The interaction of 8-anilino-1-naphthalenesulfonate with polylysine and polyarginine

Comparative studies on the interaction of 8-anilino-1-naphthalenesulfonate (ANS) with polylysine and polyarginine have been made by equilibrium dialysis and fluorescence or circular dichroism measurements, to investigate the structural characteristics of the polypeptides. The results are summarized as follows: (i) ANS binds to either of the polypeptides primarily by electrostatic interaction while hydrophobic interaction partially facilitates the dye binding; both interactions are stronger in the polyarginine-dye binding than the polylysine-dye binding. (ii) The fluorescence of ANS is more intensified when the dye binds to polyarginine than to polylysine regardless of the value of r (number of bound dye per amino-acid residue) of polypeptide-dye complexes, although the intensification depends on the r value and becomes maximum at r = 0.25–0.35 for both cases. (iii) The binding of ANS to each polypeptide is cooperative at r < 0.4. (iv) The circular dichroism is more efficiently induced in the spectral region of ANS by binding to polyarginine than to polylysine. From these results, it was concluded that, compared to polylysine, polyarginine suffers some structural change by ANS binding into a more compact molecular configuration having some regularity with a lower dielectric environment.     

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.     

Spectral changes induced in Cibacron Blue F3GA by salts,organic solvents,and polypeptides: Implications for Blue dye interaction with proteins

The interactions of Cibacron Blue F3GA with organic solvents, salts, oligopeptides, and polypeptides were studied by visible difference spectroscopy. The difference spectrum of the dye in an aqueous solution of NaCl (vs water) has a characteristic positive peak at 690 nm and negative double minima at 630 and 585 nm. Such a “salt-like” spectrum is also obtained for interaction of the dye with polycations such as oligolysines, polylysine, polyarginine, and protamine. In contrast, the difference spectrum of the dye in binary aqueous solvents containing dioxan or t-butyl alcohol at moderately high concentrations, measured against water, displays a positive peak and shoulder at 655 and 610 nm, respectively, with a small negative contribution below 550 nm. This spectrum is attributed to a nonpolar interaction of the dye with organic cosolvent molecules. The spectrum of the dye in 7 M urea is changed little from that in water, indicating similar interactions of the dye with water or urea molecules. The spectral characteristics described here for the interaction of the polyaromatic polysulfonate dye with positively charged groups, polar groups, and nonpolar moieties of neutral molecules provide a basis for describing the details of the interactions of Cibacron Blue F3GA with several proteins and for characterizing the dye binding environments in the proteins.     

Immunoglobulin E decapeptide-induced 5-hydroxytryptamine release from rat peritoneal mast cells. Comparison with corticotropin-(1-24)-peptide, polyarginine, polylysine and antigen.

A synthetic basic decapeptide from the C4 domain of human immunoglobulin E, corticotropin-(1-24)-peptide, polyarginine and polylysine stimulated up to 90% net release of 5-hydroxytryptamine from mast cells in rat peritoneal-cell suspensions. Concentration-response curves to all four polypeptides were parallel. Polyarginine and polylysine (EC50 congruent to 0.05 microM) were approximately 100-fold more potent than immunoglobulin E decapeptide and corticotropin-(1-24)-peptide (EC50 congruent to 5 microM). Polypeptide-induced release was complete within 5-10s. Immunoglobulin-E-decapeptide-induced 5-hydroxytryptamine release was additive to that induced by low concentrations of polyarginine, but non-additive to that induced by high concentrations of polyarginine. In contrast, release induced by antigen was additive along the whole length of the concentration-response curve to polyarginine. Benzalkonium chloride inhibited immunoglobulin-E-decapeptide- and polyarginine-induced 5-hydroxytryptamine release but enhanced release induced by immunological stimulation.     

