Presence Of Protamine Research Articles

Restructuring space ordering of (DNA-protamine) complexes in liquid crystalline dispersions under proteolytic enzyme treatment

Abstract Complexes of DNA with the protamines stellin A and stellin B, in polymer-containing solutions, form both liquid crystalline phases and liquid crystalline dispersions. The ‘non-specific’ organization of the (DNA-protamine) phase is determined by the presence of protamine ‘cross links’ between the DNA molecules and not by the inherent anisotropy (cholesteric) double-stranded DNA molecules. Elimination of these ‘cross links’ by proteolytic enzyme action causes an increase in the distance between the DNA molecules which results in the appearance of an intense band in the CD spectrum and a ‘fingerprint’ (cholesteric) texture.

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Effects of heparin on the inhibitory action of protamine on endothelium‐mediated vasorelaxation

The precise mechanism(s) of inhibitory action of protamine on endothelium-mediated vasorelaxation has not been fully elucidated. In addition, no information is available regarding the effects of a heparin-protamine complex on the endothelium-mediated relaxation. Employing isometric tension recording methods, we studied the effects of heparin, an anionic substance, on the protamine-induced inhibition of acetylcholine (ACh)-induced vasorelaxation in isolated rabbit small mesenteric artery. Protamine (> or = 50 micrograms/ml) inhibited ACh (0.03-10 microM)-induced relaxation under a norepinephrine (10 microM)-stimulated condition (P < 0.05). The ACh relaxation, even 20 min after washout of protamine (150 micrograms/ml), was still significantly inhibited as compared to the control (before protamine) ACh relaxation, and further, it was not significantly different from the ACh relaxation maximally inhibited in the presence of protamine. Preapplication of heparin (700 U/ml) almost abolished the protamine inhibition (50 & 150 micrograms/ml) of the ACh relaxation. However, heparin (700 U/ml), applied on washout of protamine (150 micrograms/ml), had no effect on the prolonged protamine inhibition. In conclusion, a heparin-protamine complex had no direct effect on the endothelium-mediated relaxation, and the inhibitory action of protamine on the endothelium-mediated relaxation might be due to its polycationic property. The prolongation of protamine inhibition and the lack of effects of heparin on the prolonged protamine inhibition may suggest a toxic effect of protamine on the endothelium.

Precipitation of insulinotropin in the presence of protamine: effect of phenol and zinc on the isophane ratio and the insulinotropin concentration in the supernatant.

The purpose of this study is to determine the minimum quantity of protamine required for the completion of insulinotropin precipitation under different precipitation conditions. The experiments involved combining insulinotropin with varying concentrations of protamine in pH 7.2 phosphate buffered saline and analyzing the concentrations of both proteins in the supernatant. Isophane ratio (the protamine/insulinotropin molar ratio that results in a minimum total protein concentration in the supernatant) was determined for different precipitation conditions. When neutral solutions of insulinotropin (pI 5.3) and protamine (pI 13.8) were combined, precipitation did not occur. However, in the presence of phenol and/or zinc, insulinotropin co-precipitated with protamine. In the presence of phenol, the isophane ratio and the insulinotropin concentration in the supernatant were determined to be 0.08 and 0.18 mg/ml, respectively. In the presence of zinc, the isophane ratio increased with zinc concentration, apparently from the precipitation of protamine in the presence of zinc. The isophane ratio and the insulinotropin concentration in the supernatant were 0.13 and 0.13 mg/ml, respectively, when the zinc/insulinotropin molar ratio was one. In the presence of phenol and zinc with the zinc/insulinotropin molar ratio of 1.0, the isophane ratio and the insulinotropin concentration in the supernatant were 0.11 and 1 microgram/ml, respectively. A method to determine the isophane ratio of protamine/insulinotropin precipitation was developed to determine the minimum quantity of protamine required for the completion of insulinotropin precipitation under different precipitation conditions. A synergistic effect between phenol and zinc on the precipitation of insulinotropin in the presence of protamine was found.

