1) give the stable entanglement mass as a function of rotor speed and concentration and, (2) for a given concentration predict the rotor speed at which the entanglement mass grows without limit. Numerical results for small concentrations of T2 DNA sedimenting in solutions of T7 DNA are presented.     

Filtration,diffusion, and molecular sieving through porous cellulose membranes

1. A study has been made of the diffusion and filtration of a graded series of molecules (including tritium-labelled water, urea, glucose, antipyrine, sucrose, raffinose, and hemoglobin) in aqueous solution through porous cellulose membranes of three degrees of porosity. 2. Experimental results were in close agreement with predictions based on the membrane pore theory of Pappenheimer et al. (1,2). Restriction to molecular diffusion is a function of pore radius and molecular radius described by equation (11) in the text. Molecular sieving during ultrafiltration is a function of total pore area per unit path length, pore radius, molecular radius, and filtration rate given by equations (16) and (19). 3. Estimates of average pore radius made by means of this theory were considerably larger than estimates made by the method of Elford and Ferry (3) (Table II). Sources of error in the latter method are discussed and a new method of membrane calibration is proposed in which the total cross-sectional area of the pores is measured by direct diffusion of isotope-labelled water. 4. Steady-state osmotic pressures of solutions of sucrose and raffinose measured during molecular sieving through cellulose membranes were found to be close to the "ideal" osmotic pressures calculated by van't Hoff's law. Thus the present experimental data support the methods used by Pappenheimer et al. in their studies on living capillary walls as well as their theory of membrane pore permeability.     

Novel Ribonucleic Acid Species Accumulated in the Dark in the Blue-Green Alga Anacystis nidulans

In the dark, the obligately photoautotrophic blue-green alga Anacystis nidulans accumulates large relative amounts of two novel stable ribonucleic acid species (RNAs). These species are also made in illuminated cells but are unstable in them. When darkened cells are reilluminated, these RNAs are rapidly degraded; degradation is inhibited by chloramphenicol. Upon denaturation with heat or urea, one novel species (0.33 x 10(6) daltons) dissociates into two fragments that comigrate with the second novel species (0.16 x 10(6) daltons) on polyacrylamide gels. Both RNAs are associated with particles sedimenting between 30S and 50S through sucrose gradients and are removed from these particles at low magnesium concentration. The function(s) of these RNAs remains unknown.     

Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis.

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented.     

Nectar feeding by the ant Camponotus mus: intake rate and crop filling as a function of sucrose concentration

In independent assays, workers of the ant Camponotus mus were conditioned to visit an arena where they found a large drop of sucrose solution of different concentrations, from 5 to 70% weight on weight (w/w). Single ants were allowed to collect the sucrose solution ad libitum, and feeding time, feeding interruptions, crop load, and intake rates were recorded. Feeding time increased exponentially with sucrose concentration, and this relationship was quantitatively described by the increase in viscosity with concentration corresponding to pure sucrose solutions. Ants collecting dilute solutions (5 to 15% w/w) returned to the nest with partial crop loads. Crop filling increased with increasing sucrose concentration, and reached a maximum at 42.6% w/w. Workers collecting highly concentrated solutions (70% w/w) also returned to the nest with a partially-filled crop, as observed for dilute solutions. Nectar intake rate was observed to increase with increasing sucrose concentration in the range 5 to 30% sucrose. It reached a maximum at 30.8%, and declined with increasing sucrose concentration. Results suggest that both sucrose concentration and viscosity of the ingested solution modulate feeding mechanics as well as the worker's decision about the load size to be collected before leaving the source.     

Analytical sedimentation studies of turkey gizzard myosin light chain kinase and telokin.

