Low Shear Viscometry Research Articles

Purification of an 80 kDa Ca2+-dependent actin-modulating protein, which severs actin filaments, from bovine adrenal medulla.

A protein with a molecular weight of 80 kDa, which binds Ca2+-dependently to actin, was purified chromatographically from bovine adrenal medulla by using Sephacryl S-300, DEAE-Sepharose, actin-DNase I Sepharose, and Sephacryl S-200. This protein was retained on an actin-DNase I affinity column only in the presence of Ca2+, and could be eluted from this column by EGTA. The 80 kDa protein is a monomer and binds to G-actin in a Ca2+-dependent manner at an equimolar ratio. It caused fragmentation of actin filaments at more than 4 X 10(-7) M free Ca2+ concentration, as determined by low-shear viscometry and electron microscopy. Saturating amounts of tropomyosin showed a slight protective effect on the fragmentation of actin filaments by the 80 kDa protein. Considering the mode of action on actin filaments, the 80 kDa protein reported here seems to be a gelsolin-like protein. Gel electrophoresis of this protein revealed changes in mobility depending upon the concentration of Ca2+. This result also indicates that the 80 kDa protein itself is a Ca2+-binding protein.

Tropomyosin Reverses Cross-Linking of F-Actin with Microtubule-Associated Protein-21

Brain microtubule-associated protein 2 (MAP2) is known to cross-link muscle F-actin in vitro into a gel or discrete bundles of actin filaments. Previous reports indicate that this cross-linking reverses in the presence of millimolar ATP, while MAP2 molecules remain attached along single filaments of F-actin. Therefore, it is likely that the actin filament has two sets of surface areas with ATP-sensitive and insensitive affinities for MAP2. Using purified preparations of brain MAP2 and skeletal muscle F-actin and tropomyosin, we have studied the effects of tropomyosin on the MAP2-actin interaction by dark-field light microscopy, electron microscopy, sedimentation assay, and low shear viscometry. The results show that cross-linking of F-actin with MAP2 reverses upon addition of a stoichiometric amount of tropomyosin, although MAP2 remains bound to F-actin complex with tropomyosin. The ternary complex does not dissociate noticeably when exposed to a millimolar concentration of ATP. On the basis of these findings, it is concluded that ATP-insensitive MAP2-binding of F-actin is not sterically blocked by tropomyosin, while the ATP-sensitive binding is blocked by it.

Calspectin (fodrin or nonerythroid spectrin)-actin interaction: A possible involvement of 4.1-related protein

The calspectin/actin complex extracted from the bovine brain membrane crosslinks F-actin, resulting in the increasing viscosity of F-actin determined by low-shear viscometry. We demonstrated the presence of a protein factor in this complex, which regulated the calspectin-F-actin interaction in a Ca 2+- and calmodulin-dependent manner. Erythrocyte protein 4.1, but not synapsin I, mimics the function of this brain factor using a reconstitution system including purified calspectin, calmodulin and F-actin. In the brain complex, the Mr 120,000 and the Mr 80,000 70,000 polypeptides were detected to crossreact with anti-protein 4.1 antibody.

Interaction of actinogelin with actin. No nucleation but high gelation activity.

Nucleation activity of actin polymerization of actinogelin, a calcium-sensitive F-actin cross-linking protein from rat liver, was measured by a fluorescence enhancement method using pyrenyl-actin and by high shear viscometry. No stimulation of nucleation by the addition of actinogelin was observed under several ionic conditions using the fluorescent method. Similar results were also obtained by viscometry. Therefore, it can be concluded that actinogelin has no nucleation activity for actin polymerization. By electron microscopy, it was found that actinogelin molecule has a dumbbell shape, binds to side of F-actin through its end(s), and cross-links actin filaments by binding with its two ends. It was also found that meshwork formation occurred in low Ca2+ conditions from F-actin and actinogelin. Under non-gelling high Ca2+ conditions, binding of actinogelin along the side of F-actin with its one end was still detected in accordance with the binding assay using ultracentrifugation and protein determination. Under low Ca2+ conditions, the critical gelling concentration of actinogelin measured by low shear viscometry at 20 degrees C was 6 micrograms/ml for 250 micrograms/ml of actin. Comparing this value with those of the other actin cross-linking proteins, it was found that actinogelin was one of proteins with the highest gelation activity. On the other hand, gelation activity of actinogelin in high Ca2+ conditions was one order of magnitude lower; more than 50 micrograms/ml of the protein was required for gelation. At 37 degrees C, gelation activity of actinogelin at low Ca2+ concentration was decreased to about a quarter of that at 20 degrees C, but this was still higher than that of gizzard alpha-actinin at 20 degrees C. Thus, role of actinogelin as an efficient and Ca2+-regulated cross-linker of microfilaments was substantiated.

