Molecular distribution and charge of polylysine layers at the surface of lipid membranes and mica

Electrophoretic mobility of cardiolipin liposomes was measured in the presence of polylysines of different molecular weight at various concentrations of the background electrolyte (KCl). The electrophoretic mobility in a liposome suspension changes its sign and reaches a plateau at high polylysine content. The surface charge in the plateau region was determined according to the Gouy-Chapman model of the electrical double layer. The average charge density was found to equal 0.005 and 0.016 Coulomb/m² for the polymer length of 5 and 12 units (bases), respectively, and 0.032 Coulomb/m² for polylysines with the length of 130 and 1435 units. The molecular distribution of these polylysines was studied at the mica surface using atomic force microscopy in the 10-mM KCl solution. It was shown that pentalysine molecules covered uniformly about 90% of the surface with the layer thickness of about 0.8 nm. The high-molecular polylysines cover about 60% of the surface with the layer thickness of more than 1.5 nm. The data suggest that the polymer forms a compact layer on the membrane surface; the charge density at the outer surface is determined both by the polymer properties and by the total amount of anionic lipids, irrespective of their ionization state.     

Electrostatic effects upon adsorption and desorption of polylysines on the surface of lipid membranes of different composition

Electrokinetic measurements are carried out in suspensions of liposomes made from mixtures of charged (cardiolipin, CL) and neutral (phosphatidylcholine, PC) lipids in the presence of lysine and lysine-based polypeptides. Neither monolysine nor polylysines adsorbed on neutral (PC) membranes. In the case of negatively charged membranes (CL/PC) all polypeptides showed a sharp dependence of liposome electrophoretic mobility on the amount of polymer added to the cell. In suspension of cardiolipin liposomes the position of zero charge point coincided for all high-molecular polylysines; thus, pentalysine neutralizes the membrane surface, whereas polycations with a higher polymerization degree change a sign of the surface charge. Electrophoretic mobility of liposomes in plateau range depended on the molecular weight of polylysines and composition of liposomes; for large macromolecules the absolute value came close to its value for the initial liposomes. Adsorption of polycations on planar bilayer lipid membranes (BLM) resulted in alteration of the boundary potential measured by the method of intramembranous field compensation (IFC). The electrokinetic measurements and IFC method gave close results in the case of lysine monomers; their surface concentration could be fitted by an isotherm of the molecule distribution between the membrane surface and solution. Considerable differences of the surface and boundary potentials found in the case of pentalysine, correspond to changes in the dipole component of boundary potential induced by the adsorbed molecules. Using the IFC method, the kinetics of the adsorption process before saturation was studied. The adsorption of polylysines was markedly slower (more than hour) than that of pentalysine (tens of min) or monolysine (minutes). Washout experiments showed that adsorption of penta-and monolysine on planar BLM was reversible, while that of high-molecular polylysines was practically irreversible.     

竹叶青蛇毒血小板聚集剂的分离纯化及生化特性

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤和ＦＰＬＣＭｏｎｏＱ阴离子交换柱层析及Ｓｕｐｅｒｏｓｅ－１２凝胶过滤,从竹叶青蛇的蛇毒中纯化到一个能诱导人血小板聚集的均一组分．经ＳＤＳ－聚丙烯酰胺凝胶电泳测定其分子量为６８０００左右．等电点为４．３．测定了它的氨基酸组成,对它活化血小板的作用机理进行了初步研究,结果表明它是一种强的血小板激动剂．     

Hyperbranched polylysines modified with histidine and arginine: The optimization of their DNA compacting and endosomolytic properties

The DNA compacting and transfection properties of hyperbranched polylysines whose N-terminal amino groups were modified with histidine and arginine were studied. The histidine-modified hyperbranched polylysines were shown to provide higher efficacy of binding and transfection in comparison with unmodified or hyperbranched arginine-containing polylysines. This fact was explained by the intrinsic endosomolytic activity of the histidine-modified polymers. The dependence between the quantity of the amino acids that modified the terminal lysine residues in the hyperbranched polylysines, the efficacy of their DNA binding, and the transfection activity of the DNA complexes with the corresponding carriers was found. The possibility to increase the transfection activity of the DNA complexes with the hyperbranched polylysines by glycerin or the JTS-1 amphipathic nonapeptide was studied. At the same time, their simultaneous use was found to result in a transfection decrease.     

Adenosine cyclic 3',5'-monophosphate-dependent protein kinase from human platelets.

A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit. The apparent Km for ATP in the presence of 10 muM Mg2+ was 4 muM (plus cyclic AMP) and 4.3 muM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 muM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 muM (presence of MgATP) were exhibited by the "protein kinase-cyclic AMP complex". The enzyme required Mg2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate. In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated.     

