Colorimetric method for estimating polylysine and polyarginine

A rapid and sensitive method is described for estimating polylysine and polyarginine. The method involves the stoichiometric precipitation of these polyamino acids by excess of the anionic dye methyl orange, followed by spectrophotometric determination of unbound dye. 10 μg or less of polylysine or polyarginine can thus be estimated. The effects of various salts on the estimation are described.     

Helix-coil transition in nucleoprotein: renaturation of polylysine-DNA and polylysine-nucleohistone complexes

Thermal denaturation and renaturation of directly mixed and reconstituted polylysine–DNA, directly mixed polylysine–nucleohistone complexes, and NaCl-treated nucleohistones in 2.5 × 10^?4 M EDTA, pH 8.0 have been studied. At the same input ratio of polylysine to DNA, the percent of renaturation of free base pairs in a directly mixed polylysine–DNA complex is higher than that in a reconstituted complex. For a directly mixed complex, the renaturation of free base pairs is proportional to the fraction of DNA bound by polylysine or inversely proportional to the sizes of free DNA loops. A of large amount of renaturation of free base pairs has also been observed for 0.6 M and 1.6 M NaCl-treated nucleohistones. The binding of polylysine to nucleohistone enhances the renaturation of histone-bound base pairs. The percent of renaturation of polylysine–bound base pairs is high and is approximately independent of the extent of binding on DNA by polylysine. This is true in polylysine–DNA complexes prepared either by reconstitution or by directly mixing. It also applies for polylysine–nucleohistone complexes. The model where polylysine-bound base pairs collapse at T_m′ with two complementary strands still bound by polylysine is favored over the model where polylysine is dissociated from DNA during melting. The low renaturation of histone-bound base pairs in nucleo-histone indicates that either histones do not hold two complementary strands of DNA tightly or that histones are fully or partially dissociated from DNA when the nucleo-histone is fully denatured.     

Polylysine induces a rapid Ca2+ release from sarcoplasmic reticulum vesicles by mediation of its binding to the foot protein

The addition of polylysine to a heavy fraction of sarcoplasmic reticulum (SR) vesicles produces a rapid Ca2+ release with no appreciable lag period. The polylysine concentration for half-maximal activation (C1/2) is approximately 0.99 micrograms/ml, or 0.3 microM, the lowest C 1/2 for Ca2+ release-inducing reagents reported in the literature. The time course and the [Ca2+] dependence of polylysine-induced release are similar to those of caffeine-induced Ca2+ release. At higher concentrations of polylysine (e.g., 10 micrograms/ml), however, little or no Ca2+ release occurs. Upon photolysis of SR vesicles with the photocrosslinkable radiolabeled polylysine derivative, [3H]succinimidyl azido benzoate polylysine, 0.28 and 0.52-1.2 mol polylysine were bound to 1 mol of the 400-kDa foot protein at activating (3 micrograms/ml) and inhibitory (10 micrograms/ml) concentrations of polylysine, respectively. On the other hand, the amounts of polylysine bound to the other SR proteins (mol/mol) were negligible (e.g., less than or equal to 0.0127 mol polylysine/mol calsequestrin). This suggests that the binding of polylysine to the foot protein is responsible not only for the induction of release but also for inactivation. These results provide direct evidence that the receptor for the chemical trigger of Ca2+ release is localized within the foot protein. Ruthenium red, which inhibits polylysine-induced Ca2+ release, does not inhibit polylysine binding to the foot protein, suggesting that the polylysine binding domain of the foot protein is different from the channel domain.     

Polylysine activates membrane-bound adenylyl cyclase from Xenopus laevis oocytes through the Gs transducing protein.

1. The activity of the adenylyl cyclase found in the membranes of Xenopus laevis can be affected by polylysine and other polycations. 2. The activity of the catalytic subunit measured with forskolin is inhibited by polylysine and polyarginine at concentrations above 10 microM and by spermine above 3 mM. 3. The adenylyl cyclase activity stimulated by GTP-gamma-S or F- through the stimulatory G protein (Gs) can be increased by polylysine, polyornithine and spermine but not by polyarginine. 4. Polylysine stimulation of Gs dependent activity is due to the increase in the apparent affinity for GTP-gamma-S and to a lowering of the requirement for Mg2+ concentration.     

