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PubMed2002

A direct-transfer polymerization model explains how the multiple profilin-binding sites in the actoclampin motor promote rapid actin-based motility.

The high actin-based motility rates observed in nonmuscle cells require the per-second addition of 400-500 monomers to the barbed ends of growing actin filaments. The chief polymerization-competent species is profilin.actin.ATP (present at 5-40 microM intracellular concentrations), whereas G-actin.ATP is much less abundant ( approximately 0.1-1 microM). While earlier studies unambiguously demonstrated that profilin.actin is highly concentrated within the polymerization zone, profilin-actin localization on the motile surface cannot increase the local solution-phase concentration of polymerizable actin. To explain these high rates of actin polymerization, we present and analyze a novel polymerization model in which monomers are directly transferred to growing filament ends in the actoclampin motor. This direct-transfer polymerization mechanism endows the polymerization zone with properties unavailable to bulk-phase actin monomers, and our model also indicates why profilin is the ideal mobile carrier for actin monomers.

Actins↗
PubMed1997

Phosphatidic acid elicits calcium mobilization and actin polymerization through a tyrosine kinase-dependent process in human neutrophils: a mechanism for induction of chemotaxis.

Phospholipids mediate important effects as extracellular messengers in diverse biological systems. We investigated the effects of phosphatidic acid, a biologically active phospholipid potentially involved in the inflammatory process, on calcium mobilization and actin polymerization in human neutrophils and correlated these effects with induction of chemotactic migration. Intermediate-chain length phosphatidic acid (DiC10-PA) induced a biphasic increase in intracellular Ca2+ characterized by a rapid rise commencing immediately upon addition of stimulus followed by a secondary increase which, unlike the initial response, was eliminated by chelation of extracellular Ca2+. Neither of these responses were induced by C10-lysophosphatidic acid or diacylglycerol. The tyrosine kinase inhibitor herbimycin-A (5-10 microg/ml) completely blunted the initial but not the delayed response effected by DiC10-PA. Long-chain phosphatidic acid (DiC18:1) induced only an initial rapid increase in intracellular Ca2+ and this response was similarly markedly attenuated by herbimycin-A. Among several physiologically relevant phospholipids, only phosphatidic acid was able to induce Ca2+ mobilization; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol -- used individually or in mixed liposomes -- were without effect. Phosphatidic acid conferred calcium-mobilizing activity upon inactive liposome preparations and phosphatidic acid-enriched cellular plasma membranes possessed similar calcium-mobilizing activity. Both DiC10-PA and DiC18:1-PA induced actin polymerization in neutrophils at rates which mirrored the influence of each agent on Ca2+ mobilization. Herbimycin-A blunted the initial increase in actin polymerization effected by phosphatidic acid but had no effect on the delayed, EGTA-sensitive phase. DiC10-PA and DiC18:1-PA also induced neutrophil migration along a concentration gradient. Phospholipids that failed to induce a calcium transient, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol, likewise failed to induce either actin polymerization or chemotactic migration. Unlike chemotaxis induced by zymosan-activated human serum, phosphatidate-induced chemotaxis was strongly inhibited by pretreatment of cells with herbimycin-A. Consistent with these observations, phosphatidic acid induced the tyrosine phosphorylation of several proteins as early as 10 s after stimulation. Phosphorylation of two distinct proteins with approximate molecular sizes of 72 and 82 kDa was inhibited by levels of herbimycin A used to effectively inhibit calcium mobilization, actin polymerization and chemotaxis. Thus, in neutrophilic leukocytes, extracellular phosphatidic acid induces a unique tyrosine kinase-based signalling pathway that results in calcium mobilization and actin polymerization. These processes may promote directed cellular migration as a consequence of the interaction of phosphatidic acid with neutrophil plasma membranes.

Actins↗
PubMed1997

Effect of major surgery on neutrophil chemotaxis and actin polymerization in neonates and children.