The direct effects of protamine sulfate on myocyte contractile processes

The use of protamine sulfate in patients has been associated with circulatory collapse and is suspected to directly depress left ventricular function. However, the cellular basis for these changes that occur after protamine administration are unknown. Accordingly, the first objective of this study was to determine the direct effects of protamine on isolated myocyte contractile function. Myocytes were isolated from porcine hearts and contractile function was examined at baseline and then after the administration of protamine in concentrations of 20, 40, or 80 micrograms/ml. These concentrations were chosen because they reflect the serum concentrations of protamine commonly obtained in patients. The presence of protamine resulted in a dose-dependent decline in myocyte contractile function. For example, in the presence of a 20 microgram/ml concentration of protamine myocyte contractile function did not change significantly from baseline values, whereas an 80 microgram/ml protamine concentration caused myocyte percent and velocity of shortening to fall by more than 35% from baseline values. In light of the fact that protamine directly depressed myocyte contractile function, a second objective of this study was to examine potential cellular mechanisms responsible for this effect. Accordingly, in the next series of experiments, the effects of protamine on the myocyte sarcolemmal beta-adrenergic receptor system were examined by measuring myocyte contractile function with the beta-adrenergic agonist isoproterenol (25 nmol/L), as well as with the concomitant addition of protamine and isoproterenol. In the presence of protamine, myocyte beta-adrenergic responsiveness was significantly reduced. For example, in the presence of an 80 microgram/ml dose of protamine, both myocyte percent and velocity of shortening fell by greater than 50% when compared with isoproterenol alone values (p < 0.05). To determine the reversibility of these protamine effects, we performed additional experiments in the presence of heparin. Incubation with heparin before protamine addition prevented the negative effects of protamine on myocyte function. However, the addition of heparin after protamine incubation failed to reverse the negative effects of protamine on myocyte function. In a final set of experiments, the effects of protamine on isolated myocyte electrophysiologic properties were examined using microelectrode techniques at baseline and with either 40 or 80 micrograms/ml doses of protamine. Myocyte resting membrane potential changed from baseline with the addition of a 40 micrograms/ml dose of protamine (-79.2 +/- 0.5 versus -75.2 +/- 0.8 mV (p < 0.05), with no further change at an 80 micrograms/ml dose of protamine (-73.0 +/- 1.3 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

The phospho‐opsin Phosphatase from Bovine Rod Outer Segments

The vertebrate visual transduction system involves a cycle of phosphorylation and dephosphorylation of a transmembranous photoreceptor (rhodopsin). Upon illumination, the activated photoreceptor (metarhodopsin-II) is phosphorylated by a specific kinase on up to seven serine and threonine residues. A dephosphorylation process must then be undertaken to return the photoreceptor to its ground state. Initial work, along with studies using the rabbit skeletal muscle catalytic subunit of protein phosphatase 2A, indicated that the phosphatase responsible was a member of the type-2 family. The work has been further extended and using 1000 bovine retinae, the catalytic subunit and a holoenzyme form of phospho-opsin phosphatase were purified 2100-fold and 550-fold respectively. The stimulation of the activities of both these fractions with protamine sulphate and the inhibition by okadaic acid are consistent with the fact that these phosphatases belong to the type-2A family. Western blotting using a variety of specific antibodies established that the catalytic subunit (36 kDa, C subunit) was indeed of type 2A, while the holoenzyme was a heterotrimer comprising the preceding catalytic subunit complexed to two other polypeptides of 55 kDa (B subunit) and 65 kDa (A subunit), both of which were of alpha subtype; phospho-opsin phosphatase may thus be described as a trimeric enzyme containing the ABC subunits of type-2A protein phosphatase, i.e. PP2A1. The dephosphorylation of phospho-opsin by both fractions was found to be stimulated (4-8-fold) by the presence of protamine sulphate (250 micrograms/ml; 50 microM). However, when phospho-peptides corresponding to the C-terminal region of opsin were used, these were maximally dephosphorylated without requiring the presence of protamine; at equivalent concentrations of substrates the phospho-peptides were dephosphorylated (in the absence of protamine) at rates which were approximately equal to those obtained with phospho-opsin (in the presence of protamine). It was shown that type-1 phosphatases had little activity against these phospho-peptides. Furthermore, if phospho-opsin was treated with protamine, the activity of the phosphatase assumed an elevated level and was not significantly stimulated by the addition of exogenous protamine. This effect could be reversed by washing the protamine-treated substrate with 1 M NaCl, whence the protamine-dependent stimulation returned to normal levels. To this end, studies revealed that protamine was binding to the particulate substrate in a ratio of protamine/opsin of 0.7:1. The cumulative finding may be rationalised by suggesting that the effect of protamine is a substrate-directed phenomenon and a hypothetical mechanism for this effect is considered.

