NWChem: Past, present, and future.

Specialized computational chemistry packages have permanently reshaped the landscape of chemical and materials science by providing tools to support and guide experimental efforts and for the prediction of atomistic and electronic properties. In this regard, electronic structure packages have played a special role by using first-principle-driven methodologies to model complex chemical and materials processes. Over the past few decades, the rapid development of computing technologies and the tremendous increase in computational power have offered a unique chance to study complex transformations using sophisticated and predictive many-body techniques that describe correlated behavior of electrons in molecular and condensed phase systems at different levels of theory. In enabling these simulations, novel parallel algorithms have been able to take advantage of computational resources to address the polynomial scaling of electronic structure methods. In this paper, we briefly review the NWChem computational chemistry suite, including its history, design principles, parallel tools, current capabilities, outreach, and outlook.

Model of calcium oscillations due to negative feedback in olfactory cilia.

We present a mathematical model for calcium oscillations in the cilia of olfactory sensory neurons. The underlying mechanism is based on direct negative regulation of cyclic nucleotide-gated channels by calcium/calmodulin and does not require any autocatalysis such as calcium-induced calcium release. The model is in quantitative agreement with available experimental data, both with respect to oscillations and to fast adaptation. We give predictions for the ranges of parameters in which oscillations should be observable. Relevance of the model to calcium oscillations in other systems is discussed.

5'-O-fluorosulfonylbenzoyl esters of purine nucleosides as potential inhibitors of NTPase/helicase and polymerase of Flaviviridae viruses.

Synthesis and interactions of guanosine, inosine and ribavirin 5'-fluorosulfonylbenzoyl esters with hepatitis C virus (HCV) and Flaviviruses NTPase/helicase and polymerase are described.

The proteins of the Hepatitis C virus: their features and interactions with intracellular protein phosphorylation.

Chronic infection with Hepatitis C virus (HCV) often results in cirrhosis and enhances the probability of developing hepatocellular carcinoma (HCC). The underlying mechanisms that lead to malignant transformation of infected cells, however, remain unclear. Observations made with isolated HCV antigens and/or with HCV subgenomic replicon systems demonstrated that the products encoded in the HCV genome interfere with and disturb intracellular signal transduction, often by phosphorylation of cellular proteins. Moreover, some of the HCV-encoded proteins seem to serve as substrates for host cell protein kinases. These phosphorylations affect the biological functions of the antigens. In many cases it could be demonstrated that only short stretches of the linear sequence of the viral or cellular proteins are involved and play a crucial role for these phosphorylation events. The identification of these small polypeptide elements and the subsequent development of strategies to inhibit protein-protein interactions involving them may be the first step towards reducing the chronicity and/or of the carcinogenicity of the virus. This review summarizes current knowledge of intracellular phosphorylation processes that are affected by HCV.

Metal (Cu, Zn, Fe, Pb) concentrations in human placentas.

The concentrations of some metals (Cu, Zn, Fe, Pb) in human placentas at term in two populations living in polluted (Krakow, n = 10) and non-polluted (Bieszczady, n = 13) areas were investigated by means of graphite furnace--or flame atomic absorption spectrometry (GF-AAS or F-AAS). The concentrations of Cu, Fe and Pb were higher in Krakow vs. Bieszczady, while Zn concentration was lower, but these differences were not significant. The following results were obtained for the whole studied group: Cu 1.17 +/- 0.25 microg/g w.w., Zn 8.44 +/- 2.10 microg/g w.w., Fe 115.0 +/- 31.9 microg/g w.w., Pb 51.6 +/- 18.0 ng/g w.w. The inverse accumulation of Zn and Pb is in accord with previous observations. In the whole group of placenta specimens the statistically significant correlation was also found between concentrations of Cu and Pb. The correlations between metal concentrations and placental or maternal features were the strongest for lead.

Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme.

The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH(2) terminus by microsequencing, and a binding assay with 5'-[(14)C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with k(cat) values of 133 and 5.5 x 10(-3) s(-1), respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg(2+) requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5'-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine and O(6)-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value.