Colorimetric determination of microgram quantities of polylysine by trypan blue precipitation

A simple and sensitive method for the determination of polylysine in solution is described. Polylysine is quantitatively precipitated with trypan blue. The absorbance of unbound dye in the supernatant is inversely proportional to the concentration of this polyamino acid. The precipitation is identical for all sizes of polylysine of molecular weight 13,000 or higher, and is prevented by the addition of either polyanions or serum. The measurable range of polylysine hydrobromide is between 1 and 10 micrograms/ml, which is about 10-fold lower than that by the published methyl orange precipitation method.     

Different effects of polylysine and polyarginine on the transition to a condensed state of DNA in polyethyleneglycol/salt solution.

Circular dichroism spectroscopy has been used to investigate the influence of polylysine and polyarginine on the transition to a condensed state of DNA brought about by high concentrations of polyethyleneglycol and salt. From the dependence on DNA concentration of the CD signals, the anomalous CD of free DNA in polyethyleneglycol/salt solution was attributed to the intermolecular association of DNA molecules. The CD spectral changes in polyethyleneglycol/salt solution of the DNA - polylysine complex were indistinguishable from those of free DNA while the DNA-polyarginine complex suffered much smaller spectral changes as compared with free DNA, at low DNA concentrations where time-independent CD spectra were observed in polyethyleneglycol/salt solution for both the complexed and free DNA. The repression of the spectral change by the latter complex was more remarkable at higher ratios of polyarginine to DNA. The facts indicate that, whereas polylysine binding has little influence on the intermolecular structural transition of double-stranded DNA into a compact molecular configuration in polyethyleneglycol/salt solution, polyarginine binding has an effect of inhibiting the transition.     

Increased nuclear size in BHK21 cells after treatment with non-toxic levels of calf thymus histones.

The effect of treatment with either whole calf thymus histones, or individually isolated histones, or polyarginine, or polylysine, on the nuclear size of BHK21 cells has been investigated. Incubation of the cells with mixed histones (12.5--44 microgram/ml) for 1 h considerably increased nuclear size. Increasing the histone concentration and/or the incubation time resulted in a decrease in the effect and could result in no change in nuclear size. Treatment of the cells with polyarginine or polylysine did not affect nuclear size. Experiments with individually isolated histones showed that the nuclear size effect was almost exclusively due to the histone H4. It is argued that the changes observed most likely resulted from interaction of H4 with the nucleus, and could reflect the properties of this particular histone molecule.     

The influence of chronic ethanol feeding to rats on the integrity of liver mitochondrial membrane as assessed with the Mg2+-stimulated ATPase enzyme.

The mouse submaxillary proteases (A + D), the isolation and properties of which were previously described by us, hydrolyze only arginyl bonds in proteins. Formol titrations and peptide mapping on digests of polyarginine, polylysine, lysozyme, histone, and insulin suggested this specificity. Amino acid compositions of peptides from lysozyme and insulin showed that most but not all arginyl bonds were hydrolyzed but that no lysyl bonds were split. The proteases should be useful in the sequencing of proteins.     

Specifity of DNA-basic polypeptide interactions. Influence of neutral residues incorporated into polylysine and polyarginine.

An approach is described for evaluation of the specificity of basic polypeptides concerning the base pair composition of DNA. The polypeptides were covalently bound to CNBr activated agarose and two DNAs strongly different in base composition but of equal molecular weight were loaded and detached by a NaCl gradient. The difference in the NaCl concerntrations between the elution maxima of the two DNAs was taken as a measure for the recognition specificity. The results obtained confirmed the known AT- und GC-specificity of polylysine and polyarginine, respectively. Neutral residues incorporated into polylysine generally reduce the interaction affinity and also the AT-specificity of their host. This behavior is very pronounced with three homogeneous fractions of clupeine containing about one third of neutral aliphatic amino acids within clusters of arginine; the base pair specificity of these arginine copolymers was found to be practically nil.     