Sedimentation equilibrium and velocity studies were performed with turkey gizzard myosin light chain kinase (MLCK) and telokin, a small protein apparently corresponding to the sequence of the COOH-terminal end of MLCK. The measurements carried out with MLCK give values for the monomer molecular weight (M(r)), sedimentation coefficient (S20 degrees,w), and virial coefficient (A2) of 108,000, 3.74 S, and -1.95 x 10(-4) mol.ml.g-2, respectively. In the case of telokin, M(r) = 18,500; S20 degrees, w = 1.63 S; and A2 = 5.81 x 10(-4)mol.ml.g-2. Combination of the results of the two kinds of experiment shows that MLCK is a rod-shaped molecule (a/b = 18.9) with a Stoke's radius of 69 A. Telokin is also elongated (a/b = 8.3) with a Stoke's radius of 29 A. MLCK apparently exhibits self-association, with 15% of the protein sedimenting as a dimer in the experiments.     

RNA overproducing revertants of an alanyl-tRNA synthetase mutant of Escherichia coli

Summary One class of revertants of a temperature-sensitive alanyl-tRNA-synthetase mutant of Escherichia coli is characterized by a highly increased RNA content. Both stable RNA species, tRNA and rRNA contribute to the increase of the RNA/protein ratio. The RNA oversynthesis by these revertants is independent from the condition of the rel and alaS allels.Genetic analysis of the site responsible for RNA overproduction by means of a strain in which reversion to temperature-resistant growth was accompanied by the inability to grow at temperatures below 30°C (cold-sensitive phenotype) showed a chromosomal location between aroE and str. Zone centrifugation through sucrose gradients revealed that the revertants analysed contain besides a small amount of 70s ribosomal particles a high amount of particles sedimenting in a 41s–43s position in 10 mM magnesium and give rise to slower sedimenting particles in 0.1 mM magnesium ion concentration. The results provide evidence that a regulatory mechanism of RNA synthesis is affected by the ribosomal mutations which is independent from that under the control of the rel gene. Increase of both the tRNA and rRNA ratios relative to protein indicate that both stable RNA species are regulated in a similar manner.     

Honeybees prefer warmer nectar and less viscous nectar,regardless of sugar concentration

The internal temperature of flowers may be higher than air temperature, and warmer nectar could offer energetic advantages for honeybee thermoregulation, as well as being easier to drink owing to its lower viscosity. We investigated the responses of Apis mellifera scutellata (10 colonies) to warmed 10% w/w sucrose solutions, maintained at 20–35°C, independent of low air temperatures, and to 20% w/w sucrose solutions with the viscosity increased by the addition of the inert polysaccharide Tylose (up to the equivalent of 34.5% sucrose). Honeybee crop loads increased with nectar temperature, as did the total consumption of sucrose solutions over 2 h by all bees visiting the feeders. In addition, the preference of marked honeybees shifted towards higher nectar temperatures with successive feeder visits. Crop loads were inversely proportional to the viscosity of the artificial nectar, as was the total consumption of sucrose solutions over 2 h. Marked honeybees avoided higher nectar viscosities with successive feeder visits. Bees thus showed strong preferences for both warmer and less viscous nectar, independent of changes in its sugar concentration. Bees may benefit from foraging on nectars that are warmer than air temperature for two reasons that are not mutually exclusive: reduced thermoregulatory costs and faster ingestion times due to the lower viscosity.     

Sedimentation study of a catalytically active form of rabbit muscle phosphofructokinase at pH 8.55

The enzymatic active form of rabbit muscle phosphofructokinase (PFK) was observed directly by using the method of reacting or active enzyme centrifugation (AEC). These studies were performed in two assay systems: a coupled enzyme and a pH-dependent dye-linked system in glycylglycine buffer at pH 8.55 and 23 +/- 1 degree C. The sedimenting band of PFK was stabilized by three solvent systems: 50% (v/v) D2O, 10% (w/v) sucrose, and 4% (v/v) or 10% (v/v) glycerol. The active PFK species sediments as a single component with a sedimentation coefficient of 12.4 +/- 0.5 S, after correcting for protein--solvent interactions. Although PFK may undergo association--dissociation, there is no observable change in the value of s20,w over a 57-fold range of protein concentration. Throughout this range only a single active species of PFK was observed, and within an experimental uncertainty of +/- 10%, the enzymatic activity observed in the sedimentation studies accounts for the total enzymatic activity observed in the steady-state kinetics. Partially purified PFK was subjected to AEC analysis. Results reveal the presence of again a single active form sedimenting at the same rate as the purified enzyme. Results from sedimentation velocity studies indicate that the stabilizing solvents employed in AEC enhance the self-association of PFK. However, such an enhancement alone cannot account for the observation of a single active species with a sedimentation coefficient of 12.4 S. The interactions between solvent additives and PFK were studied by density measurements and by the application of multicomponent theory. Results from such a preferential solvent interaction study indicate that PFK is preferentially hydrated in the presence of sucrose or glycerol. The enhancement of PFK self-association is most likely due to a nonspecific solvent--protein interaction.     

Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase. II. The influence of surface potential upon the activating ion equilibria

Sarcoplasmic reticulum Ca2+-ATPase has been reconstituted with excess lipid (25-150 g egg phosphatidylcholine per g sarcoplasmic reticulum protein) by a procedure combining the use of a non-ionic detergent with cholate dialysis. The reconstituted vesicles were analyzed by sucrose density fractionation and freeze-fracture electron microscopy. At the lowest lipid to protein ratios some vesicles containing aggregated protein were observed. At a lipid to protein ratio of 150:1 (w/w) only 30-40% of the reconstituted protein sedimented through 7% (w/v) sucrose. The remainder of the latter preparation was characterized by a high Ca2+-uptake capacity and a coupling ratio of 1.6 mol Ca2+ transported per mol ATP hydrolyzed. Intramembranous particles in this preparation occurred isolated in the membrane. In most cases only one particle could be seen on a fracture face. Cross-linking with cupric phenanthroline indicated that protein-protein contacts were drastically reduced by reconstitution. It is concluded that aggregation of intramembranous particles is not required for optimal Ca2+-transport function. The dispersed preparation obtained by a combined reconstitution and sucrose density fractionation procedure is useful for further characterization of the Ca2+ pump.     

Critical micelle concentrations of gangliosides

The micellar properties of mixed, bovine gangliosides and purified galactosyl-N-acetylacetylgalactosaminyl (N-acetylneuraminyl) galactosylglucosylceramide were studied by gel filtration, equilibrium dialysis, and band and boundary centrifugation in sucrose gradients. The dissociation of micelles is very slow (days) in water and required us to approach equilibrium by association of monomers rather than by the dissociation of micelles. The gangliosides were therefore first converted into very low molecular weight aggregates (1-3 molecules) by dissolving them in Me2SO. Galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylceramide was then diluted into aqueous sucrose gradients and sedimented by the boundary centrifugation technique. This gave a sedimenting micelle and a nonsedimenting monomer concentration of (1-2) x 10-10 M (or less) which corresponds to the critical micelle concentration value. The mixed gangliosides revealed two micellar sizes (i.e., 10 and 4.5 S), the slower sedimenting species being formed from the larger one with time (days). The critical micelle concentration of the mixed gangliosides was found to be approximately 10-8 M by a gel filtration, equilibrium dialysis, and band centrifugation.     

Ultracentrifuge studies of histone fractions from calf thymus deoxyribonucleoprotein

Molecular weights and sedimentation coefficients of four major fractions of calf thymus histones were measured. The minimum molecular weights were determined in concentrated solutions of guanidine hydrochloride. The results indicate that, with the possible exception of fraction F3, the fractions are heterogeneous. Comparisons in 0.1m-sodium chloride suggest that fraction F1 does not aggregate and show that fractions F2(a) and F3 aggregate to form larger complexes than does fraction F2(b). The degree of aggregation of each fraction is independent of pH in the range pH1-7. Detailed studies with fraction F2(b) have confirmed that the change in sedimentation coefficient observed as the sodium chloride concentration of the solution is increased results from increases in the apparent molecular weight of the sedimenting units. It has been found that the molecules of fraction F2(b) are present as single molecules only in sodium chloride solutions of 33mm or less. At these low concentrations the effects of charge greatly increase the concentration dependence of the sedimentation rate; the results can, however, be interpreted by using the theory developed by Alexandrowicz & Daniel (1963) and Daniel & Alexandrowicz (1963).     