Inhibition of the helicase activity of HCV NTPase/helicase by 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5 '-triphosphate (ribavirin-TP).

In the presented study the ribavirin-TP--an established inhibitor of the NTPase activity of the superfamily NTPase/helicases II--was investigated as an inhibitor of the unwinding activity of the hepatitis C virus (HCV) NTPase/helicase. The kinetics of the reaction revealed that ribavirin-TP reduces the turnover number of the helicase reaction by a mechanism that does not correspond to that of the inhibition of the NTPase activity. Our results suggest that derivatives of ribavirin-TP with enhanced stability towards hydrolytic attack may be effective inhibitors of the enzyme.

ATP-binding domain of NTPase/helicase as a target for hepatitis C antiviral therapy.

To enhance the inhibitory potential of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) vs hepatitis C virus (HCV) NTPase/helicase, ribavirin-5'-triphosphate (ribavirin-TP) was synthesized and investigated. Ribavirin-TP was prepared with the use of modified Yoshikawa-Ludwig-Mishra-Broom procedure (cf. Mishra & Broom, 1991, J. Chem. Soc., Chem. Commun, 1276-1277) involving phosphorylation of unprotected nucleoside. Kinetic analysis revealed enhanced inhibitory potential of ribavirin-TP (IC50=40 microM) as compared to ribavirin (IC50 > 500 microM). Analysis of the inhibition type by means of graphical methods showed a competitive type of inhibition with respect to ATP. In view of the relatively low specificity towards nucleoside-5'-triphosphates (NTP) of the viral NTPase/helicases, it could not be ruled out that the investigated enzyme hydrolyzed the ribavirin-TP to less potent products. Investigations on non- hydrolysable analogs of ribavirin-TP or ribavirin-5'-diphosphate (ribavirin-DP) are currently under way.

A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC.

A synthetic peptide corresponding to the amino acid sequence Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487-1500)] with a Km of 11 microM and Vmax of 24 micromol x min(-1) x mg(-1). HCV(1487-1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50 corresponding to the Km values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487-1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe1499]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]HCV(1487-1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and Pi allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that[Phe1499]HCV(1487-1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.

Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus.

The RNA-stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP-binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N- and C-terminal sequencing and by its capability to bind 5'-fluorosulfonylbenzoyladenosine (FSBA). The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 microM. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP-binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides. Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 microM), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 microM). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP-binding domain.

Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) inhibits the nuclear transport and the enzymatic activity of the catalytic subunit of protein kinase A. This inhibition is mediated by an arginine-rich domain localized between amino acids 1487-1500 of the HCV polyprotein. The data presented here indicate that the arginine-rich domain, when embedded in recombinant fragments of NS3, interacts with the catalytic site of protein kinase C (PKC) and inhibits the phosphorylation mediated by this enzyme in vitro and in vivo. Furthermore, a direct binding of PKC to the NS3 fragments leads to an inhibition of the free shuttling of the kinase between the cytoplasm and the particulate fraction. In contrast, a peptide corresponding to the arginine-rich domain (HCV (1487-1500)), despite also being a PKC inhibitor, did not influence the PKC shuttling process and was transported to the particulate fraction by the translocating kinase upon activation with tetradecanoylphorbol-13-acetate. Using the tetradecanoylphorbol-13-acetate -stimulated respiratory burst of NS3-introduced neutrophils as a model system, we could demonstrate that NS3 is able to block PKC-mediated functions within intact cells. Our data support the possibility that NS3 disrupts the PKC-mediated signal transduction.

Identification and characterization of a histone binding site of the non-structural protein 3 of hepatitis C virus.