A potent platelet aggregation inducer purified from Trimeresurus mucrosquamatus snake venom

A non-coagulant platelet aggregation inducer (called platelet 'aggregoserpentin') was isolated from Trimeresurus mucrosquamatus snake venom by CM-Sephadex chromatography and purified by gel filtration. It was homogeneous as judged by the ultracentrifugal analysis and electrophoresis on polyacrylamide gel and cellulose acetate membrane. The molecular weight was estimated to be 68 000 as judged by the SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-75. The ultracentrifugal analysis gave 3.19 Svedberg units. It was a protein-polysaccharide complex containing 340 amino acid residues and 50% carbohydrate per molecule. The isoelectric point was pH 5.4. It did not possess any of the hydrolase enzymatic properties which were found in the crude venom. The minimal concentration of 'aggregoserpentin' necessary to induce platelet aggregation was 10 ng/ml, about one four-hundredth of that of the crude venom. It did not cause lysis of platelets because lactate dehydrogenase was not found in supernatant after complete aggregation. An intravenous injection of 'aggregoserpentin' (35 microgram/kg) into rabbit ear marginal vein caused marked decrease of platelet number to approx. 10-20% of that of the control.     

Binding of coagulation factor XI to washed human platelets

The binding of human coagulation factor XI to washed human platelets was studied in the presence of zinc ions, calcium ions, and high molecular weight kininogen. Significant factor XI binding occurred at physiological levels of these metal ions when high molecular weight kininogen was present. Binding required platelet stimulation and was specific, reversible, and saturable. Scatchard analysis of the binding yielded approximately 1500 binding sites per platelet with an apparent dissociation constant of approximately 10 nM. Since the concentration of factor XI in plasma is about 25 nM, this suggests that in plasma factor XI binding sites on stimulated platelets might be saturated. Calcium ions and high molecular weight kininogen acted synergistically to enhance the ability of low concentrations of zinc ions to promote factor XI binding. The similarity between the concentrations of metal ions optimal for factor XI binding and those optimal for high molecular weight kininogen binding, as well as the ability of high molecular weight kininogen to modulate these metal ion effects, implies that factor XI and high molecular weight kininogen may form a complex on the platelet surface as they do in solution and on artificial negatively charged surfaces.     

Some properties of human epidermal growth factor (hEGF)-like immunoreactive material originating from platelets during blood coagulation

During blood coagulation, a considerable amount of human epidermal growth factor (hEGF)-like immunoreactive material (designated as platelet hEGF-LI) was liberated from platelets. The molecular nature of the platelet hEGF-LI was examined. The molecular weight of platelet hEGF-LI estimated by gel filtration was approximately 60,000-70,000. On chromatofocusing chromatography, platelet hEGF-LI was eluted mainly at pH 4.75 as a sharp peak with a minor peak at 4.30, like urine EGF. By treatment with 2-mercaptoethanol, the factor was converted into a material with molecular weight of 35,000-40,000. These results suggest that the majority of hEGF-LI in platelets may exist either in a covalently bound-form with some protein(s) in platelets or as a dimer intermolecularly cross-linked by an S-S linkage. Details of the biological properties and the physiological significance of the platelet hEGF-LI remain to be clarified.     

Purification and characterization of a phosphatidylinositol transfer protein from human platelets

We report the purification of a phospholipid transfer protein from human platelets. This protein preferentially transfers phosphatidylinositol, with phosphatidylcholine and phosphatidylglycerol being transferred to a lesser extent. Phosphatidylethanolamine is not transferred. Transfer activity is detected by measuring the transfer of radiolabeled phospholipids between two populations of small unilamellar vesicles. The protein was purified approximately 1000-fold over the platelet cytosol by chromatography on Sephadex G-75, sulfooxyethyl cellulose, and hydroxylapatite. The molecular weight of this protein appears to be 28 000 as determined by gel filtration chromatography. When the purified protein is analyzed on sodium dodecyl sulfate-polyacrylamide gels, two major components and several minor ones are observed. The molecular weight of the two major bands are 28 600 and 29 200. Isoelectric focusing of the platelet cytosol yielded phosphatidylinositol and phosphatidylcholine transfer activity at pH 5.6 and 5.9. The platelet phospholipid transfer protein is able to catalyze the transfer of phosphatidylinositol and phosphatidylcholine between vesicles and human platelet plasma membranes. One possible physiological role for this transfer protein is an involvement in the rapid turnover of inositol-containing lipids which occurs upon exposure of platelets to various stimuli.     