Ice Recrystallization Behavior of Corn Starch/Sucrose Solutions: Effects of Addition of Corn Starch and Antifreeze Protein III

Knowledge of the behavior of corn starch during frozen storage is necessary to understand more complex systems. In the present study, ice recrystallization in corn starch (0.3% and 3%, w/w)/sucrose (40%, w/w) solution was investigated at −10 °C based on the theory of Ostwald ripening. The addition of corn starch to the sucrose solution increased the ice recrystallization (IR) rate constant. To explore the mechanism causing higher IR rate constant, fluorescence microscopy was used to analyze the distribution of corn starch molecules. Fluorescence micrograph showed corn starch distributed homogenously in the freeze-concentrated phase. Ice crystal size distribution assessment showed that at the same average radius, the addition of corn starch increased the standard deviation of ice crystal size distribution. The findings revealed that the addition of corn starch widened the distribution of ice crystal size, which may be the mechanism causing higher IR rate constant. To inhibit the ice recrystallization process, antifreeze protein type III (AFP III) was added to sucrose solutions with and without corn starch. In the presence of corn starch, 0.01-mg/mL AFP III was enough to significantly reduce the IR rate. Conversely, the samples without corn starch did not show a significant reduction in IR rate constant at the same AFP III concentration. The outcomes revealed that corn starch enhanced the activity of AFP III. The results of this study showed that corn starch increased the IR rate constant, and AFP III supplemented with corn starch was synergistically more efficient in retarding IR rate constant.

     

Sedimentation velocity of DNA in isokinetic sucrose gradients: calibration against molecular weight using fragments of defined length.

The relationship between sedimentation coefficient and molecular weight for DNA sedimenting in preformed alkaline and neutral sucrose gradients was determined using absolute molecular weight standards (restriction fragments of plasmid pBR322 and phage lambda DNA). The range of calibration for alkaline gradients was extended to small DNA fragments (652 base-pairs) for the first time. The exponent b in the equation S20 degrees, w = aMb was found to be 0.380 in neutral gradients and 0.410 in alkali. The latter value differs significantly from previous estimates. The gradients were isokinetic, and the distance sedimented was shown to be directly proportional to the sedimentation coefficient at all times.     

Heterogeneity of chondrocyte mitochondria: A study of the Ca2+ concentration and density banding characteristics of normal and rachitic cartilage

Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca²⁺ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5–2.0 M) in the presence of inhibitors of Ca²⁺ transport (ruthenium red and 5,5′-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca²⁺ concentration (309 nmol/mg protein). Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca²⁺ concentration (up to 5700 nmol/mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca²⁺ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca²⁺ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca²⁺ concentration necessary for initiation of the calcification mechanism was 4 μmol/mg protein.     

The effect of low ionic strength extracellular solutions on the resting potential in skeletal muscle fibers

Intracellular measurements of the resting potential were made in fibers of the frog sartorius muscle in solutions of varying salt composition and concentration to determine the effects of low ionic strength extracellular solutions on the resting potential. Changes in the glass microelectrode tip potential in low ionic strength solutions were minimized by adding ThCl₄ to the extracellular solution. These experimental conditions allowed measurement of the relationship of the resting potential to the concentration of the salt in the extracellular solution by replacing it with the nonionic substance, sucrose. Substitution of sucrose for the extracellular NaCl produced a stable depolarization which was logarithmically related to the NaCl concentration. Substitution of sucrose for choline Cl, instead of NaCl, produced the same degree of depolarization. When Na salts of anions less permeable than chloride (Br, I, NO₃) were used, the resting potentials in 116 mM solutions were close to those with chloride (±3mv). The depolarizations produced in low ionic strength solutions of these salts were significantly less than those with chloride.     

X-ray scattering study of the shape of the DNA region in nucleosome core particle with synchrotron radiation.