BACKGROUND: Chronic hepatitis resulting from the hepatitis C virus (HCV) infection leads to cirrhosis in at least half the infected patients and increases the risk of hepatocellular carcinoma. There are indications that this pathogenic effect may result from the disturbance of intracellular signal cascades caused by the interaction with viral antigens. Although a great amount of data has been accumulated about functional regions in HCV proteins, relatively little is known about their intracellular targets. Previously, we have demonstrated that the full-length non-structural protein 3 of HCV (NS3) (Borowski P, Heiland M, Feucht H, Laufs R. Characterisation of non-structural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC. Evidences for action on the level of substrate and enzyme. Arch Virol 1999a; 144) and its NH2- and COOH-terminal truncated form (Borowski P, Heiland M, Oehlmann K, Becker B, Kornetzky L, Feucht HH, Laufs R. Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase. Eur J Biochem 1996;237:611-618) associate to stable complexes with core histones H2B and H4. The changes of the properties of histones as substrate for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) were found as a direct consequence of the interaction. OBJECTIVE: In the present study we further these observations, localize the histone binding domain of NS3 and investigate the mechanisms by which NS3 affects the functions of the histones in vitro. STUDY DESIGN: HCV protein exhibiting the mentioned histone binding activity was produced in a bacterial expression system, purified and binding to histones was biochemically characterized. The region of NS3 involved in the interaction with histones was defined by proteolytic fragmentation, microsequencing and a specific histone binding assay. Furthermore, a functional test to quantify the interaction of histones with DNA was established and the binding of DNA to histone as a function of NS3 concentration was analysed by means of graphical methods. RESULTS: The investigated fragment of HCV polyprotein consisting of amino acid residues 1189-1525 (HCV-polyprotein-(1189-1525)) displayed significant histone binding activity. The binding occurred at a molar ratio 1:1 of histone to HCV-polyprotein-(1189-1525) and was mediated by a linear stretch of amino acids located between the residues 1343 and 1379 of the HCV polyprotein. To demonstrate that HCV-polyprotein-(1189-1525) affects the binding of DNA to histones we used two independent methods: overlay assay and binding assay on Sepharose beads. Graphic analysis of the binding kinetics revealed an uncompetitive type of inhibition. CONCLUSIONS: Our results provide the first evidence that NS3 binds and affects the functions of core histones. The mechanism by which the NS3 interferes with the histone functions involves conformational changes of histone molecule.

Protein kinase C-alpha produces reciprocal effects on the phorbol ester stimulated tyrosine phosphorylation of a 50 kDa kinase in Jurkat cells.

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly(Glu4,Tyr1). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the alpha-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the alpha-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with 32Pi indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein underin vivo conditions. These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by alpha-PKC.

Characterisation of non-structural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC: evidences for action on the level of substrate and enzyme.

Generally, the maximum activities of the protein kinases A (PKA) and C (PKC) show an optimum value for their substrate concentrations rather than a saturation curve; at high substrate concentrations, the kinase activity is completely abolished. The C- and N-truncated form of the non-structural protein 3 (NS3) of hepatitis C virus (HCV) (HCV-polyprotein-(1,189-1,525)) abolishes the inhibiting effect of the substrate, yielding saturable Michaelis-Menten kinetics of PKA and its catalytical domain (C subunit). In contrast, HCV-polyprotein-(1,189-1,525) activates PKC with increasing Vmax, while it abolishes the substrate inhibition of its catalytical domain (M-kinase) through a mechanism analogous to that of PKA and C subunit. PKC isoforms alpha, beta and gamma investigated are similarly activated by HCV-polyprotein-(1,189-1,525). Our data suggest that NS3 attenuates the substrate inhibition through a generalized mechanism operating mainly on the substrate level that directly results from a specific protein-protein interaction. In the case of the PKC, an additional kinase activating mechanism operates on the enzyme level. Both actions of NS3, the attenuation of the substrate inhibition and the activation of PKC, could not be explained by classical means that predict autophosphorylation to enhance the rate of substrate phosphorylation. The results are discussed in view of similar activities displayed by matchmakers and some molecular chaperones.

Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C.

The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72(syk). Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (alpha > betaI = betaII > gamma) of CDp72(syk) phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.

Properties of the proteolytically generated catalytic domain (42 kDa kinase) of epidermal growth factor receptor: comparison with holoenzyme.