Studies on the accessibility of deoxyribonucleic acid in deoxyribonucleoprotein to cationic molecules

The binding of deoxyribonucleoprotein to Toluidine Blue, to cetylpyridinium chloride and to polylysine of various molecular weights was studied to determine the percentage of free DNA phosphate groups in deoxyribonucleoprotein. Binding was measured by addition of these reagents to deoxyribonucleoprotein at a range of concentrations such that complete precipitation of the deoxyribonucleoprotein occurred. With Toluidine Blue the binding corresponded to about 48% of the DNA phosphates in deoxyribonucleoprotein. The dye did not cause appreciable displacement of protein from the DNA. With cetylpyridinium chloride the binding corresponded to about 41% of the DNA phosphates. With polylysine preparations of molecular weight 1250 and 7790 the binding values for deoxyribonucleoprotein were 46 and 38% respectively. The results suggest that the free phosphates lie in stretches sufficiently long to accommodate most of each polylysine molecule. With polylysine of molecular weight 62000 cross-linking of free stretches of DNA on different deoxyribonucleoprotein molecules probably occurs. It is concluded that although most of the free phosphates are probably ;hidden' beneath covering histone, corresponding perhaps to runs of non-basic residues in the latter, they are surprisingly accessible to very large molecules. The relevance of this finding to the problem of gene repression is discussed.     

The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme.

The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either alone or in combination with the recombinant non-catalytic beta subunit. Calmodulin is not phosphorylated by the CK2 holoenzyme, in either the native or the reconstituted form, unless polylysine is added. In the presence of polylysine, it becomes a good substrate for CK2 (Km 14.2 microM, Kcat 4.6 mol.min-1.mol CK2-1). The recombinant alpha subunit, however, spontaneously phosphorylates calmodulin, this phosphorylation being actually inhibited rather than stimulated by polylysine. The calmodulin tridecapeptide, RKMKDTDSEEEIR, reproducing the phosphorylation site for CK2, is spontaneously phosphorylated by either CK2 holoenzyme or the recombinant alpha subunit with 5.8-fold and 2.8-fold stimulation by polylysine, respectively. The recombinant beta subunit of CK2 is itself a good exogenous substrate for the enzyme, its phosphorylation, however, is inhibited rather than enhanced by polylysine. On the contrary, the phosphorylation of the nonapeptide, MSSSEEVSW, reproducing the beta-subunit phosphoacceptor site, is dramatically stimulated by polylysine. Using a variety of small peptide substrates, it was shown that phosphorylation rate is diversely stimulated by polylysine. The observed stimulation, moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the beta subunit, since the recombinant alpha subunit is much less responsive than CK2 holoenzyme, either native or reconstituted. While the phosphorylation of the peptide RRRDDDSDDD by CK2 is stimulated 2.8-fold, with 15 nM polylysine being required for half-maximal stimulation, a stimulation of only 1.9-fold, with 80 nM polylysine required for half-maximal stimulation, is attained with recombinant alpha subunit. The concentration of polylysine required for half-maximal stimulation is comparable to CK2 concentration and increases by increasing CK2 concentration, suggesting that polylysine primarily interacts with the enzyme, rather than with the peptide substrate.     

The effect of added H1 histone and polylysine on DNA synthesis and cell division of cultured mammalian cells

Addition of H1 histone or polylysine (10 μg/ml) to cultured Friend erythroleukemia cells or to two mouse lymphoma cell lines (el-4 and S-49) increased levels of cell division in these cultures. There is a stimulation of incorporation of labeled thymidine into DNA in cultures containing H1 histone and polylysine. DNA fiber autoradiographic experiments revealed that replicon size is decreased in the cells cultured with H1 histone and polylysine at later periods of culture.     

A rapid method for separating small vesicles from suspension.