The authors have examined the effect of major surgery in neonates and older children on neutrophil (PMN) chemotaxis and on actin polymerization, an essential early step in PMN movement. Isolated PMNs from the following subjects were studied: healthy adult volunteers (n = 28), healthy newborns (n = 21), newborns undergoing major surgery (n = 7), and older infants and children undergoing major surgery (n = 14). Chemotaxis was measured by a micropore filter assay, and actin polymerization was measured by flow cytometry. Blood samples from surgical patients were obtained preoperatively, hourly during the procedure, immediately postoperatively, and 48 hours after surgery. Mean preoperative newborn PMN chemotaxis was similar to that of healthy newborn PMN, and mean preoperative PMN chemotaxis in children was similar to that of healthy adults. There were no significant alterations in PMN chemotaxis during or after major surgery in neonates or children. Peak PMN actin polymerization, after stimulation with formyl methionyl leucyl phenylalanine (FMLP) (10 nm), was significantly diminished in healthy neonates compared with adults (P < .005). Preoperative surgical neonates had similar peak PMN actin polymerization levels to those of healthy newborns, and older preoperative children had similar levels to adults. PMN actin polymerization did not significantly change during or after major surgery. Despite reductions in PMN chemotaxis and actin polymerization in healthy neonates, there is no further impairment of these PMN functions during or after major surgery. Our data suggest that PMN chemotactic function is resistant to the stress of uncomplicated major surgery in neonates and children.

Actins↗
PubMed2000

Resin shear bond strength to porcelain and a base metal alloy using two polymerization schemes.

STATEMENT OF PROBLEM: Fractures in ceramometal restorations can occur and need to be repaired because replacements are not an economic solution. PURPOSE: This study evaluated the shear bond strengths of 4 porcelain repair systems (Metabond C&B [ME], Silistor [SI], Clearfil Lustre [CL], and Scotchbond Multipurpose Plus [SQ]) to a base metal alloy and porcelain in relation with the polymerization shrinkage of a visible light-cured composite superstructure and compared with the ceramometal bond strength (Vita VMK 68). MATERIAL AND METHODS: Thirty-two samples were prepared for each bonding system: 16 for resin-metal bond strength test, and 16 for resin-porcelain bond strength test. For each group, bonding agent was applied to 8 substructures and the resin superstructure was polymerized onto the bonding agent; and for the remaining 8 specimens, prepolymerized resin superstructures were bonded with bonding agent. All specimens were subjected to 500 cycles between 5 degrees C and 55 degrees C with 20 seconds dwell time. Tests were performed in a mechanical testing machine with a 0.5 mm/min crosshead speed. RESULTS: All materials showed an increase in shear bond strength when prepolymerized resin superstructures were used. However, the effect of polymerization shrinkage of resin superstructure was statistically significant only for CL group (P <. 05). The highest metal-resin bond was obtained from ME group with prepolymerized resin superstructures (35.27 +/- 2.40 MPa), and the lowest value was obtained for the SI group in which resin superstructures were polymerized in situ (8.71 +/- 1.03 MPa). The highest porcelain-resin bond was obtained from SC group with prepolymerized resin superstructures (20.71 +/- 1.13 MPa) and the lowest was obtained from SI group (9.99 +/- 1.52 MPa). CONCLUSION: Higher bond strength values were obtained with prepolymerized resin superstructures compared to in situ polymerized superstructures. Metabond C&B provided the best results for both prepolymerized and in situ polymerized resin superstructure preparation techniques at the failures where metal was exposed. The best results in situations in which the fracture is limited into porcelain were obtained with the use of Scotchbond Multipurpose Plus material. However, a variety of in vivo and in vitro tests are required before a final judgment is made.

Analysis of Variance↗
PubMed2003

Effect of two types of latex gloves and surfactants on polymerization inhibition of three polyvinylsiloxane impression materials.

STATEMENT OF PROBLEM: Polymerization inhibition of polyvinylsiloxane impression materials has been reported when in sustained contact with some types of latex gloves. PURPOSE: This study examined the polymerization inhibition of 3 polyvinylsiloxane impression materials placed in contact with surfaces subjected to prior contact with gloves or commonly used surfactants. MATERIAL AND METHODS: A 2 x 3 x 4 x 2 design was used (n = 20), with 2 types of gloves (powdered and unpowdered), 3 types of polyvinylsiloxane impression materials (Aquasil, Extrude, and Affinis), 4 surfactant conditions (water, soap/water-rinse, alcohol, and unexposed), and 2 ambient temperatures of 22 degrees C and 36 degrees C. After glove exposure to surfactants, a glass surface was subjected to rubbing contact with the treated glove for a standardized time. After drying, automixed polyvinylsiloxane impression materials were dispensed onto the treated surface. Specimens were removed and evaluated for polymerization inhibition at the manufacturer's recommended polymerization time (36 degrees C), or after 15 minutes at 22 degrees C. Specimens were rated as polymerized, or as inhibited if any polyvinylsiloxane residue remained on the slab. A chi-square analysis was used to evaluate the results (alpha=.05). RESULTS: Setting inhibition was found only with one of the polyvinylsiloxane materials when alcohol was used as a surfactant. At 22 degrees C, the inhibition rate ranged from 95% to 100% for both glove types; at 36 degrees C inhibition ranged from 40% (unpowdered gloves) to 75% (powdered gloves), respectively. CONCLUSION: Under these in vitro conditions, glove exposure to alcohol resulted in polymerization inhibition of 1 of 3 tested polyvinylsiloxane impression materials (Extrude).