A comparative x-ray study of a nucleoprotamine and DNA complexes with polylysine and polyarginine

The high-humidity X-ray pattern of oriented fibers prepared from salmon spermheads strongly resembles that of DNA in the B form. However, the nucleoprotamine pattern has a more intense first layer line and increased lateral unit-cell dimensions. Complexes of DNA with poly-L -lysine and poly-L -arginine were prepared and photographed at various relative humidities. The most crystalline patterns were obtained at 92% and also indicate DNA in the B form. However, whereas polylysine–DNA, like the spermheads, has a primitive hexagonal cell, polyarginine–DNA, like NaDNA, has three molecules in the unit cell. Polylysine–DNA, but not polyarginine–DNA, also resembles spermheads in having a strong first layer line. All three complexes show increasing intermolecular distance with increasing humidity, but with sharp maxima when photographed in water, which indicates cross-linking between the molecules. Lowering the humidity causes the polylysine–DNA, but not polyarginine–DNA, to change conformation from the B to the C form. The structural implications of these results are discussed in the light of model-building studies and a comparison of calculated and observed X-ray intensities.     

Infrared linear dichroism investigations of deoxyribonucleic acid complexes with poly(L-arginine) and poly(L-lysine).

Complexes between DNAs from various sources and poly(L-lysine) and poly(L-arginine) were studied by means of infrared linear dichroism. The measurements of dichroic ratios allowed us to determine the orientation of the phosphate group of DNA in the complexes with basic polypeptides. At high relative humidities (higher than 90%, B form), the bisector of the less than OPO in the complexes forms an angle with respect to the helical axis which has a value lower by about 4 degrees than in the corresponding DNA sample. This change of orientation of the phosphate group of DNA indicates a modification of the B form upon binding of polylysine or polyarginine. The structural transitions B leads to A and B leads to C measured as a function of relative humidities were not affected by formation of complexes with both basic polypeptides. Similar results were obtained for complexes prepared by direct mixing or by salt gradient dialysis. The presence of A and C forms was observed in complexes of DNA with poly(L-lysine) and poly(L-arginine) at lower relative humidity. Thus, the conformational flexibility of DNA in complexes with polylysine and polyarginine is not changed despite a substantial increase in the Tm (melting temperature). These results are considered as a model for the understanding of interactions between DNA and histones particularly of the binding of the N-terminal fragment, lysine or arginine rich.     

Interaction of human erythrocyte multicatalytic proteinase with polycations

The multicatalytic proteinase from human erythrocytes (macropain, proteasome) is a large enzyme composed of at least six distinct subunits ranging in molecular masses from 20 to 30 kDa. As its name implies, this proteinase appears to contain multiple catalytic sites with differing specificities toward peptide substrates. Several polycationic substances, including polylysines, polyarginine, protamine and histone H1 markedly stimulated caseinolytic activity of the proteinase. Activation was instantaneous, and involved increasing the Vmax of the proteinase for casein. Prolonged preincubation with polylysine at 37 degrees C resulted in autolytic inactivation of the proteinase. The polylysine concentrations required for half-maximal activation or autolytic inactivation were the same. A 23 kDa subunit of the proteinase disappeared at the same rate as loss of catalytic activity, and with the same pH dependence and polylysine concentration dependence. These results suggest that polylysine perturbs the structure of the multicatalytic proteinase, resulting in increased catalytic activity toward substrates; and, with prolonged exposure, allowing autoproteolytic inactivation to occur. The 23 kDa subunit appeared to be required for expression of caseinolytic activity, and may therefore be a catalytic subunit of the complex having activity against casein.     