Participation of 200K or 150K subunit of neurofilament in construction of the filament core with 70K subunit and promotion of tubulin polymerization by incorporated 200K subunit.

We have already reported that neurofilaments are capable of stimulating microtubule assembly and causing gelation. After separation of each of the triplet proteins of neurofilaments it was demonstrated that only the 200K subunit shows the activity to promote tubulin polymerization (Minami, Y. & Sakai, H. (1983) J. Biochem. 94, 2023-2033). The separation of each subunit protein led us to attempt the reconstitution of filaments from the 200K and 70K subunits or from the 150K and 70K subunits. It was found that both the 200K and 150K subunits independently contribute to the formation of intermediate-sized filaments, provided that each subunit was combined with the 70K subunit before removing urea by dialysis for reconstitution. On the other hand, the 200K subunit alone formed a very short thread-like structure after removal of urea, and the 150K subunit formed a filamentous structure, both incapable of being incorporated into filaments made of the 70K subunit alone. These observations suggest that the 200K and 150K subunits are not peripherally attached to a filament core made of 70K protein, but they take part in the formation of the core. Moreover, both proteins can co-polymerize with the 70K protein at a weight ratio of about 1 : 1 at least, which is in excess of that of the intact neurofilament. We investigated whether or not the 200K subunit incorporated with the 70K subunit into filaments could also stimulate tubulin polymerization. Low-shear viscometry measurements suggested that the 200K subunit retains the activity to initiate tubulin polymerization. This was confirmed by measuring viscosity changes with an Ostwald-type viscometer. In contrast, filaments reconstituted from the 70K and 150K proteins were incapable of increasing low-shear viscosity when mixed with tubulin. These observations suggest that the domain of the 200K protein embedded in the core of intermediate-sized filament is separate from the site responsible for promotion of tubulin polymerization.

Association of actin with the platelet membrane

Human platelet membrane-actin associations were studied by means of differential extraction of purified membranes and low-shear viscometry of membrane-F-actin mixtures. As indicated by resistance to extraction with 0.6 M potassium iodide, a significant amount of platelet actin appears to be tightly associated with the membrane. When tested by falling-ball viscometry, both whole and KI-extracted membranes increased the low-shear viscosity of preformed rabbit skeletal muscle F-actin at physiologically reasonable pH and ionic conditions. This membrane-associated actin gelation activity was dependent upon low free calcium concentration (10− 8-10− 7 M). The results are consistent with specific associations between actin and platelet membranes and may be relevant to membrane-cytoskeletal interactions believed to occur in the intact cell.

Properties of smooth muscle vinculin.