Alcohols↗
PubMed1990

Enhanced polymerization of polar macromolecules by an applied electric field with application to mitosis.

Numerous cellular processes are characterized by the rapid polymerization of protein molecules to form rod-like structures. Examples include the formation of spindle fibers from tubulin during cell division and the polymerization of actin into the actin filaments of the pseudopod in chemotaxis. It has been proposed that these proteins possess an electric dipole moment and that the onset of an internal electric field triggers polymerization. In this theoretical study, the relative probability of polymerization of a polar protein species is calculated in the presence and absence of an electric field. There is a significant enhancement of polymerization in the presence of an electric field, which increases as the size of the attachment site decreases. We conclude that a cytoplasmic pool of suitable proteins will rapidly polymerize if an electric field is applied, while remaining in a random configuration in the absence of a field. This mechanism is applied to the mitotic spindle structure, and by assuming that the spindle poles become oppositely charged during mitosis, a finite difference method is used to calculate the spindle structure at metaphase. Good agreement is obtained with experiment data.

Actins↗
PubMed1997

Thrombin interaction with fibrin polymerization sites.

Thrombin is central to hemostasis, and postclotting fibrinolysis and wound healing. During clotting, thrombin transforms plasma fibrinogen into polymerizing fibrin, which selectively adsorbs the enzyme into the clot. This protects thrombin from heparin-antithrombin inactivation, thus preserving the enzyme for postclotting events. To determine how the fibrin N-terminal polymerization sites of A alpha 17-23 (GPRVVER) and B beta 15-25 (GHRPLDKKREE) and their analogs may interact with thrombin, amidolysis vs. plasma- and fibrinogen-clotting assays were used to differentiate blockade of catalytic site vs. other thrombin domains. Amidolysis studies suggest GPRVVER inhibition of thrombin catalytic site through hydrophobic interaction, and GPRVVER inhibited clotting. Neither GPRP nor VVER nor the B beta 15-25 homologs inhibited amidolysis. Contrary to heparin, acyl-DKKREE promoted plasma-clotting, but inhibited fibrinogen-clotting. In addition, acyl-DKKREE reversed the anticoagulant effect of heparin (0.1 U/ml) in plasma. The results suggest fibrin B beta 15-25 interaction with thrombin, possibly by blocking the heparin-binding site. Together with the reported fibrin A alpha 27-50 binding to thrombin, polymerizing fibrin appears to initially bind to thrombin catalytic site and exosite-1 through A alpha 17-50, and to another thrombin site through B beta 15-25. As these fibrin sites are also involved in polymerization, competition of the polymerization process with thrombin-binding could subsequently dislodge thrombin from fibrin alpha-chain. This may re-expose the catalytic site and exosite-1, thus explaining the thrombogenicity of clot-bound thrombin. The implications of these findings in polymerization mechanism and anticoagulant design are discussed.

Amino Acid Sequence↗
PubMed2002

Reduction of polymerization contraction stress for dental composites by two-step light-activation.

OBJECTIVES: The goal of this study was to assess the reduction of polymerization contraction stress of composites during a two-step light-activation process and to relate this reduction to the process of polymerization shrinkage and specimen thickness. METHODS: Three test procedures were performed to compare two-step light-activation with delay with one-step continuous irradiation of composites: polymerization contraction stress using a closed-loop servohydraulic testing instrument, polymerization shrinkage by a mercury dilatometer, and degree of conversion by FTIR. For the one-step continuous curing method, the samples were light-activated for 60s at 330 mW/cm(2). For the two-step curing method, a 5s light exposure at 60 mW/cm(2) was followed by 2 min without light exposure, and then a second light exposure for 60s at 330 mW/cm(2). The same light parameters were used for measurements of stress, shrinkage, and degree of conversion. Three composites, Heliomolar, Herculite and Z100 were evaluated. The contraction stress experiments were repeated with varying thickness for Herculite using the one-step and two different two-step techniques. RESULTS: Polymerization contraction stress 10 min after light-activation was significantly reduced (P<0.05) by the two-step method: 29.7% for Heliomolar, 26.5% for Herculite, and 19.0% for Z100. Total volumetric shrinkage and degree of conversion were not significantly different for composites cured by the two different techniques. Increasing the thickness of the composite sample reduced the measured contraction stress, especially for one of the two-step curing methods. SIGNIFICANCE: A combination of low initial energy density followed by a lag period before a final high-intensity light irradiation provides a reduction of polymerization contraction stresses in dental composites. The stress reductions cannot be attributed to reductions in degree of conversion or unrestrained volumetric shrinkage.