Modeling the influence of slurry concentration on Saccharomyces cerevisiae cake porosity and resistance during microfiltration

Filtration of an isotonic suspension of baker's yeast through a 0.45‐μm membrane was studied at two different pressures, 40 and 80 kPa, for yeast concentrations ranging from 0.14 to 51 kg/m³ (dry weight). For a yeast volume fraction above 0.06 (～21.8 kg/m³), the porosity of the yeast cake is less dependent on the suspension concentration. For highly diluted suspensions, the specific cake resistance approaches a minimum that depends on the filtration pressure. Correlation functions of cake porosity and specific cake resistance were obtained for the concentration range investigated showing that the Kozeny–Carman coefficient increases when the applied pressure increases. Both filtration pressure and slurry concentration can be process controlled. In the range of moderate yeast concentration, the filtrate flux may be increased by manipulating the filtration pressure and the slurry concentration, thereby improving the overall process efficiency. The complex behavior of yeast cakes at high slurry concentration can be described by a conventional model as long as part of yeast cells are assumed to form aggregates, which behave as single bigger particles. The aggregation effect may be accounted for using a binary mixture model. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

Boophilus microplus anticoagulant protein: an antithrombin inhibitor isolated from the cattle tick saliva

An anticoagulant was isolated from saliva of the cattle tick Boophilus microplus. Crude saliva prolonged both recalcification time and prothrombin time in assays with bovine plasma. It also inhibited thrombin, but not fXa, amidolytic activity. We purified the antithrombin activity by a combination of gel filtration, anion exchange, and affinity chromatography. The purified inhibitor has a molecular weight of 60,000 Da, determined by SDS-PAGE. The anticoagulant IC50 varied from 100 nM to 1.1 microM, depending on the thrombin concentration and substrate used (fibrinogen or platelet receptor). The excess of inhibitor in relation to thrombin indicates that it is not a tight-binding inhibitor. Chromogenic assays using a panel of five serine-proteinases suggest that the inhibitor is specific against thrombin.     

Studies on the physical states of human platelet myosin in crude extracts

The physical properties of human platelet myosin in crude extracts were studied by means of Sepharose 4B gel filtration and sucrose density gradient centrifugation in the presence or absence of Mg-ATP. Platelet myosin extracted with a buffer containing 0-0.15 M KCl gave a Stokes radius of about 12.0-12.5 nm irrespective of the presence or absence of Mg-ATP. The sedimentation coefficients obtained in the presence of Mg-ATP were about 10-11 and 8.5S at 0.05-0.10 and 0.15 M KCl, respectively, whereas the values obtained in the absence of Mg-ATP were about 16, 9-12, and 8.5S at 0.05, 0.10, and 0.15 M KCl, respectively. The apparent molecular weight in the presence of Mg-ATP, therefore, was about 500,000 and 420,000 at 0.05-0.10 and 0.15 M KCl, respectively, while the molecular weight in the absence of Mg-ATP was about 790,000, 460,000-620,000, and 440,000 at 0.05, 0.10, and 0.15 M KCl, respectively. The purified monomeric platelet myosin that had been solubilized with Mg-ATP at 0.10 M KCl had a Stokes radius of about 12.5 nm, a sedimentation coefficient of about 9S, and an apparent molecular weight of 460,000. On the other hand, while crude platelet myosin extracted at 0.6 M KCl with Mg-ATP gave a Stokes radius of about 20 nm, a sedimentation coefficient of about of 6S, and an apparent molecular weight of about 490,000, each of these physical parameters obtained in the absence of Mg-ATP was much larger than that obtained in the presence of Mg-ATP because the myosin was associated with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Filtration profile in isolated zone 1 and zone 3 dog lungs at constant high alveolar pressure

We measured the rate of liquid filtration in isolated dog lung lobes inflated to a constant alveolar pressure of 25 cmH2O and with all open vessels filled with plasma. We measured lung weight gain at vascular pressures ranging from 5 to 40 cmH2O relative to pleural pressure. We confirmed that under zone 1 conditions the "arterial" and "venous" extra-alveolar segments have essentially the same filtration characteristics. Using the combined extra-alveolar vascular system, we determined when recruitment of filtration surface area occurred as we increased vascular pressure from 0 to 40 cmH2O. Based on an abrupt increase in filtration rate as vascular pressure approached the zone 1/3 boundary, we infer that a sudden recruitment of exchange surface area occurred at that point. Based on the slopes of the zone 1 and zone 3 filtration profiles, we conclude that extra-alveolar vascular segments contribute approximately 25% of total to filtration in the lung under zone 3 conditions, although the exact vessels filtering under zone 1 conditions have yet to be determined. Our analysis of the data supports the concept that there is a difference in the perimicrovascular pressure around alveolar and extra-alveolar vessels, which in part may account for the apparent high filtration fraction apportioned to extra-alveolar vessels.     

