Determination of pseudocontact shifts of low-populated excited states by NMR chemical exchange saturation transfer.

Despite the critical roles of excited states in protein functions, they remain intractable for most structural studies because of their notably low populations. Chemical shifts for "invisible" states in slow exchange with the ground state are intuitively observed using nuclear magnetic resonance (NMR) chemical exchange saturation transfer (CEST) experiments. Here, we present a CEST NMR spectroscopy study for the observation of protein pseudocontact shifts (PCSs) of excited states, which carry valuable angular and distance information about these states. We first validate this approach, dubbed PCS-CEST, in the slow-exchange system of Abp1p SH3-Ark1p labeled with lanthanide ions, where the PCSs of the minor states resemble those of the holo-form ground state as expected. We further demonstrate that pre-existing folding transitional conformations of an FF domain exhibit remarkably lower PCS values than the ground state, which suggests that the low-populated ensemble is unfolded or largely unfolded. A higher resolution of PCSs of the minor states is achieved using our 1D selective CEST experiments. Thus, PCS-CEST provides an exquisite structural probe into the minor but functionally essential excited states.

Effects of highly hygroscopic excipients on the hydrolysis of simvastatin in tablet at high relative humidity.

Effects of highly hygroscopic sorbitol, citric acid, sodium carboxymethyl cellulose or polyvinylpolypyrrolidone, on the hydrolysis of simvastatin in tablets at 25°/90% RH were studied. The simvastatin tablets were prepared by direct powder compression. Simvastatin and its hydrolyte, simvastatin acid, were quantitatively analysed by high performance liquid chromotography. The hygroscopicity, water swelling ratio, water solubility and pH of the four hygroscopic excipients were investigated. During the investigation period, the weight gain of sorbitol or citric acid increased faster than that of polyvinylpolypyrrolidone or sodium carboxymethyl cellulose at 25°/90% RH, accordingly, the moisture sorption of the tablets containing citric acid or sorbitol (T-3 or T-6) were more than that of the tablets containing sodium carboxymethyl cellulose or polyvinylpolypyrrolidone (T-4 or T-5). The increase of simvastatin acid content with time at 25°/90% RH for the tablets was in the following order: T-6 < T-4 < T-3 < T-5. The effects of the four excipients on the hydrolysis of simvastatin in tablet were related to not only their hygroscopicity but also their other properties, such as moisture retention capacity and pH. Sorbitol as hygroscopic excipient in tablet can most effectively prevent the hydrolysis of simvastatin in tablet.

Large exchange bias after zero-field cooling from an unmagnetized state.

Exchange bias (EB) is usually observed in systems with an interface between different magnetic phases after field cooling. Here we report an unusual phenomenon in which a large EB can be observed in Ni-Mn-In bulk alloys after zero-field cooling from an unmagnetized state. We propose that this is related to the newly formed interface between different magnetic phases during the initial magnetization process. The magnetic unidirectional anisotropy, which is the origin of the EB effect, can be created isothermally below the blocking temperature.

Exploration of pressure-induced dissociation of pyruvate oxidase.

The dissociation of pyruvate oxidase (PO) caused by pressure up to 220 MPa at various conditions was explored by measuring the intrinsic fluorescence spectra and polarization. At 5 degrees C and pH 7.6 the standard volume change (deltaV0) and free energy upon dissociation of the enzyme is -220 ml/mol and 29.83 kCal/mol, respectively. It was found that FAD was irreversibly removed during the pressure-dissociation of the enzyme. A much smaller standard volume change (-153 ml/mol) and lower free energy (24.92 kCal/mol) of apo-pyruvate oxidase (apo-PO) compared with the native enzyme indicated that FAD played very important role in stabilizing the enzyme and significantly influenced the standard volume change. The substrate pyruvic acid can significantly stabilize the enzyme against pressure in spite the standard volume for the enzyme in this case has a big increase relative to the native enzyme. The comparison of the intrinsic fluorescence of the native and the activated enzyme obtained by limited proteolysis indicated that the physical separation of alpha-peptide from the enzyme only occurred when the subunits were dissociated from each other under pressure.

Exploration of RNA 3' terminal locations in rat ribosome.

The locations of the 3' ends of RNAs in rat ribosome were studied by a fluorescence-labeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3' ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe - fluorescein 5-thiosemicarbazide (FTSC), indicating that the 3' termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3' terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3' end of 5 S RNA was located on the interface of two subunits. However, no fluorescence-labeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3' end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3' ends of ribosomal RNAs including oxidation with NaIO4, reduction with KBH4 and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3' ends of RNAs were not involved in the translation activity of ribosome.

Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts.

Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti-inflammatory and anti-neoplastic actions in human. Its derivative, aspirin, is the most commonly used anti-inflammatory and analgesic drug. Aspirin and sodium salicylate (salicylates) have been reported to have multiple pharmacological actions. However, it is unclear whether they bind to a cellular protein. Here, we report for the first time the purification from human fibroblasts of a approximately 78 kDa salicylate binding protein with sequence identity to immunoglobulin heavy chain binding protein (BiP). The Kd values of SA binding to crude extract and to recombinant BiP were 45.2 and 54.6 microM, respectively. BiP is a chaperone protein containing a polypeptide binding site recognizing specific heptapeptide sequence and an ATP binding site. A heptapeptide with the specific sequence displaced SA binding in a concentration-dependent manner whereas a control heptapeptide did not. Salicylates inhibited ATPase activity stimulated by this specific heptapeptide but did not block ATP binding or induce BiP expression. These results indicate that salicylates bind specifically to the polypeptide binding site of BiP in human cells that may interfere with folding and transport of proteins important in inflammation.

Protein folding pathways of adenylate kinase from E. coli: hydrostatic pressure and stopped-flow studies.

Adenylate kinase (AKe) from E. coli is a small, single-chain, monomeric enzyme with no tryptophan and a single cysteine residue. We have constructed six single-Trp mutants of AKe to facilitate optical studies of these proteins and to specifically examine the interrelationship between their structure, function, dynamics, and folding reactions. In this study, the effects of hydrostatic pressure on the folding reactions of AKe were studied. The native structure of AKe was transformed to a non-native, yet pressure stable, conformation by hydrostatic pressure of about 300 MPa. This pressure lability of AKe is rather low for a monomeric protein and presumably may be attributed to substantial conformational flexibility and a correspondingly large volume change. The refolding of AKe after pressure-induced denaturation was reversible under ambient conditions. At low temperature (near 0 degrees C), the refolding process of pressure-exposed AKe mutants displayed a significant hysteresis. The observation of a slow refolding rate in the 193 region and a faster folding rate around the active site (86, 41, 73 regions) leads us to suggest that in the folding process, priority is afforded to functional regions. The slow structural return of the 193 region apparently does not hinder the more rapid return of enzymatic activity of AKe. Circular dichroism studies on the pressure-denatured Y193W mutant show that the secondary structure (calculated from far-UV spectra) returned at a rapid rate, but the tertiary structure alignment (calculated from near-UV spectra) around the 193 region occurred more slowly at rates comparable to those detected by fluorescence intensity. Denaturation of AKe mutants by guanidine hydrochloride and subsequent refolding experiments were also consistent with a much slower refolding process around the 193 region than near the active site. Fast refolding kinetic traces were observed in F86W, S41W, and A73W mutants using a fluorescence detection stopped-flow rapid mixing device, while only a slow kinetic trace was observed for Y193W. The results suggest that the differences in regional folding rates of AKe are not derived from the specific denaturation methods, but rather are inherent in the structural organization of the protein.

Eukaryotic elongation factor 2 can bind to the synthetic oligoribonucleotide that mimics sarcin/ricin domain of rat 28S ribosomal RNA.

Eukaryotic elongation factor 2 (eEF2) catalyzes the translocation of peptidyl-tRNA from the A site to P site by binding to the ribosome. In this work, the complex formation of rat liver eEF2 with a synthetic oligoribonucleotide (SRD RNA) that mimics sarcin/ricin domain of rat 28S ribosomal RNA is invested in vitro. Purified eEF2 can specifically bind SRD RNA to form a stable complex. tRNA competes with SRD RNA in binding to eEF2 in a less extent. Pretreatment of eEF2 with GDP or ADP-ribosylation of eEF2 by diphtheria toxin can obviously reduce the ability of eEF2 to form the complex with the synthetic oligoribonucleotide. These results indicate that eEF2 is likely to bind directly to the sarcin/ricin domain of 28S ribosomal RNA in the process of protein synthesis.

Non-specific deadenylation and deguanylation of naked RNA catalyzed by ricin under acidic condition.