The structure of the DNA region in rat thymus nucleosome core particle has been studied by synchrotron X-ray scattering analysis and the contrast-variation technique has been applied to determine the contribution of the DNA to the total scatterings. Small-angle contrast-matching measurements show that the entire core particle and isolated histone octamers are contrast-matched by solvents containing 64 and 54% (w/w) sucrose, respectively. At a contrast of 54% sucrose, where the scattering of the DNA dominates, the scattering data extending to higher angle of about 0.05 A-1 have been collected from relatively concentrated solutions (10 mg/ml) of core particles and interpreted on the basis of the regular helical model for the DNA region. The model calculations show that the shape of the DNA around the histone core is approximately by 1.8 turns of regular helix of 42 A radius and 28 A pitch. These values for helical parameters of our model are in good agreement with those of the structure of DNA in crystallized nucleosome cores shown by earlier diffraction studies.     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

The alpha particulate liver glycogen. A morphometric approach to the kinetics of its synthesis and degradation.

Electron micrographs of rat hepatocytes with a glycogen content between 0.36 and 2.55% (w/w) were submitted to morphometrical analysis. From the number and size of glycogen profiles, the distribution of radius and volume of glycogen alpha particles were computed. The 7-fold difference in glycogen content was accompanied by an only 1.8-fold increase in the mean volume of the particles while their number increased by a factor of 4. On the basis of these observations, it is proposed that the population of glycogen particles can be divided in two groups. The first one is made of growing particles, still associated with glycogen synthase; they are the only particles present at low glycogen concentration and their number is limited. Application of a simple mathematical model allows to estimate their number in hepatocytes as 49 X 10(12) particles . ml-1. The second group is made of glycogen particles which have reached their maximal size and the number of which is in principle unlimited. The maximal particle size is estimated to be 0.36 X 10(-15) ml, corresponding to an average molecular weight of 178 X 10(6). The average molecular weight of glycogen, as measured from the actual size of the particles, varied from 89 X 10(6) to 161 X 10(6).     

Platelet contractile proteins: separation and characterization of the actin and myosin-like components.

Solution of thrombosthenin, the contractile protein complex isolated from pig platelets, have been studied by analytical ultracentrifugation and zone sedimentation in sucrose density gradients. Freshly prepared thrombosthenin in 0.6 M KCl shows a prominent peak in the ultracentrifuge with S degrees 20w about 5.5 and higher molecular weight aggregates (greater than 100S) sedimenting quickly to the bottom of the cell. Short term storage of high ionic strength solutions of thrombosthenin induces actomyosin-like gel formation and these gels dissociate with ATP and Mg2+ ions into two components of S degrees 20w 8.0 and S degrees 20w50. The supernatant, after actomyosin gel removal, contains only the S degrees 20w5.5 protein. From results of Ca2+ ATPase activity measurements and SDS polyacrylamide gel electrophoretic mobilities of dissociated thrombosthenin separated into fractions in sucrose density gradients, it is concluded that the S degrees20w5.5 protein species is the myosin-like protein of thrombosthenin. The S degrees 20w8.0 protein is not fibrinogen but also has myosin-like properties and is believed to be myosin dimer. Species of higher S values seen in the presence of ATP and Mg2+ in the analytical ultracentrifuge and located in the higher density zones of the sucrose gradients all gave in SDS polyacrylamide gel electrophoresis a single band of molecular weight 46-47,000 daltons. These subunit proteins appear to be derived from a range of polymeric variants of the F-actin-like protein of the contractile complex. All these higher density F-actin-like proteins readily form superprecipitates and display syneresis when combined with rabbit skeletal muscle myosin or platelet myosin. They are also all capable of conferring upon these two myosins a Mg2+ activated ATPase activity. It is suggested that in thrombosthenin solutions a myosin monomer-dimer equilibrium state exists which can be directionally influenced by a number of factors. The coexistence in the solution of F-actin and Mg2+ ATP, for example, increases the propensity of the myosin-like protein to form the higher molecular weight aggregate. Such aggregation may be the initiating mechanism for the intracellular organization of the thick filaments of the actomyosin complex, preparatory to a contractile event.