Techniques for rapidly aggregating suspensions of small vesicles made either of natural biological membrane or of phospholipid or phospholipid-protein mixtures by addition of one of the basic polypeptides, polylysine or protamine, have been investigated. Either filtration or centrifugation may be used to rapidly (15 to 30 s) and completely (over 98%) separate the vesicular aggregate from the suspending medium. At low values of the polylysine-to-vesicle weight ratio, aggregation is observed to increase with increasing polylysine concentration. At high values of this same ratio, aggregation decreases with increasing polylysine concentration. With protamine, like polylysine, aggregation increases with protamine concentration at low values of the weight ratio. At high values of the ratio, aggregation induced by protamine does not rapidly decrease with increasing protamine concentration as it does with polylysine. Explanations are given for these observations. While leakage induced by polylysine may be troublesome under some conditions with phospholipid vesicles or reconstituted systems, protamine aggregation has been found to induce much less leakage than polylysine aggregation. The leakage rate induced by protamine was not found to be significantly different from the control leakage rate for the first few minutes after addition of protamine under any of the conditions tested. Since this provides ample time for filtration, the protamine aggregation-filtration technique seems to be the method of choice for separation of many types of small vesicles from the suspending medium. It combines the advantages of rapid separation, complete separation, low rate of vesicle leakage, and versatility in being able to separate the vesicles from uncharged molecular components such as sugars where ion-exchange techniques will not work (1), as well as from ionic components. Low cost and simplicity are further advantages of the technique.     

The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.     

Methylation of chromatin DNA.

E. coli DNA methylase has been used to methylate chromatin DNA in vitro. At saturation only 50% of the chromatin DNA becomes methylated. The methylated regions of chromatin correspond to that fraction of the chromatin which is sensitive to staphylococcal nuclease. Using in vitro methylated chromatin followed by nuclease digestion movement of chromatin proteins along the DNA can be detected. By this criterion, sonication of chromatin or precipitation with MnCl2 causes 10% of the previously uncovered methylated regions to become covered by protein. Reconstitution of methylated chromatin results in the randomization of the chromatin proteins. Using nuclei which were methylated in vitro we have demonstrated that a small degree of protein sliding does occur during the preparation of chromatin from nuclei. Finally, we have prepared open region DNA by polylysine titration. This procedure does not cause displacement of chromatin proteins.     

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.     

Mechanism of inhibition of the proximal tubular isotonic fluid absorption by polylysine and other cationic polyamino acids.

The present study was initiated with the hope of clarifying the role of negative charges in the luminal brush border membrane in the overall process of trans-epithelial isotonic sodium and water absorption. Using micropuncture techniques, cationic polyamino acids such as polylysine (mol wt 100,000, 17,000 and 1,500-5,000, 1 mg/ml), tetralysine, polyornithine (mol wt 100,000, 1mg/ml), polyethyleneimine (2 mg/ml), polymyxin B (2 mg/ml), protamine sulfate (25 mg/ml) and histone (0.5 mg/ml) were perfused through the segments of rat kidney proximal tubule for 30 sec to 2 min. The rate of isotonic fluid absorption was measured before and after each perfusion with the Gertz's split drop method using Ringer's solution as a shrinking drop. Polylysine 100,000 and 17,000 and polyornithine were the most potent, inhibiting isotonic reabsorption by 93%. The sequence of inhibitory effect was: polylysine 100,000 congruent to polyornithine 100,000 congruent to polylysine 17,000 greater than polyethyleneimine greater than polylysine 1,500-5,000 congruent to polymyxin B greater than protamine sulfate congruent to histone. In contrast, tetralysine (2 mg/ml) showed no inhibitory effect. Electrical potential difference (p.d.) of the proximal tubular cells was destroyed within 10 sec of luminal perfusion with polylysine 100,000 (1 mg/ml). Simultaneously with the drop in p.d., electrical resistance of the luminal brush border membrane was nearly totally eliminated, whereas transepithelial input resistance remained unaltered. Furthermore, trypan blue dye was taken up by polylysine 100,000-perfused tubular cells but not by normal cells. Expanding drop analysis (mannitol solution as a split drop) was performed as a screening test to examine if the permeability for water and sodium in the lateral paracellular pathway is altered by polylysine 100,000. No significant difference was observed in the velocity of split drop expansion between untreated and polylysine-perfused tubules. A lower concentration of polylysine 100,000 (0.1 mg/ml) showed a much less inhibitory effect on fluid absorption and on cell p.d. These observations indicate that the strong inhibition on proximal tubular fluid absorption exerted by polylysine and perhaps also by other cationic polyamino acids is due not to modification of membrane negative charges but to the lysis of tubular cells by these polycations.     

