Vanadium stimulates human bronchial epithelial cells to produce heparin-binding epidermal growth factor-like growth factor: a mitogen for lung fibroblasts.

The bronchial epithelium is a potential source of growth factors that could mediate airway fibrosis during the progression of diseases such as asthma and chronic bronchitis. We report that conditioned medium (CM) from normal human bronchial epithelial cells (NHBECs) contains mitogenic activity for human lung fibroblasts that is blocked by the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitor AG1478 and by neutralizing antibodies raised against heparin-binding epidermal growth factor-like growth factor (HB-EGF). Neutralizing antibodies against other EGF-R ligands (EGF and transforming growth factor-alpha) or other antibodies against growth factors (platelet-derived growth factors, insulin-like growth factor-1) had no affect on the mitogenic activity of NHBEC-CM. HB-EGF messenger RNA (mRNA) expression in NHBEC was detected by reverse transcriptase/polymerase chain reaction and Northern blot analysis. HB-EGF protein was detected by enzyme-linked immunosorbent assay. Vanadium pentoxide (V2O5), a fibrogenic metal associated with occupational asthma, caused a several-fold increase in HB-EGF mRNA expression and protein, whereas the inert metal titanium dioxide had no effect on HB-EGF expression. V2O5-induced HB-EGF mRNA expression was inhibited by the EGF-R tyrosine kinase inhibitor AG1478, the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580, and the MAP kinase kinase inhibitor PD98059. Finally, HB-EGF induced the production of fibroblast growth factor (FGF)-2 by human lung fibroblasts and anti-FGF-2 antibody partially blocked the mitogenic activity of NHBEC-CM on fibroblasts. These data suggest that HB-EGF is a fibroblast mitogen produced by NHBECs and that induction of an FGF-2 autocrine loop in fibroblasts by HB-EGF accounts for part of this mitogenic activity.

Asparagine-344 is a key residue for ligand binding in keratinocyte growth factor receptor.

The membrane proximal, immunoglobulin- (Ig-) like domain 3 of KGFR shows significant sequence similarity to the Ig light chain variable (V) domain. According to our model, based on this similarity, the F-G loop in KGFR corresponds to the complementarity determining region (CDR) 3 of the Ig V domain. The F-G loop in the membrane proximal domain of the keratinocyte growth factor receptor has previously been shown to participate in determining the FGF ligand binding specificity of KGFR [Gray, T. E., Eisenstein, M., Shimon, T., Givol, D., & Yayon, A. (1995) Biochemistry 34, 10325-10333]. Here, we report the effects of additional mutations in this F-G loop. Both a single mutant KGFR Q348-->I and a double mutant KGFR Q348-->I, Q351-->H are found to have relatively mild effects on ligand binding, as was previously found for three other F-G loop mutant receptors. In contrast, a single mutation N344-->A in the F-G loop of KGFR is sufficient to abolish essentially all affinity of this receptor for its primary ligand KGF, while some affinity for aFGF is retained. Asparagine-344 is, therefore, essential for ligand binding by KGFR. We discuss the likelihood of this effect being due to global or local structural changes or to the removal of a specific interaction with the ligand, in relation to various known and model structures. Taking into account the mild effects of other mutations in the region and various other considerations, we tend to favor the idea that asparagine-344 is a key residue in determining the local conformation of the F-G loop.

Quantitation of mucin RNA by PCR reveals induction of both MUC2 and MUC5AC mRNA levels by retinoids.

The polydispersity of most human secretory mucin messages has made them difficult to detect specifically and quantitatively, impeding the evaluation of the relative expression of the various mucin genes and their role in normal and pathological conditions. For this reason, we developed competitive reverse transcriptase-polymerase chain reaction (PCR) methods to measure the airway mucins MUC2 and MUC5AC. Oligonucleotide pairs were designed that specifically detect MUC2 and MUC5AC, as demonstrated by the size and sequence of the PCR product and the expected tissue distribution. The mucin oligonucleotide primers were used to synthesize internal competitive standards, called MIMIC. Using this assay, the relative expression of these messages was analyzed in retinoid-replete or -deprived cultures of normal human tracheobronchial epithelial (NHTBE) cells. Retinoid deficiency induces squamous metaplasia in vivo and in vitro. Consistent with these observations and in contrast to a previous report, retinoid-deprived cultures produced at least an order of magnitude less MUC2 and MUC5AC message than retinoid-replete cultures. In summary, this paper describes methodology that can be applied to the specific and quantitative measurement of mucin messages and demonstrates that, in NHTBE cells, the level of MUC2 and MUC5AC mRNA is increased by retinoids.

Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells.