Treatment of A431 cell membranes with trypsin or Streptomyces griseus proteinase results in degradation of the EGF-R and the concomitant generation of an active kinase with a molecular mass of 42 kDa (42 kDa kinase). To investigate the biochemical properties of the 42 kDa kinase, the EGF-R was immunoaffinity-purified from the A431 cell membranes and the kinase proteolytically generated. The proteolysis of EGF-R changes both the Vmax and the Michaelis constants of substrates. These substrates determine the extent of the changes of the parameters. The 42 kDa kinase is less responsive to polyions as regulators of kinase activity and is less efficiently inhibited by genistein and tyrphostin. The experiments described here point to a role of the extracatalytic domains in determining the substrate specificity and regulation of kinase activity.

Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase.

Chronic hepatitis resulting from hepatitis C virus (HCV) infection develops into cirrhosis in at least half of infected patients and increases the risk of hepatocellular carcinoma. The pathogenic effects of a number of viruses result from the disturbance of intracellular signal cascades caused by viral antigens. Therefore, we investigated the interaction of nonstructural protein 3 (NS3) of HCV with the cyclic AMP-dependent signal pathway. We found a similarity between the HCV sequence Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg localized in NS3 and the general consensus sequence of protein kinase A (PKA). Consequently, the catalytic (C) subunit of PKA bound to a bacterially expressed fragment of HCV polyprotein containing amino acid residues 1189 to 1525. When this fragment was introduced into cells, it inhibited the translocation of the C subunit into the nucleus after stimulation with forskolin. The result of this inhibition was significantly reduced histone phosphorylation. Therefore, the presence of NS3 in the cytoplasm of infected cells may affect a wide range of PKA functions and contribute to the pathogenesis of the diseases caused by HCV.

Characterization of the C-terminal domain of ras-GTPase-activating protein (ras-GAP) as substrate for epidermal growth factor receptor and p60c-src kinase.

We describe in vitro tyrosine phosphorylation of the C-terminal 334 amino acids of ras-GTPase-activating protein (ras-GAP)1 that contains the activity domain for ras interaction. To date, there have been no other phosphorylation sites determined than the reported in N-terminal domain of ras-GAP Tyr-460, which is considered to be the major phosphorylation site of ras-GAP. In our assays some differences of the kinetic parameters were observed when the reaction was catalyzed by EGF-R compared to p60c-src. Enzyme specific regulation of activity is associated with autophosphorylation which leads to reduced (in case of EGF-R) or increased (in case of p60c-src) phosphorylation of the C-terminal 334 amino acids of ras-GAP (GAP334). Because of the characteristics of these investigated reactions the phosphorylation of GAP334 seems to be-independent from the presence of SH2 or SH3 domains-triggered off by complex mechanisms different from those regulating the phosphorylation at Tyr-460.

Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase.

Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 , located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189-1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189-1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.

Regulation of epidermal growth factor receptor kinase activity by polyions.

We investigated polyionic agents with regard to their effects as modulators of epidermal growth factor receptor (EGF-R) kinase activity. Many synthetic polypeptides containing glutamine as well as casein were phosphorylated, while polycationic compounds with tyrosine residues were not phosphorylatable and thus inhibited the EGF-R activity. Polyarginine, protamine sulfate, spermidine, heparin, and poly-L-lysine with a chain length of < 20.5 kDa triggered the phosphorylation of poly(Tyr1, Glu4). On the other hand, dextran sulfate and poly-L-lysine with chain lengths of > 20.5 kDa inhibited the EGF-R kinase activity. Alteration of the state of autophosphorylation of EGF-R is not in agreement with the activity of EGF-R kinase towards poly(Tyr1, Glu4). Casein and histone H1 both increased the autophosphorylation of EGF-R in a concentration-dependent manner, but only casein increased the activity of the enzyme towards an exogenous substrate. The compounds enhancing the EGF-R activity, such as poly-L-lysine, protamine, and poly-L-arginine, down-modulated the autophosphorylation reaction. We discuss the consequences of these effects as to in vivo conditions.