Influence of Membrane-Associated Heparan Sulfate on the Internalization of the Small Proteoglycan Decorin

The small dermatan sulfate proteoglycan decorin is efficiently internalized by a variety of cells of mesenchymal origin. Previous studies had implicated the involvement of 51- and 26-kDa receptor proteins in this uptake process. The surface localization of these proteins has now been demonstrated by labeling with a membrane-impermeant, biotinylating reagent. The human keratinocyte cell line HaCaT exhibited only about 5% of the clearance rate of fibroblasts for exogeneously added decorin, although it was not deficient in the 51-and 26-kDa proteins. Evidence is presented that plasma membrane-associated heparan sulfate influences receptor trafficking and contributes to the low internalization rate of the receptors in keratinocytes: (i) Heparitinase digestion of intact keratinocytes led to an ∼10-fold increase in the efficiency of decorin endocytosis. (ii) Endocytosis of decorin was increased more than 10-fold in keratinocytes in the presence of protamine, a cationic, heparan sulfate-binding protein. This effect is considered to be caused by competition between protamine and the endocytosis receptor for cell surface-associated heparan sulfate. (iii) Preincubation of keratinocytes with heparan sulfate-degrading enzymes at 37°C led to a decrease of receptor proteins localized at the cell surface as judged by subsequent surface labeling at 0°C. (iv) An alteration of the biosynthesis of heparan sulfate proteoglycans by p -nitrophenyl-β-xyloside was accompanied by an increased yield of intracellularly located receptor proteins. Plasma membrane-associated heparan sulfate from keratinocytes differed from the corresponding species of fibroblasts in quantity and quality. It is, therefore, suggested that the intracellular trafficking of the decorin receptor proteins is influenced by the amount and/or the composition of membrane-associated heparan sulfate.

Characterization of Casein Kinase II, and of p98 as One of Its Effective Phosphate Acceptors in Sea Urichin Eggs1

CK-II has been partially purified from a 1.5 M KCl extract of unfertilized sea urchin eggs by means of DEAE-cellulose column chromatography, gel filtration on Sephacryl S300, and heparin-agarose column chromatography, successively. This partially purified CK-II was associated with a 98 kDa polypeptide (designated as p98), which was then separated from the kinase by Mono Q column chromatography (HPLC). The biochemical characteristics of CK-II purified from unfertilized eggs were similar to those of CK-IIs from various mammalian cells: requirements of divalent cations (Mg2+ and Mn2+) and phosphate acceptors (casein and p98), response to basic polypeptides and heparin, subunit structure and molecular size. Moreover, it was found that: (i) p98 (apparent pI 10.0) has DNA-binding ability and functions as an effective phosphate acceptor for CK-II in vitro; (ii) phosphorylation of p98 by the kinase was highly stimulated by histone-like sperm protein from sea urchin sperm, protamine (salmon sperm), and poly Arg; and (iii) selective phosphorylation of p98 by CK-II was detected when the DEAE-cellulose fraction from unfertilized eggs was incubated with [gamma-32P]ATP in the presence of protamine. Some biochemical characteristics of p98 from the eggs were similar to those of transcriptional factor Sp1. The evidence obtained suggests that (i) arginine-rich sperm proteins function as potent activators for CK-II in unfertilized eggs and (ii) specific phosphorylation of p98 by the activated kinase may play an important role in the transcriptional regulation in the eggs accompanying fertilization.