Acrylic Resins↗
PubMed2004

The influence of storage and indenter load on the Knoop hardness of dental composites polymerized with LED and halogen technologies.

OBJECTIVES: The mechanical properties of light cured dental composites are greatly influenced by the light curing unit (LCU) used for the polymerization. Previous studies have shown that for some composites lower mechanical properties were obtained if light emitting diode (LED) LCUs were used for the polymerization instead of halogen LCUs. Previous studies have also shown that light cured composites improve their mechanical properties through a post-curing process after the initial illumination with the LCUs. Therefore, this study investigated the post-curing process, to ascertain if it can compensate for the lower mechanical properties of composites polymerized with LED LCUs. METHODS: The Knoop hardness was measured for four dental composites (Z100, Spectrum, Definite, Solitaire2) polymerized with an LED LCU (LED63 prototype) or a halogen LCU (Trilight), directly after the curing process and after 5 days of storage. In addition, the load on the indenter was varied from 200 to 400 gf to investigate the influence of the load on the measured hardness on the top and bottom of the 2 mm thick samples. RESULTS: In general the Knoop hardness at the bottom of the stored samples, cured with the LED LCU, was the same or statistically significantly greater than for the samples cured with the halogen LCU. A statistically significantly lower (p<0.0001) Knoop hardness was obtained on the top of the samples if the composite Definite was polymerized with the LED LCU instead of the halogen LCU. The load of 200 or 400 gf on the indenter had a statistically significant influence (p<0.0001) on the measured Knoop hardness for the composite Z100. The Knoop hardness measured with an indenter load of 400 gf increased statistically significantly (p<0.0001) for all composites after the 5 days' storage, whether cured with the LED LCU or halogen LCU. SIGNIFICANCE: The post-curing effect cannot compensate for the lower hardness of composites containing co-initiators if polymerized with an LED LCU instead of a halogen LCU. The indenter load had a statistically significant influence on the measured Knoop hardness of composites and has the potential to falsify results if not selected carefully.

Air↗
PubMed2003

A three-dimensional finite element model of the polymerization process in dental restorations.

Restoration of dental restorations with resin composite is hampered by shrinkage of the material during the polymerization process. In this study, we simulated the polymerization process in a detailed three-dimensional finite element model of a human upper premolar with a cusp-replacing restoration. It was analyzed how the stress patterns changed during polymerization and it was assessed whether immediate failure of the restoration is likely to occur and if so, which locations within the restoration are at highest risk and what the clinical implications of these findings are for clinical practice. It was found that the stresses increased rapidly during polymerization and decreased again in the post-polymerization phase. At the interface, tensile stresses relaxed to a higher degree than the shear stresses. Stress values in the composite material and at the interface with the tooth tissue were lower than the reported strength values suggesting that immediate failure is unlikely. The safety factor against mechanical failure of the interface, however, was relatively low indicating that the interface between the composite material and the tooth tissue is at a higher risk for failure than the bulk of the restoration. Stress relaxation was less effective in areas where the interface surface was irregular. In common practice, irregular interface surfaces are used to increase the retention of the restoration. This study indicates that the increased retention may be compromised to some extent by higher shrinkage stresses. The fact that stresses considerably decreased during the post-polymerization period suggests that mechanical loading should be limited during the first few hours after restoration.

Bicuspid↗
PubMed2001

Effect of ultrasonic frequency on polymerization of styrene under sonication.