Polyamines and basic proteins stimulate activation by cAMP and catalytic activity of Mucor rouxii cAMP-dependent protein kinase

Partial activation of Mucor rouxii cAMP-dependent protein kinase by cAMP was obtained when kemptide was used as substrate, but complete activation was attained with cAMP plus protamine or histone. Full activation could not be achieved by increasing kemptide or cAMP concentration. Complete activation by cAMP could be obtained by addition of 10 microM polylysine, 10 microM lysine-rich histone or 0.5 mM spermine plus spermidine. The degree of stimulation could be up to 5-fold, depending on the amount of enzyme in the assay. The same concentrations of polycations increased 1.5-2.3-fold the Vmax of kemptide phosphorylation by the free catalytic subunits of both Mucor and bovine heart protein kinases; 10 microM polyarginine inhibited completely the activity of both enzymes.     

Interactions of basic polypeptides and proteins with calmodulin.

Low concentrations (less than 10 microgram/ml) of a number of highly basic polypeptides inhibit the calmodulin-stimulated cyclic nucleotide phosphodiesterase. Inhibitory compounds include synthetic polypeptides [polylysine (D and L) and polyarginine] and basic proteins (protamine, histones H1, H2A, H2B, H3 and H4 and myelin basic protein). Polylysine of mol.wt. about 2000 or higher was inhibitory, but pentalysine did not inhibit. Other basic proteins and compounds did not inhibit, including bradykinin, spermine and putrescine. In mixtures of calmodulin and basic protein, complexes were formed whether Ca2+ was present or not. This was true for polylysine, myelin basic protein and histone H2B. These interactions suggest that the inhibition of the phosphodiesterase is due to interaction of these basic proteins with calmodulin. The wide variety of basic polypeptides and proteins that affect the calmodulin stimulation of phosphodiesterase indicates that these interactions are not specific.     

Helix–coil transition in nucleoprotein—theory and applications

A general theory of helix–coil transition of irreversibly complexed nucleoproteins is presented. The equations are tested by experimental results in basic polypeptide–DNA complexes, nucleohistone I and pea bud nucleohistones. They show good agreement between theory and experiments. The theory provides direct measurement of a fraction of DNA base pairs covered by proteins, yielding a value of about 75% histone-covered base pairs in pea bud nucleohistone. It also provides a measurement of an average number of amino acid residues per nucleotide in protein-bound regions. This number varies from 1.0 to 1.4 in DNA–polylysine or DNA–polyarginine and from 2.9 to 3.3 in nucleohistone Ia, Ib, f1, and pea bud nucleohistone.     

Detection of hybrid formation between peptide nucleic acids and DNA by fluorescence polarization in the presence of polylysine.

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9-13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.     

A lymph node neutral proteinase acting on myelin basic protein

A neutral protease present in inguinal and popliteal lymph nodes of rats with acute experimental allergic encephalomyelitis (EAE), rats injected with Freund's adjuvant, and rats that are normal has been found to hydrolyze basic protein present in purified brain and spinal cord myelin. The enzyme has been enriched by ammonium sulfate precipitation, and its properties have been studied. The protease activity toward different substrates was very specific and decreased in the following order: Protamine sulfate = polylysine (MW 183,000) > myelin basic protein > histone > polylysine (MW 2000) > polyarginine > cytochrome c. Other proteins including casein, freshly denatured hemoglobin, egg albumin, bovine serum albumin, and ribonuclease were ineffective as substrates. The pH curve showed a peak at pH7 for rat myelin, isolated beef basic protein, and histone. A possible role for this enzyme in demyelination in acute experimental allergic encephalomyelitis is suggested.     

Effect of poly-basic amino acids on the phosphorylation of various substrate proteins by cytosolic protein-tyrosine kinase from porcine spleen

Cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) is strongly activated by poly-L-lysine using bovine serum albumin, ovalbumin, phosphorylase b, calmodulin and H1 histone as substrate proteins. However, this polyamine inhibited the enzyme activities when myelin basic protein, tubulin and H2B histone were used as substrate proteins. These stimulatory and inhibitory effects on CPTK-40 are not specific for polylysine, but polyarginine and polyornithine have similar effects on this phosphorylation reaction. Effect of poly-basic amino acids on CPTK-40 seems to be mainly on the substrate proteins, rather than on the enzyme itself.