Evaluation of ultrafiltration membranes with biological macromolecules

Eighteen ultrafiltration membranes ranging in molecular weight cutoff ratings from 500 to 300,000 were tested with water, 0.5M NaCl solution, and, in some cases, with macromolecules and urea in a 3-in. stirred filter cell. Approximately half of the membranes showed a significant decrease in filtration rate during the first 24-hr period. The steady-state rates were less than the manufacturers' rating for about two thirds of the membranes, the discrepancy being greater for the membranes with high molecular weight cutoffs. The filtration rates were linearly dependent on applied pressure over the range at least as great as 15 to 55 psig. The rate decreased as the concentration of macromolecules such as transfer ribonucleic acid (tRNA) increased; the rate for a concentration of 3 mg tRNA/per ml was one-fourth of that observed when no tRNA was present. Some increase in rate (～33 to 50%) was obtained by increasing the stirring speed from 100 rpm to 1000 rpm. The membranes were effective for desalting and concentration of macromolecules but not for separation of large molecules from each other, such as tRNA from bovine serum albumin. Easily denatured molecules such as catalase were not deactivated by filtration at 4°C.     

High-speed gel filtration of polypeptides in sodium dodecyl sulfate

The separation of polypeptides treated with SDS was studied using G3000SW packing prepared from silica for high-speed gel filtration. The peaks of ovalbumin, chymotrypsinogen A, cytochrome c, aprotinin, and insulin B chain were completely separated in the presence of 0.1% SDS and 0.05 M sodium phosphate buffer (pH 7.0). A plot of the logarithm of molecular weight of polypeptides versus Kd was linear over a molecular weight range of 3,000 to 50,000 at the above concentrations of SDS and sodium phosphate. The slopes of the plots of log molecular weight versus Kd depend to a significant extent on the concentration of the sodium phosphate buffer (pH 7.0).     

Small molecular weight heat shock-related protein, HSP20, exhibits an anti-platelet activity by inhibiting receptor-mediated calcium influx

We have shown that Hsp20, one of small molecular weight heat shock protein, which is present at a high concentration both in vascular smooth muscle cells and in circulating blood in patient with vascular disease, strongly inhibits platelet aggregation in vitro and ex vivo. To clarify the mechanism, we investigated the effect of Hsp20 on free calcium concentration in human platelet cytoplasm using fura 2. Hsp20 inhibited thrombin-induced calcium influx without affecting calcium release from intracellular calcium stores. The degree of inhibition is well-correlated with that of suppression of thrombin-induced platelet aggregation by this substance. Hsp20 also inhibited the elevation of cytoplasmic free calcium level triggered by collagen, but not that by A-23187. In contrast, Hsp28, another type of small molecular weight Hsp, failed to affect the cytoplasmic free calcium level. These findings suggest that Hsp20 inhibits the receptor-mediated calcium influx of platelets without affecting calcium release from intracellular calcium stores, leading to its anti-platelet activity.     

Latex phagocytosis by polymorphonuclear leukocytes: Role of sialic acid groups

Polystyrene latex particles are rapidly phagocytized by rabbit polymorphonuclear (PMN) leukocytes. The uptake is influenced by macromolecules which have the effect of altering the surface charge of the latex particle. The influence of polylysines of varying chain length on the surface charge of latex particles and of PMN cells was studied by micro-electrophoresis. Charge reversal at the latex surface was found to occur at concentrations considerably below that at which the surface charge of the PMN cells is reversed. Phagocytosis of latex by PMN cells is enhanced in the presence of low concentrations of long-chain polylysines. The enhancement of phagocytosis is strongly reduced if PMN cells are treated with neuraminidase. This suggests participation of siliac acid groups in a stage of particle-cell interaction which precedes engulfment.     

Physicochemical studies on tryptic digestion of bovine fibrinogen.

The high molecular weight fragments observed during tryptic digestion of bovine fibrinogen and the variation of their relative proportion with time has been studied. Separation of the different molecular species was carried out by gel filtration and the molecular weights of the isolated fragments were determined by sedimentation equilibrium and from their electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels. The fibrinogen is degraded by trypsin into distinct fragments, with molecular weights of 270 000, 170 000, 90 000 and 50 000 accompanied by a series of smaller fragments whose properties were not investigated. The relative proportion of the components was estimated from area measurements on scans of the stained gels obtained after electrophoresis in the presence of sodium dodecylsulfate. The relative concentration and the molecular weight of each component established its molar concentration in each of the digestion mixtures obtained after varying incubation times (1-60 min). These data were used for a kinetic analysis of the process. The kinetic model derived on the basis of the trinodular model of fibrinogen (see Appendix) gave a very good representation of all the experimental results.     

A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.