The effect of ultrasonic frequency on polymerization of styrene under sonication at 50 degrees C was studied at the frequencies of 23.4, 45.7, 92, 518 kHz and 1 MHz. Polymerization under sonication was carried out at the ultrasonic intensity that gives the same reaction rate of decomposition of porphyrin. The magnitude of the polymerization rate increases in the order of 92, 45.7 and 23.4 kHz. At the high frequencies of 518 kHz and 1 MHz, no polymerization was observed. These facts mean that there is an optimum frequency in the range from 92 to 518 kHz for effective polymerization. The average-number molecular weights at the sonication time of 3 h are 5.5 x 10(4), 8.0 x 10(4) and 11.5 x 10(4) for the irradiated frequencies of 92, 45.7 and 23.4 kHz, respectively. Sonication for 3 h at 92 kHz gives polystyrene with very high polydispersity, about 5.0, in comparison with the results obtained at 23.4 and 45.7 kHz. These observations indicate that polymerization under sonication is influenced by the irradiated frequency.

Journal Article↗
PubMed1998

Comparison of absorption of nutrients and secretion of water between oligomeric and polymeric enteral diets in pigs.

In patients who require enteric tube-feeding the osmolality of the formulas is assumed to play an important role. There is the dilemma that osmolality increases as the digestibility of formulas is enhanced by means of degradation of the nutrients. Hitherto there have been no reports of whether there are differences in nutrient absorption and water fluxes between iso-osmotic polymeric and hyperosmotic oligomeric diets. We therefore investigated absorption of nutrients and net fluxes of water during perfusion of a 1.5 m jejunal segment with oligomeric, polymeric and commercial oligopeptide diets either in the absence of pancreatic juice or with concomitant infusion of pancreatic enzymes. In the absence of pancreatic juice the absorption rates of the polymeric diet and the commercial oligopeptide diets reached 58.0 and 84.5% respectively of that of a completely-hydrolysed hyperosmotic oligomeric diet. The concomitant infusion of pancreatic enzymes with the polymeric and oligopeptide diets significantly increased the absorption rates of nutrients and energy. The highest absorption rate of energy occurred with the commercial formula Survimed (Fresenius, Bad Homburg, Germany), probably due to an optimal composition of the macronutrients. The increase in absorption due to the degradation of nutrients by pancreatic enzymes was associated with an increase in net water secretion and flow-rate, reaching similar values to those with the hyperosmotic oligomeric diet. It may be concluded that iso-osmotic oligopeptide formulas require further pancreatic hydrolysis for optimum absorption. In patients with normal pancreatic secretion, oligopeptide formulas have no advantage over polymeric diets. In patients with reduced pancreatic secretion, either completely-hydrolysed hyperosmotic oligomeric diets or polymeric diets supplemented with pancreatic enzymes are appropriate.

Animals↗
PubMed1999

Amino acid order in polymeric dipeptide surfactants: effect on physical properties and enantioselectivity.

The effect of amino acid order on chiral selectivity in polymeric dipeptide surfactants, as well as the physical properties of the surfactants, is investigated. An understanding of enantioselectivity of such dipeptide surfactants is crucial to the design of more efficient polymeric surfactants and has implications in other areas of research such as enantioselective interactions of amino acid based compounds (i.e., enzymes, hemoglobin, antibodies, etc.). It should be noted that such polymeric surfactants are not easily crystallized. Therefore, in a manner similar to the study of proteins, fluorescence spectroscopy is a powerful tool used to study the structure-function relationship of these polymeric surfactants. The microenvironments inside the core of 18 polymeric surfactants were characterized using the environmentally sensitive probes pyrene and 6-propionyl-2-(dimethylamino)naphthalene (Prodan). The surfactants examined in this study include all possible dipeptide combinations of the L-form of alanine, valine, and leucine and the achiral amino acid glycine (except glycine-glycine) as well as the single amino acid surfactants of alanine, valine, and leucine. The results of the fluorescent probe studies led to a proposed structure of the polymeric dipeptide surfactants in solution. The implications of the proposed structure for chiral selectivity were tested with two model atropisomers, (+/-)1,1'-bi-2-naphthol and (+/-)1,1'-bi-2-naphthyl-2,2'-diyl hydrogen phosphate, using capillary electrokinetic chromatography.

Amino Acid Sequence↗
PubMed1993

Interaction and polymerization of the G-actin-myosin head complex: effect of DNase I.