Protamines, not histones, are the predominant basic proteins in sperm nuclei of solitary ascidian tunicates

AbstractIn this work we study the protein content in sperm nuclei from 10 species of ascidian tunicates. The 7 species that we have investigated cytochemically show type 3 intermediate sperm basic proteins, as observed previously for the solitary tunicate Styela montereyensis (Chiva, Rosenberg, and Kasinsky, J. Exp. Zool., 253:7–19, 1990). We have focused this study on two specific points: the presence of protamines and the presence of somatic histones. In 4 species belonging to the suborder Phlebobranchiata, sperm nuclei contain only a protein more basic than histones (protamine). This protamine has an electrophoretic mobility similar to core histone H4. The nuclei of sperm in the species belonging to the suborder Phlebobranchiata are also characterized by their complete lack of somatic histones. In 5 species from the suborder Stolidobranchiata, sperm nuclei contain a similar protamine. However, 2 species from the genus Styela contain additionally other protamines with different electrophoretic mobilities. For all species belonging to the suborder Stolidobranchiata, we also have found very small quantities of residual somatic histones. We conclude that during the process of spermiogenesis in ascidian tunicates protamines replace histones either completely (Phlebobranchiata) or almost completely (Stolidobranchiata). In this respect spermiogenesis in tunicates (class Ascidiacea) is similar to that in vertebrates which, in general, also display the substitution of somatic histones by protamines. On the other hand, this model is different from that in echinoderms, where the protein content of sperm nuclei consists mainly of somatic histones and sperm‐specific variants of histones. © 1992 Wiley‐Liss, Inc.

Protamine-induced permeability changes in the neutrophil plasma membrane as the basis of activation of exocytosis.

Protamine induces a gradual change in plasma membrane permeability in rabbit neutrophils, which is evident from the increase of indol fluorescence, and the leakage of quin2 from quin2-loaded neutrophils. The influx of extracellular Ca2+ into the neutrophil provides an explanation for exocytosis which occurs in the presence of Ca2+ and protamine. The dependence of exocytosis on Ca2+ concentration follows the same pattern as is observed in neutrophils permeabilized by other means. In the absence of Ca2+, and in the presence of protamine, La3+ has an activating effect on exocytosis. At higher concentrations La3+ inhibits exocytosis that occurs in the presence of Ca2+ and protamine, as do some other metal ions. The resemblance between the membrane effects of a number of toxins, as reported in literature, and protamine-induced membrane damage suggests that they occur via the same mechanism.

Alteration of the kinetic properties of the epidermal growth factor receptor tyrosine kinase by basic proteins.

Previous studies have shown that lysine- and arginine-rich proteins can enhance the activity of tyrosine and serine/threonine protein kinases. However, the kinetics and mechanism of this activation are not fully understood. Therefore we investigated the ability of poly(amino acids) and the arginine-rich protein, protamine, to alter the kinetic properties of epidermal growth factor (EGF) receptor protein-tyrosine kinase activity using immunoaffinity-purified receptor isolated from human epidermoid carcinoma (A431) cells. Poly(L-lysine), poly(L-arginine) and protamine stimulated EGF receptor kinase activity by 3-5-fold at non-saturating doses of ATP and peptide substrate, while poly(L-glutamate) had no effect. Initial kinetic studies demonstrated an increase in the maximum velocity and a decrease in the apparent Km for the peptide substrate angiotensin II in the presence of the basic effectors. Further analysis of the kinetic mechanism by product inhibition revealed that protamine altered the pattern of ADP inhibition towards the peptide substrate but not towards ATP. The change was indicative of the receptor's ability to form an enzyme-angiotensin II-ADP ternary complex in the presence of protamine but not in its absence. In addition, the basic effectors had a substantially decreased influence on the kinase activity of a C-terminally truncated form of the EGF receptor. Thus the changes in kinase activity may be partially mediated by the C-terminal region of the receptor, which contains the sites of receptor self-phosphorylation. These results suggest that the basic domains of proteins can interact with the EGF receptor to induce changes in its kinetic properties, especially with regard to reactant recognition and binding.