Polylysine titration of rat liver chromatin fractions after DNase II digestion.

The concentration of free phosphate groups is measured in rat liver chromatin after DNase II digestion using polylysine titration. The unsheared chromatin completely precipitates at lysine/DNA phosphate ratios of 0.5 to 0.6. Digestion of the chromatin reduces the lysine/DNA phosphate ratio of complete precipitation by about 0.2 units suggesting the removal of free phosphate groups. The two chromatin fractions: MgC12 insoluble (template-inactive) and Mg12 soluble (template-active) chromatins precipitate at about the same lysine/DNA phosphate ratio. Some 15% of the MgC12 soluble chromatin remains in solution at any polylysine concentration. The removal of histone H 1 FROM THE MgC12 insoluble chromatin increases the lysine/DNA phosphate ratio by about 0.2 units suggesting that 20% of the DNA phosphate groups in nucleosomes are masked by histone H 1.     

O2- generation and lipid peroxidation during the oxidation of a glycated polypeptide, glycated polylysine, in the presence of iron-ADP

Oxidation of glycated polylysine, a model compound of glycated protein, caused O2- production even at physiological pH, which could be accelerated by Fe3(+)-ADP. An enediol structure in glycated polylysine and related compounds, which could be confirmed by I2 uptake, was related to their oxidizability. Glycated polylysine was easily coordinated with Fe3+ even in the presence of phosphate at pH 7.4 and the formation of the iron complex was prevented by desferrioxamine. The exposure of unsaturated phospholipid liposomes to glycated polylysine-Fe3(+)-ADP system caused the production of a thiobarbituric acid-reacting substance, which was completely inhibited by 5 microM alpha-tocopherol or 150 microM desferrioxamine and slightly by 0.5 microM SOD. Catalase (20 micrograms/ml) and 10 mM sodium-benzoate did not affect the iron-glycated polylysine-induced lipid peroxidation, indicating no participation of an OH. in this reaction. A ferrous ion-coordinated glycated polylysine may act as an initiator of phospholipid peroxidation in the presence of oxygen. A possible mechanism of the iron-glycated polylysine-induced lipid peroxidation was discussed.     

Conjugation in Tetrahymena pyriformis. The effect of polylysine, concanavalin A, and bivalent metals on the conjugation process

The polycation polylysine, at different degrees of polymerization, was found to cause a marked inhibition of the conjugation process. Inhibition of conjugation by polylysine was highly dependent on the molecular weight of the polymer. When polylysine of a mol wt of 1,250 (degree of polymerization=6) was used, a concentration of 1.6 X 10(-5) M was required for a complete inhibition of conjugation, while only 2 X 10(-7) M of polylysine of a mol wt of 71,000 (degree of polymerization=340) was needed for the same effect. Polyaspartic acid prevented the inhibition of conjugation by polylysein. Chelators of bivalent metals such as O-phenanthroline (10(-3) M), EDTA (10(-3) M), and EGTA (5 X 10(-3) M) strongly inhibit the conjugation process in Tetrahymena pyriformis. The inhibition was partially prevented when bivalent metals such as Zn++, Fe++, and Ca++ were added together with the chelators. The lectin concanavalin A (25 mug/ml) completely prevented the conjugation process, while other lectins, such as phytohemagglutinin (500 mug/ml), soybean agglutinin (75 mug/ml) and wheat germ agglutinin (250 mug/ml) had no effect. Inhibition of conjugation by concanavalin A is completely reversible by 40 mM of alpha-methyl-D-mannoside.     

Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye

The electrical sheet resistance between living cells grown on planar electronic contacts of semiconductors or metals is a crucial parameter for bioelectronic devices. It determines the strength of electrical signal transduction from cells to chips and from chips to cells. We measured the sheet resistance by applying AC voltage to oxidized silicon chips and by imaging the voltage change across the attached cell membrane with a fluorescent voltage-sensitive dye. The phase map of voltage change was fitted with a planar core-coat conductor model using the sheet resistance as a free parameter. For nerve cells from rat brain on polylysine as well as for HEK293 cells and MDCK cells on fibronectin we find a similar sheet resistance of 10 MOmega. Taking into account the independently measured distance of 50 nm between chip and membrane for these cells, we obtain a specific resistance of 50 Omegacm that is indistinguishable from bulk electrolyte. On the other hand, the sheet resistance for erythrocytes on polylysine is far higher, at approximately 1.5 GOmega. Considering the distance of 10 nm, the specific resistance in the narrow cleft is enhanced to 1500 Omegacm. We find this novel optical method to be a convenient tool to optimize the interface between cells and chips for bioelectronic devices.     

Thermal denaturation of calf thymus DNA: existence of a GC-richer fraction

In 2.5 x 10(-4)M EDTA buffer, the derivative melting curve of calf thymus DNA shows a major band at 47 degrees with a shoulder at about 54 degrees . The fraction of melting area of this shoulder is about 13%. For reconstituted polylysine-calf thymus DNA complexes, in addition to the melting of free DNA regions at about 50 degrees (T(m)) there is another melting at about 106 degrees (T(m)) of polylysine-bound regions. The melting band of the complex at T(m) is not symmetrical. As more polylysine is bound to DNA the melting amplitude is diminished greatly on the major band at 47 degrees but only slightly on the shoulder at 54 degrees . The insensitivity of this shoulder appears to result from the existence of a 13% fraction of calf thymus DNA containing 55% GC. It is not favorably bound by polylysine. It remains in the supernatant after centrifugation and melts at about 54-56 degrees . This conclusion is further supported by two facts: the reconstitution method provides a condition for selective binding of polylysine to AT-rich DNA, and it yields a fully symmetric melting band at T(m) for complexes of polylysine with homogeneous bacterial DNA such as the one from M. luteus.     

A chemotaxonomic appraisal of the distribution of lipomannans within the genus Micrococcus

Abstract Bactericidal effects of polylysine on different bacterial species were measured. Marked differences in sensitivity were observed. Based on the concentration of polylysine required to reduce cell viability by 50%, Mycobacterium smegmatis and Mycobacterium tuberculosis were found to be the most sensitive and Escherichia coli the most resistant. In addition, two Gram-positive organisms, Staphylococcus epidermidis and Streptococcus salivarius exhibited significant differences in sensitivity which suggests that the relationship between sensitivity towards polylysine and bacterial cell type is not necessarily a function of the overall cell envelope structure. The high sensitivity of mycobacteria suggests the possible use of polylysine. or a conjugate of polylysine and another agent in anti-mycobacterial drug design.     

Influence of carbamoylation on some analytical properties of basic polypeptides

The effect of carbamoylation on the assay or identification of histones and polylysine was investigated. Incubation with sodium cyanate decreased the positive charge on these polypeptides as judged by changes in the binding of methyl orange or the electrophoretic mobility. Histones in chromatin appeared less accessible to carbamoylation than isolated histones. Carbamoylation of proteins under conditions in which there was little or no effect on the Lowry procedure could affect their assay by methods utilizing metachromasia with Coomassie Blue G. The Bradford assay has low sensitivity for Hl histone and polylysine but this can be increased by preincubation with sodium cyanate. More extensive carbamoylation of polylysine caused decreased sensitivity which was the only response seen with core nucleosomal histones and bovine serum albumin when preincubated with sodium cyanate. It was concluded that the sensitivity for Hl histone and polylysine in assays dependent on metachromasia with Coomassie Blue G may be changed by factors which decrease the positive charge on these polypeptides.     

An adhesion-based method for plasma membrane isolation: Evaluating cholesterol extraction from cells and their membranes

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-β-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-β-cyclodextrin plasma membrane extraction properties.