Ricin A-chain catalyzes the hydrolysis of the N-glycosidic bond of a conserved adenosine residue at position 4324 in the sarcin/ricin domain of 28S RNA of rat ribosome. The GAGA tetraloop closed by C-G pairs is required for recognition of the cleavage site on 28S ribosomal RNA by ricin A-chain. In this study, ricin A-chain (reduced ricin) exhibits specific depurination on a synthetic oligoribonucleotide (named SRD RNA) mimic of the sarcin/ricin domain of rat 28S ribosomal RNA under neutral and weak acidic conditions. Furthermore, the activity of intact ricin is also similar to that of ricin A-chain. However, under more acidic conditions, both enzymes lose their site specificity. The alteration in specificity of depurination is not dependent on the GAGA tetraloop of SRD RNA. A higher concentration of KCl inhibits the non-specific N-glycosidase activity much more than the specific activity of ricin A-chain. In addition, characterization of depurination sites by RNA sequencing reveals that under acidic conditions ricin A-chain can release not only adenines, but also guanines from SRD RNA or 5S ribosomal RNA. This is the first report of the non-specific deadenylation and deguanylation activity of ricin A-chain to the naked RNA under acidic conditions.

Pressure-exploration of the 33-kDa protein from the spinach photosystem II particle.

The 33-kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high-pressure protein-unfolding. The protein has a very low free energy as previously reported by chemical unfolding studies, suggesting that it must be easy to modulate its unfolding transition by rather mild pressure. Moreover, the protein molecule consists of only one tryptophan residue (Trp241) and eight tyrosine residues, which can be conveniently used to probe the protein conformation and structural changes under pressure using either fluorescence spectroscopy or fourth derivative UV absorbance spectroscopy. The different experimental methods used in the present study indicate that at 20 degrees C and pH 6, the 33-kDa protein shows a reversible two-state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most proteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (DeltaGo) and in volume (DeltaV) for the transition at pH 6.0 and 20 degrees C are -14.6 kJ.mol-1 and -120 mL.mol-1, respectively, in which the DeltaGo value is consistent with that obtained by chemical denaturation. We found that pressure-induced protein unfolding is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of DeltaGo (only a minor variation was observed for the DeltaV value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in DeltaV without any significant changes in DeltaGo. It was also found that NaCl significantly protects the protein from pressure-induced unfolding. In the presence of 1 M NaCl, the pressure needed to induce the half-unfold of the protein is shifted to a higher value (shift of 75 MPa) in comparison with that observed without NaCl. Interestingly, in the presence of NaCl, the value of DeltaV is significantly reduced whilst that of DeltaGo remains as before. The unfolding-refolding kinetics of the protein has also been studied by pressure-jump, in which it was revealed that both reactions are a two-state transition process with a relatively slow relaxation time of about 102 s.

Solution structure of the second extracellular loop of human thromboxane A2 receptor.

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.

Non-specific depurination activity of saporin-S6, a ribosome-inactivating protein, under acidic conditions.

Among five ribosome-inactivating proteins tested only saporin-S6 could efficiently release the adenine from adenosine 20 of the synthetic oligoribonucleotide (SRD RNA) mimic of the sarcin/ricin domain of rat 28S rRNA with a Km of 9 microM and a kcat of approximately 0.4 min(-1) at pH 7.6. The optimal pH for the depurination activity of saporin-S6 is 5.0. However, saporin-S6 lost its site-specificity of depurination on SRD RNA around the optimal pH. The non-specific depurination activity of saporin-S6 was dependent on the enzyme concentration and pH conditions. These results are valuable to understand the diversity and the depurination mechanism of ribosome-inactivating proteins.

Identification of the substrate interaction site in the N-terminal membrane anchor segment of thromboxane A2 synthase by determination of its substrate analog conformational changes using high resolution NMR technique.

The present studies describe an investigation for the interaction of N-terminal membrane anchor domain of thromboxane A(2) synthase (TXAS) with its substrate analog in a membrane-bound environment using the two-dimensional NMR technique. TXAS and prostaglandin I(2) synthase (PGIS), respectively, convert the same substrate, prostaglandin H(2) (PGH(2)), to thromboxane A(2) and prostaglandin I(2), which have opposite biological functions. Our topology studies have indicated that the N-terminal region of TXAS has a longer N-terminal endoplasmic reticulum (ER) membrane anchor region compared with the same segment proposed for PGIS. The differences in their interaction with the ER membrane may have an important impact to facilitate their common substrate, PGH(2), across the membrane into their active sites from the luminal to the cytoplasmic side of the ER. To test this hypothesis, we first investigated the interaction of the TXAS N-terminal membrane anchor domain with its substrate analog. A synthetic peptide corresponding to the N-terminal membrane anchor domain (residues 1-35) of TXAS, which adopted a stable helical structure and exhibited a membrane anchor function in the membrane-bound environment, was used to interact with a stable PGH(2) analog,. High resolution two-dimensional NMR experiments, NOESY and TOCSY, were performed to solve the solution structures of in a membrane-mimicking environment using dodecylphosphocholine micelles. Different conformations were clearly observed in the presence and absence of the TXAS N-terminal membrane anchor domain. Through combination of the two-dimensional NMR experiments, completed (1)H NMR assignments of were obtained, and the data were used to construct three-dimensional structures of in H(2)O and dodecylphosphocholine micelles, showing the detailed conformation change upon the interaction with the membrane anchor domain. The observation supported the presence of a substrate interaction site in the N-terminal region. The combination of the structural information of and was able to simulate a solution structure of the unstable TXAS and PGIS substrate, PGH(2).