Protamine sulfate as an effective alternative to polybrene in retroviral-mediated gene-transfer: implications for human gene therapy

The polycation protamine sulfate was compared to polybrene, the usual agent employed, for its ability to increase the efficiency of retroviral infection. The murine retroviral vector SAX, which contains the neo R gene and the human adenosine deaminase (ADA) cDNA, was used as a marker of cell infection. SAX viral supernate was titered on NIH 3T3 cells in varying concentrations of polycation. The highest infection efficiency for protamine was seen at 5 μg/ml and was 7-fold greater than infections performed in the absence of polycation. Infection efficiency using protamine averaged 92% ±11 (SEM) of the highest efficiency obtained with polybrene. Total ADA activity attained when human-ADA deficient T cells were exposed to SAX supernate in the presence of protamine was 83% of that attained with polybrene. The infection rate of mouse bone marrow early progenitor cells (CFU-S) was similar with each polycation. In summary, for supernate infections, concentrations of 5–10 μg/ml of protamine provided essentially the same infection efficiency as polybrene with low toxicity on a range of cell types. Since protamine is approved for human use by the U.S. Food and Drug Administration it provides an effective alternative to polybrene when developing human gene therapy protocols.

Prevention of thrombin/antithrombin III reaction in the presence of protamine or polybrene

The rate of the thrombin/antithrombin III (AT III) reaction was decreased in the presence of free polybrene or protamine. The reaction rate was also decreased in protamine-coated tubes and tubes on which polybrene was absorbed nonspecifically. The reaction was also prevented when either thrombin or AT III was transfered into noncoated tubes after instantaneous contact with protamine-coated tubes or tubes with polybrene. These facts suggest that the thrombin/AT III reaction rate is determined with concentrations of reactive protein species.

Influence of protamine on the susceptibility of Pseudomonas aeruginosa to cefotaxime, sulphadimethoxine, polymyxin B and some β-lactam antibiotics

Protamine, a basic protein, was shown to influence the activity of antibacterial agents. When added to tryptic soy broth it enhanced the bactericidal activity of cefotaxime, sulphadimethoxine, ticarcillin, piperacillin and carbenicillin against Pseudomonas aeruginosa. In contrast, the activity of polymyxin B against this species was markedly reduced in the presence of protamine.

Hyposmotic swelling leads to the expression of trypsin-like activity by rat peritoneal fluid mast cells.

Rat peritoneal fluid mast cell present parallel increases in cell area (swelling), and in hydrolytic activity on the trypsin substrate p-tosyl arginine methyl ester (TAME), when placed in Tris buffers of concentrations between 0.15 and 0.03 M. Under these conditions, cells do not degranulate and preserve their trypsin-like enzyme activity after low speed centrifugation. Exposure to more dilute Tris buffers, between 0.015 and 0.003 M, leads to cell rupture accompanied by progressive degranulation and loss of activity on TAME. Protamine, a heparin antagonist prevented this loss when added to mast cells prior to hyposmotic lysis, or lysis by sonication or repeated periods of freezing and thawing. Enzyme activity released in the presence of protamine was fully recovered in supernates of cell lysates submitted to low speed centrifugation. Controlled swelling of mast cells propitiates the expression of trypsin-like activity, possibly by facilitating enzyme-substrate interaction. Cell lysis on the contrary, leads to inactivation of such activity, possibly by enzyme binding to heparin in exposed mast cell granules.

Mode of action towards oligopeptides and proteins of hydrolase H, a high-molecular-weight aminoendopeptidase from rabbit skeletal muscle.

The mode of action towards oligopeptides and proteins of hydrolase H purified from rabbit skeletal muscle was studied. The presence of protamine or alpha-N-benzoylarginine p-nitroanilide (an endopeptidase substrate) changed both the Km and V values of the enzyme towards Leu-beta-naphthylamide (an aminopeptidase substrate). This indicates that the binding site for an endopeptidase substrate is different from that for an aminopeptidase substrate. Hydrolase H as an aminopeptidase displayed broad specificity. The enzyme hydrolyzed various dipeptides readily except the dipeptides containing Pro or an amino acid with a hydrophobic beta-branched chain at the NH2 terminus. Pro and Val at the NH2 terminus of tripeptides were also difficult to release, whereas Ile and Val of tetrapeptides were easily released in contrast with those of dipeptides. The longer the peptide chain of Glyn (n = 2, 3, 4), the more susceptible was it to hydrolase H. Hydrolase H behaved as an endopeptidase only towards protamine among the proteins tested. The other proteins, casein, bovine serum albumin, myofibrils, troponin, hemoglobin, sarcoplasmic proteins, and myoglobin were probably attacked only by the aminopeptidase activity of the enzyme.