The properties of polymerization and interaction of the G-actin-myosin S1 complexes (formed with either the S1(A1) or the S1(A2) isoform) have been studied by light-scattering and fluorescence measurements in the absence and in the presence of DNase I. In the absence of DNase I, the G-actin-S1(A1) and G-actin-S1(A2) complexes were found to be characterized by different limiting concentrations (l.c.), defined as the complex concentrations above which the polymerization occurs spontaneously within 20 h at 20 degrees C in a "no salt" buffer (l.c. = 0.42 and 8.8 microM for G-actin-S1(A1) and G-actin-S1(A2), respectively). The occurrence of a limiting concentration for either complex together with the kinetic properties of the polymerization led us to conclude that the G-actin-S1 polymerization occurs via a nucleation-elongation process. Fluorescence titrations and proteolysis experiments revealed that G-actin interacts with S1 with a 1:1 stoichiometry (independently of the presence of ATP) with dissociation constants, in the absence of nucleotide, of 20 and 50 nM for the G-actin-S1(A1) and G-actin-S1(A2) complexes, respectively. In the presence of at least a 1.5-fold excess of DNase I, the polymerization of the G-actin-S1 complexes was blocked even at high protein concentration or in the presence of salts. In addition, the affinity of either S1 isoform to actin was reduced 4-5-fold by DNase I, while the stoichiometry of the G-actin-S1 complexes was not changed. However, since the dissociation constants remain in the submicromolar range, we could demonstrate the existence of ternary DNase I-G-actin-S1 complexes stable under polymerizing conditions. Finally, the study of the effect of nucleotides and of various salts on the G-actin-S1 interaction further showed significant differences between the G-actin-S1 and F-actin-S1 interactions.

Actins↗
PubMed1993

Use of polymerized mixed liposomes to study interactions of phospholipase A2 with membranes.

Polymerized liposomes of thiol-based phospholipids, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (BLPC) and -phosphoglycerol (BLPG) were used to study interactions of several phospholipases A2 (PLA2) with membranes. Large liposomes (an average diameter of 100 +/- 10 nm) prepared from BLPC or BLPG were readily hydrolyzed by PLA2. Once polymerized, however, these liposomes were resistant to the PLA2 hydrolysis. When liposomes were prepared from a mixture of 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphocholine (pyrene-PC) (5 mol%) and BLPC, fluorescence measurements of resulting polymerized mixed liposomes showed that the pyrene-PC molecules exist solely as monomers without forming a patch and were selectively hydrolyzed by PLA2. Progress of the hydrolysis can be readily monitored by measuring the change in fluorescence emission at 380 nm in the presence of bovine serum albumin. Rapid and selective hydrolysis of inserted phospholipids in polymerized mixed liposomes supports the notion that facile migration of a phospholipid substrate from membrane to the active site of enzyme is a critical step in the catalysis of PLA2. On the basis of these findings, various combinations of polymerized mixed liposomes were prepared and their hydrolysis by PLA2 measured. When compared to the substrate specificity of PLA2s determined using Triton X-100/phospholipid mixed micelles, results from polymerized mixed liposomes indicate that electrostatic interactions between the interfacial binding site of PLA2 and membrane surfaces play an important role in the determination of substrate specificity of PLA2 and in the regulation of PLA2 activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis↗
PubMed1988

Reconstitution of rhodopsin and the cGMP cascade in polymerized bilayer membranes.