Effects of hydrostatic pressure on the structure and biological activity of infectious bursal disease virus.

The effects of high hydrostatic pressure on the structure and biological activity of infectious bursal disease virus (IBDV), a commercially important pathogen of chickens, were investigated. IBDV was completely dissociated into subunits at a pressure of 240 MPa and 0 degrees C revealed by the change in intrinsic fluorescence spectrum and light scattering. The dissociation of IBDV showed abnormal concentration dependence as observed for some other viruses. Electron microscopy study showed that morphology of IBDV had an obvious change after pressure treatment at 0 degrees C. It was found that elevating pressure destroyed the infectivity of IBDV, and a completely pressure-inactivated IBDV could be obtained under proper conditions. The pressure-inactivated IBDV retained the original immunogenic properties and could elicit high titers of virus neutralizing antibodies. These results indicate that hydrostatic pressure provides a potential physical means to prepare antiviral vaccine.

Topology of catalytic portion of prostaglandin I(2) synthase: identification by molecular modeling-guided site-specific antibodies.

Prostaglandin I(2) synthase (PGIS) is an eicosanoid-synthesizing cytochrome P450, located in the endoplasmic reticulum (ER) membrane. The membrane topology of the catalytic portion of PGIS is still unknown. General models of the membrane topology of microsomal P450s have been proposed in two forms: (a) large part of the polypeptide exposed on the cytoplasmic side with an NH(2)-terminal membrane anchor to the ER membrane and (b) deep immersion of the polypeptide in the membrane, as described by J. P. Miller et al. (1996, Biochemistry 35, 1466-1474). We have characterized the membrane topology of catalytic portion of PGIS using molecular modeling-guided site-specific antibodies. A 3D working model of PGIS was constructed by homology modeling using P450(BM-3) crystal structure as a template (S. K. Shyue et al., 1997, J. Biol. Chem. 272, 3657-3662). Three hydrophilic peptides corresponding to different regions of the surface portion of PGIS with residues 109-127 (P109-127), 353-368 (P353-368), and 411-431 (P411-431) predicted from the model and an NH(2)-terminal hydrophobic peptide (residues 1-28, P1-28) were synthesized and used to prepare site-specific antibodies. All three of the hydrophilic peptide antibodies have high titer and are specifically recognized human PGIS, as shown by binding assays and Western blot analysis. In contrast, the hydrophobic NH(2)-terminal peptide has a much lower titer binding to the PGIS protein. The overall arrangement of the PGIS polypeptide with respect to the endoplasmic reticulum (ER) membrane was examined by immunocytochemistry techniques in transiently transfected COS-1 cells with recombinant human PGIS cDNA and in ECV cells expressing endogenous PGIS. The immunofluorescence staining for the cells with selective permeabilization of the plasma membrane using streptolysin O indicated that all three of the hydrophilic peptide antibodies bound to the cytoplasmic surface of the ER membrane. These results provide direct experimental evidence supporting the predicted 3D protein topological model in which the segments are located on the protein surface and the membrane topological model in which PGIS is largely exposed on the cytoplasmic side of the ER membrane. It also led us to conclude that the PGIS substrate, prostaglandin H(2) (PGH(2)), produced by prostaglandin H(2) synthase (PGHS) in the ER lumenal side must pass through the ER membrane barrier to the catalytic site of the PGIS in the cytoplasmic side of the ER membrane.

Pressure effects on tryptophan and its derivatives.

The high pressure effects on fluorescence of free tryptophan (Trp) and its derivatives, N-acetyl-tryptophan (AT), N-acetyl-tryptophanamide (NATA), tryptophanamide (TA), and tryptophan, containing 6-polypeptides in aqueous solution, were investigated in a pressure range from 0.1 to 650 MPa. It was found by analyzing the center of spectral mass in the wavelength range from 300 to 450 nm that high pressure shifted the fluorescence spectra of all these species to red direction: 421 cm(-1) for Trp, 305 cm(-1) for AT, 310 cm(-1) for NATA, 265 cm(-1) for TA, and 220 cm(-1) for single tryptophan containing 6-polypeptides. All the fluorescence efficiencies (i.e., quantum yield) of the compounds were reduced with pressure except free tryptophan where its fluorescence efficiency was enhanced with pressure. Glycerol, ethanol, and pH obviously influenced the pressure effects on their fluorescence characteristics. Since the tryptophan fluorescence is usually used as a probe for protein structural investigation, these findings suggested that the intrinsic pressure effect on tryptophan (or its derivatives) must be taken in consideration to explain the phenomenon observed in high pressure study on biomolecules when using the usual fluorospectroscopic approaches. In the present investigation, the mechanisms involved for pressure effects on tryptophan and its derivatives were explored and discussed.

Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin.

The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.

Cryoinactivation and conformational drift of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle.

The cryoinactivation of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle (GAPDH-rabbit) was studied. It was found that the inactivation of GAPDH-rabbit at 0 degrees C was much faster than that of GAPDH from yeasts, and showed obvious time and concentration dependence. The spectral properties, enzyme activity and behavior under pressure, of GAPDH-rabbit treated either by cryoinactivation, or pressure-induced dissociation and reassociation, were very similar. These results provided evidence to support the idea that cryoinactivation of oligomeric proteins, might take place through a cycle of dissociation-reassociation accompanied with the so-called conformational drift postulated by King and Weber (1986).

DNA-cleaving activity of superoxide dismutase specific for circular supercoiled double-stranded DNA in vitro.

Several superoxide dismutases (SODs), N. tabacum Mn-SOD, porcine erythrocyte Cu/Zn-SOD and bovine erythrocyte Cu/Zn-SOD, have been found to exhibit the activity to cleave the circular supercoiled double-stranded DNA into nicked and further linear form in vitro. The fact that porcine erythrocyte Cu/Zn-SOD did not cleave the linear double-stranded DNA excluded the possibility that nuclease contaminated the SOD preparations and showing the cleaving activity was dependent on the supercoiled form of DNA. Porcine erythrocyte Cu/Zn-SOD inactivated by H2O2 or guanidine still remained its supercoiled DNA-cleaving activity. However, when the SOD was digested with proteases, its activity to cleave supercoiled DNA was completely abolished. These results suggested that the supercoiled DNA-cleaving activity was relative to the apoenzyme moiety, less relevant to the O2- dismutation site of SOD. The enzymatic mechanism of cleaving activity of SOD to supercoiled DNA was discussed.

Beta-adrenergic and fibroblast growth factor receptors induce neuronal process outgrowth through different mechanisms.

The mechanisms that initiate and direct neuronal process formation remain poorly understood. We have recently described a neuronal progenitor cell line, AS583-8.E4.22 (AS583-8) which undergoes neurite formation in response to beta2-adrenergic and basic fibroblast growth factor (bFGF) receptor activation [Kwon, J.H. et al., (1996) Eur. J. Neurosci., 8, 2042-2055]. In the present study, a comparison of these responses revealed that isoproterenol (ISO), a beta-adrenergic receptor agonist, induces multiple, highly branched processes within 30 min while bFGF induces fewer, unbranched processes within 24 h. In contrast to the ISO response, bFGF induces mitogen-activated protein kinase activation and c-fos expression in the cell line and results in neurite outgrowth that is dependent on new mRNA and protein synthesis. Two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cytoskeletal preparations revealed different patterns following ISO vs. bFGF exposure suggesting selective changes in protein expression and/or post-translational modifications. Immunoblot analysis of these preparations for beta-tubulin, tyrosinated alpha-tubulin and acetylated alpha-tubulin also revealed different patterns following each type of treatment. Follow-up confocal microscopy revealed that following ISO, the distribution of tyrosinated tubulin extends to the distal ends of processes whereas acetylated alpha-tubulin is diminished within distal ends. This pattern has been reported to be associated with enhanced microtubule dynamics, a state in which process outgrowth is facilitated. In contrast, following bFGF treatment the distributions of tyrosinated and acetylated alpha-tubulin were identical, a state associated with a diminution of microtubule dynamics. These results, a different time course of neurite formation, dependency on new gene expression and differential expression and cellular distribution of major cytoskeleton proteins suggest that neurite outgrowth induced by ISO vs. bFGF is mediated by two distinct intracellular effector mechanisms in AS583-8 cells. In addition, studies, using the differential distribution of post-translational modified alpha-tubulins in neurites of primary neuronal cultures as marker for the two distinct processes of neurite formation suggest, that similar mechanisms are present in vivo. Therefore, the AS583-8 cell line provides a useful model to study these signalling mechanisms that couple neurotransmitter and growth factor receptor activation to the cytoskeletal changes that mediate neurite formation.