Separation and characterization of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from chick embryo cartilage

Two distinct sulfotransferases (chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase), which catalyzed transfer of sulfate to position 6 and position 4 of acetylgalactosamine residues of chondroitin, were extracted from epiphyseal cartilage of 14-day-old chick embryos and separated by gel chromatography on Sephacryl S-200 in the presence of 3 M guanidine-HCl. When the enzyme solutions containing 3 M guanidine-HCl were dialyzed against 0.02 M Tris-HCl, pH 7.2, containing 10% glycerol, chondroitin 4-sulfotransferase became almost insoluble, whereas chondroitin 6-sulfotransferase remained soluble. Endogenous acceptors for sulfate transfer were completely removed from both enzyme preparations. Addition of basic proteins and polyamines as well as Mn 2+ to the incubation medium caused a stimulation of both sulfotransferases; the stimulation of chondroitin 6-sulfotransferase with these cations was higher than that of chondroitin 4-sulfotransferase. The K m values for 3′-phosphoadenylyl sulfate of both enzymes were much smaller in the presence of protamine or spermine than in the presence of Mn 2+. The two sulfotransferases differed in the requirement for sulfhydryl compounds; in the absence of sulfhydryl compounds, the activity of chondroitin 4-sulfotransferase was very low, whereas the activity of chondroitin 6-sulfotransferase was essentially unaffected. These observations indicate that at least two sulfotransferases are involved in the biosynthesis of chondroitin sulfate, and suggest that the production of the isomers of chondroitin sulfate in chondrocytes is affected by various factors such as the intracellular concentration of sulfhydryl compounds and basic substances.

Protamine interacts with the D-domains of fibrinogen

The mechanism of precipitation of fibrinogen in the presence of a basic protein, protamine, has been investigated. The precipitation was clearly inhibited by the addition of Fragment D. Photooxidation of fibrinogen abolished the association caused by thrombin, but it did not affect the precipitation brought about by protamine. Not only fibrinogen but also fibrin monomer precipitated in the presence of protamine. These results led to the conclusion that protamine bound directly with D-domains of fibrinogen (of fibrin monomer) to cause the precipitation.

The interaction of protein-bound heparin and antithrombin III.

When commercially prepared porcine mucosal heparin is added to human plasma, some of the heparin fractions form a complex with antithrombin III activating it to an immediate inhibitor of thrombin as well as of other serine proteases. Certain fractions of heparin may complex with other proteins such as alpha 2-macroglobulin, another progressive inhibitor of thrombin. Without complexing with antithrombin III, this protein-bound heparin fraction(s) still retains the capacity to activate it to an immediate inhibitor of thrombin. Protamine sulfate inactivates those heparin fractions that bind to antithrombin III but not those bound to alpha 2-macroglobulin. Activated antithrombin III may undergo a molecular change in the presence of protamine which not only changes it back to a progressive inhibitor but makes it resistant to activation by the protein-bound heparin fraction(s). However, it can still be reactivated by other heparin fractions in fresh whole heparin. The observations presented may help explain heparin "rebound" in patients believed adequately neutralized with protamine.

Mode of binding and internalization into mouse macrophages of heparin complexed with polycations

Heparin uptake by cultured macrophages was investigated from the standpoint of: (1) whether the increased uptake in the presence of polycations is due to charge neutralization, and (2) whether the heparin becomes internalized. Regarding the first point, our results are compatible with the notion that charge neutralization is mainly responsible for the enhanced uptake of heparin in the presence of protamine, histone, poly( DL-lysine) and poly( L-ornithine). As for the second point, chasing experiments at low and high temperatures strongly suggest that while heparin binds onto the cell membrane at both 4°C and 37°C, it undergoes internalization only at 37°C.