The successful reconstitution of rhodopsin, the rod outer segment (ROS) G protein, and the ROS phosphodiesterase (PDE) into partially polymerized bilayer membranes is described. Purified bovine rhodopsin (Rh) was inserted into performed partially polymerized lipid vesicles. Sonicated vesicles composed of approximately equal moles of dioleoylphosphatidylcholine (DOPC) (or 1-palmitoyl-2-oleoyl-phosphatidylcholine) and 1,2-bis(octadeca-2,4-dienoyl)phosphatidylcholine (DENPC) were photolyzed with 254-nm light to polymerize the DENPC and form domains of DOPC and polyDENPC in the vesicle wall. Rh-octyl glucoside (OG) micelles were slowly added to the vesicle suspension to give 15 mM OG (below the OG critical micelle concentration). The suspension was incubated and then dialyzed and purified on a sucrose gradient. Ultracentrifugation revealed a major Rh-lipid band which was harvested and found to contain a 100 +/- 10 phosphatidylcholine to rhodopsin ratio (Rh-polyDENPC/DOPC). The orientation of Rh in the membrane was determined by limited proteolytic digestion of Rh and by competitive inhibition of monoclonal antibody binding to solubilized disk membranes. Results were compared with control membranes of Rh-DOPC (1:43) prepared by insertion and Rh-phospholipid membranes prepared by detergent dialysis. Visual inspection of thermolysin proteolytic patterns of Rh indicates one major population cleaved at the carboxy terminus, as is found in disk membranes with an asymmetric arrangement of Rh. In contrast, proteolysis of a Rh-egg PC/PE (1:50/50) membrane (detergent dialysis) produced two Rh populations, which indicates a symmetric arrangement of Rh. The Rh-polyDENPC/DOPC (1:100) membranes were allowed to compete with solubilized, immobilized disk membranes for the monoclonal antibody R2-15 (specific for the amino-terminal region of Rh). They were intermediate between the asymmetric ROS disk membranes and the symmetric dialysis membranes in their ability to bind the R2-15 monoclonal antibody. The data indicate approximately 80% of the Rh's in Rh-polyDENPC/DOPC are in the normal orientation found in disks. These Rh-containing polymerized bilayer membranes demonstrated functionality as determined by chemical regeneration, kinetic spectrophotometry, and cGMP cascade reconstitution experiments. In the latter experiments the peripheral proteins, ROS G protein and PDE, bound with comparable efficiency to both the polymerized PC bilayers and egg PC bilayers. Thus the biocompatibility of the phosphatidylcholine membrane surface was maintained after polymerization of DENPC.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1980

Concentration-dependent influence of various cytochalasins and chaetoglobosins on the phalloidin-induced polymerization of G-actin in 0.6 M potassium iodide.

Phalloidin, a bicyclic peptide from the poisonous Amanita phalloides mushroom, stimulates the viscosimetrically determined polymerization of G-actin to F-actin in 0.6 m potassium iodide, a medium in which spontaneous polymerization does not occur. The cytochalasins B, D, E, and G (CB, CD, etc.) and the chaetoglobosins A, B, C, E, F, and J (Ch-A, Ch-B, etc.) have been found to influence the rat of polymerization in different ways, depending on their chemical structure and concentrations applied. Class I cytotoxins, e.g., CB, CG, Ch-C, Ch-E, and Ch-F, which exert on F-actin a weak degradative power (DP, "Spudich effect"), increase the polymerization rate when present in a ratio of 4 mol to 1 mol of actin. Polymerization is slightly enhanced or retarded at molar ratios ranging from 0.04:1 to 0.4:1. Class III cytotoxins of strong degradative power, i.e., high affinity for actin, such as CE, Ch-B, and Ch-J, decrease the polymerization rate at a molar ratio of 4:1 during the first 30-40 min; however, they increase it at a molar ratio of 0.4:1. Members of class II (CD, Ch-A) exert an effect that can be interpreted as a combination of the effects of class I and class III cytotoxins. An explanation of this difference in behavior is offered on the basis of experiments on F-actin degradation by Ch-J and its reconstitution by phalloidin in the presence of Ch-J.

Actins↗
PubMed2004

Ca2+-calmodulin regulates fesselin-induced actin polymerization.

Fesselin is a proline-rich actin-binding protein that was isolated from avian smooth muscle. Fesselin bundles actin and accelerates actin polymerization by facilitating nucleation. We now show that this polymerization of actin can be regulated by Ca(2+)-calmodulin. Fesselin was shown to bind to immobilized calmodulin in the presence of Ca(2+). The fesselin-calmodulin interaction was confirmed by a Ca(2+)-dependent increase in 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) fluorescence upon addition of fesselin to MIANS-labeled wheat germ calmodulin. The affinity was estimated to be approximately 10(9) M(-1). The affinity of Ca(2+)-calmodulin to the fesselin F-actin complex was approximately 10(8) M(-1). Calmodulin binding to fesselin appeared to be functionally significant. In the presence of fesselin and calmodulin, the polymerization of actin was Ca(2+)-dependent. Ca(2+)-free calmodulin either had no effect or enhanced the ability of fesselin to accelerate actin polymerization. Ca(2+)-calmodulin not only reversed the stimulatory effect of fesselin but reduced the rate of polymerization below that observed in the absence of fesselin. While Ca(2+)-calmodulin had a large effect on the interaction of fesselin with G-actin, the effect on F-actin was small. Neither the binding of fesselin to F-actin nor the subsequent bundling of F-actin was greatly affected by Ca(2+)-calmodulin. Fesselin may function as an actin-polymerizing factor that is regulated by Ca(2+) levels.

Actins↗