Vinculin, isolated from turkey gizzard smooth muscle, was purified by chromatography on CM-cellulose after isolation from a DEAE-cellulose column. Two-dimensional gel electrophoretic analysis of crude muscle fractions demonstrated that: 1) much of the approximately 130,000-dalton protein present in smooth muscle did not co-isoelectrically focus with the purified 130,000-dalton vinculin and 2) the purified vinculin consisted of three major, closely spaced isoelectric variants that were present only in small amounts in the original smooth muscle sample. Purified vinculin sedimented as a single peak with a sedimentation coefficient S0 20,w of 5.9. Circular dichroism spectra of purified vinculin indicated a considerable degree of secondary structure, with an alpha-helical content of approximately 50% as measured at 208 nm. The ultraviolet absorption spectrum of vinculin gave a measured E1%(278) of 4.64. Digestion of vinculin, much of which is located at the cytoplasmic surface of the cell membrane, with Ca2+-activated neutral protease purified from skeletal muscle yielded major fragments with molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 98,000, 85,000, and 26,000. The factor(s) in DEAE-cellulose-purified vinculin responsible for decreasing the low shear viscosity of actin was removed and found in a crude fraction isolated by CM-cellulose chromatography. The purified vinculin had a small, but positive effect on the MgCl2-induced polymerization of actin as measured by low shear viscometry.

Bundling of microtubules in vitro by a high molecular weight protein prepared from the squid axon.

A high molecular weight protein has been partially purified from sheaths of squid giant axons. This protein fraction was capable of restoring the membrane excitability of the squid axon which had been destroyed by internal perfusion of microtubule poison, when perfused along with microtubule proteins (Matsumoto et al. (1979) J. Biochem. 86, 1155-1158). This protein, designated as 260 K protein, was purified by gel filtration and Con A-Sepharose affinity chromatography. The apparent molecular weight of the axonal protein was estimated to be 260,000 by electrophoresis in the presence of sodium dodecylsulfate. This protein was revealed to be a glycoprotein. When phosphocellulose-purified tubulin was incubated with 260 K protein at 36 degrees C in the presence of dimethylsulfoxide, turbidity of the solution was much increased. 260 K protein co-sedimented with microtubles assembled from purified tubulin. Light microscopic and electron microscopic observations revealed that the high turbidity was due to bundling of microtubules which was caused by 260 K protein. On the other hand, the effect of this protein on the turbidity increase was not so prominent when microtubules were assembled from microtubule proteins consisting of tubulin and microtubule-associated proteins. High shear and low shear viscometry and co-sedimentation experiments revealed that 260 K protein had little effect on actin polymerization under the same medium conditions as used in tubulin polymerization.

Gel Characteristics—Structure as Related to Texture and Waterbinding of Blood Plasma Gels

ABSTRACTHeat induced denaturation and aggregation of plasma protein solutions were studied by low shear viscometry and turbidity measurements. The microstructure of blood plasma gels was evaluated by scanning electron microscopy (SEM). Relationships between gel structure, texture, and waterbinding properties of blood plasma gels prepared under various conditions such as different heating temperatures, pH and protein and salt concentrations were investigated. Generally, it was found that the degree of elasticity and waterbinding properties decreased with an increasing degree of random aggregation of the protein gel network. The degree of aggregation increased with increasing protein and salt concentration and decreasing pH from 9 to 6. With increasing heating temperature from 77° C to 92° C, a partial disruption of the gel structure due to local aggregation phenomena was demonstrated by SEM micrographs.

Chemical modification of actin. Acceleration of polymerization and reduction of network formation by reaction with N-ethylmaleimide, (iodoacetamido)tetramethylrhodamine, or 7-chloro-4-nitro-2,1,3-benzoxadiazole.

We examined the properties of rabbit skeletal muscle actin labeled at Cys-373 with N-ethylmaleimide or with (iodoacetamido)tetramethylrhodamine, and of N-ethylmaleimide-actin further modified with 7-chloro-4-nitro-2,1,3-benzoxadiazole (which primarily labels Lys-372). All three derivatives polymerize more rapidly than unlabeled actin. As measured by fluorescence photobleaching recovery and low-shear viscometry, all three also show a lower extent of network formation relative to native actin. N-Ethylmaleimide has a much smaller effect on the rate of polymerization and on network formation than do the other two derivatives. We suggest that chemical modification of actin with these compounds may stabilize nuclei, accounting for the acceleration of polymerization. Stabilization of nuclei also reduces the average filament length at equilibrium, thereby reducing the extent of network formation. We note a parallel between these results and the effects that cytochalasin and capping proteins have on the polymerization of actin.

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane.

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.

Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins.

We have used low shear viscometry and electron microscopy to study the interaction between pure actin filaments and microtubules. Mixtures of microtubules having microtubule-associated proteins (MAPs) with actin filament have very high viscosities compared with the viscosities of the separate components. MAPs themselves also cause a large increase in the viscosity of actin filaments. In contrast, mixtures of actin filaments with tubulin polymers lacking MAPs have low viscosities, close to the sum of the viscosities of the separate components. Our interpretation of these observations is that there is an interaction between actin filaments and microtubules which requires MAPs. This interaction is inhibited by ATP and some related compounds. Electron micrographs of thin sections through mixtures of actin and microtubules show numerous close associations between the two polymers which may be responsible for their high viscosity.

Viscous and Elastic Behavior Attributed to Polymer Entanglements

Abstract Polymer chains are separated and behave as individual hydrodynamic units in sufficiently dilute solutions. A minimum polymer molecular weight, dependent on concentration, is necessary to produce the characteristic rheological effects generally attributed to entanglements. The minimum polymer molecular weights and concentrations for which entanglement effects are observed are called the characteristic entanglement compositions. Undiluted polymers exhibit such effects only above some minimum molecular weight. The common observation of entanglement effects indicates that they are not due solely to chemical or structural inhomogeneities. Polymer composition, e.g., polarity and perhaps tacticity, can lead, however, to changes in frequency and strength of entanglements. Entanglements appear to govern many important polymer characteristics, thus providing a strong motivation for their study. Characteristic chain spacings between entanglements have been reported from various viscoelastic experiments, low shear viscometry, nonNewtonian flow, and from relaxation times measured by nuclear magnetic resonance. The different techniques generally give concordant values, although with a wide variation in precision. For a few polymers, e.g., polydimethylsiloxane, the characteristic entanglement spacing has been calculated by each of the four techniques. For others, e.g., polyisobutylene and polystyrene, entanglement spacings have been reported by all except NMR. Entanglement effects have been treated theoretically by analogy with theories of rubber elasticity. Other theories have been developed based on breakage and reformation of entanglements and on polymer chain slippage. Certain of these theories have been shown to have the same formalism and yield similar conclusions. In general, the entanglement hypothesis provides a consistent interpretation for a variety of rheological data on concentrated systems of amorphous polymers, this despite the fact that an entanglement has not as yet been directly “seen”. A discussion of entanglements and the first method of calculating entanglement spacings was given by Mark and Tobolsky. A review in the field of polymer viscosities for concentrated systems has been recently prepared. Experimental details and theoretical derivations are given in texts. The notations used are defined in the Appendix.

Low shear viscometry in relation to brushing

The flow of decorative paints should be capable of prediction from a rheological study of the paint. By taking into consideration the loss of solvent from a film during the early drying stages, a good estimation of its flow properties in relatively high film thicknesses can be obtained.

Acridine mutagens and DNA structure

A number of independent critical structural parameters, which have been determined by studies on sedimentation, low angle X-ray scattering, flow dichroism, flow-polarized fluorescence, and chemical reactivity, provide the evidence for intercalation of the acridines between two otherwise sequential base pairs, and require the rejection of various alternative hypotheses. The binding requires a local untwisting and extension of the double helix, which is found to be compatible with the normal structural and bonding parameters. The expectation of a substantial alteration in the chemical reactivity of intercalated molecules because of their inaccessibility to electrophilic attack has been verified by reaction rate studies. Study of the relation between viscosity enhancement in dilute DNA solutions and intercalation has been extended using very low shear viscometry. The results agree with the earlier measurements in that the enhancement is observed with the binding of intercalatable cations, while others yield a slight diminution. A pronounced stabilization of the double helix is found, corresponding to a substantially raised thermal transition temperature. Intercalation into polyribonucleotide complexes formed between poly A and poly U renders the double helix substantially more stable than the triple. While intercalation seems to be prerequisite for mutagenicity of the insertion-deletion type, the acridine structure is not essential, nor are all intercalating molecules mutagenic.