The goal of our studies was to establish procedures for subculturing normal human tracheobronchial epithelial (NHTBE) cells without compromising their ability to differentiate into mucous and ciliated cells (i.e., differentiation competence) and to study the regulation of airway secretions by epidermal growth factor (EGF) and retinoic acid (RA). Primary NHTBE cells were obtained from a commercial source and subcultured repeatedly in serum-free medium on plastic tissue culture dishes. The subcultured cells were tested after every passage for differentiation competence in air-liquid interface (ALI) cultures. The apical secretions of cultured NHTBE cells were characterized by immunoblotting, Western blotting, or enzyme-linked immunosorbent assay using a variety of antibodies. They contained mucin-like materials as well as lysozyme, lactoferrin, and secretory leukocyte protease inhibitor (SLPI). We found that an EGF concentration of 25 ng/ml, which is commonly used in airway cell cultures, adversely affected growth, mucin production, and morphology of ALI cultures and that RA was essential for mucociliary differentiation. Without RA, the epithelium became squamous and mucin secretions decreased 300- to 900-fold. In contrast, secretion of lysozyme, lactoferrin, and SLPI was significantly increased in RA-depleted cultures. Cells of passage 2 (P-2) through P-4 remained competent to differentiate into mucous and ciliated cells when grown in ALI cultures. However, mucin secretion and ciliagenesis decreased in P-3 and P-4 cell cultures and P-3 but not P-4 cell cultures exhibited bioelectric properties characteristic of airway epithelium. We concluded that P-2 and P-3 NHTBE cell cultures retain many important features of normal airway epithelium. This enables one to conduct many studies of airway cell biology with a greatly expanded (6,000-fold) cell pool.

Molecular modeling based mutagenesis defines ligand binding and specificity determining regions of fibroblast growth factor receptors.

The fibroblast growth factor receptor 2 (FGFR2) and the keratinocyte growth factor receptor (KGFR) have different ligand binding specificities despite differing only in the second half of their immunoglobulin-like (Ig-like) domain III. Three-dimensional model structures were generated for domain III on the basis of variable (V) Ig domains. The region that differs between the two receptors is predicted to include two loops: one connects beta-strands F-G and is analogous to the complementarity determining region 3 (CDR3) of immunoglobulins; the other connects beta-strands D-E. These regions were targeted for mutagenesis. Single mutations in the F-G loop were found to only slightly alter ligand binding, whereas a double mutant, KGFR Y345-->S,Q348-->I, acquired significant affinity for bFGF. Notably, the affinity of this double mutant KGFR for KGF and aFGF was essentially unaltered. A mutant FGFR2, in which the D-E beta-hairpin (T319TDKEI) is replaced with the KGFR D-E beta-hairpin (S319SNA), has 9-fold reduced affinity for bFGF. These results demonstrate that the F-G or CDR3 analogous loop in FGFRs plays a key role in determining ligand binding and specificity. In addition, however, the protein loop connecting beta-strands D and E may also be involved in ligand binding. Several point mutations in FGFR2, shown recently to give rise to multiple inherited skeletal defects, are localized according to our models to the F-G or D-E loops of domain III. Our results strongly suggest that these naturally occurring mutations specifically alter ligand binding by FGFR2.

Regulation of ciliated cell differentiation in cultures of rat tracheal epithelial cells.

The cellular pathway of ciliated cell differentiation and its regulation is poorly defined. To begin to understand the process of ciliated cell differentiation, we sought to identify factors regulating ciliated cell development in vitro. Rat tracheal epithelial (RTE) cells were cultured on collagen gel-coated membranes at an air-liquid interface in hormone- and growth factor-supplemented medium (complete medium [CM]). Under these conditions, RTE cells first proliferate and then differentiate into a pseudostratified mucociliary epithelium. Ciliated cell differentiation was measured using a monoclonal antibody, RTE3, which was shown to specifically react with the plasma membrane of ciliated cells. Cultures were immunostained in situ, and the percentage of the culture surface covered with ciliated cells was estimated using videomicroscopy and an image analysis program. If an air-liquid interface was not created and the cells were maintained in the submerged state, ciliated cell differentiation was suppressed 25-fold. Culture in the absence of mitogenic components present in CM, including epidermal growth factor (EGF), cholera toxin (CT), or bovine pituitary extract, resulted in 2- to 4-fold increases in the percentage of ciliated cells. When both EGF and CT were removed from the media, DNA synthesis and total cell number was reduced, while ciliated cell differentiation increased as much as 5-fold. These results demonstrate that submersion inhibits, while withdrawal of mitogenic compounds promotes, ciliated cell differentiation in vitro.

Ligand binding by fibroblast growth factor receptors investigated using chimeric receptor molecules.

In order to map in detail the ligand binding sites of fibroblast growth factor receptor 2 (FGFR2) and keratinocyte growth factor receptor (KGFR), we have generated receptor molecules that are chimeric within the membrane proximal sequence that varies between them. The chimeric molecules are found to bind aFGF with a greater than 5-fold difference in affinity, indicating that there is coupling between the chimeric regions with respect to aFGF binding. Further, binding of bFGF and KGF is abolished in the chimeras, showing that the binding site for these ligands requires the whole of the 48- or 50- amino acid variable sequence to be intact. Direct interactions between the different regions exchanged in the chimeras are most probably involved in forming KGF or bFGF binding sites.

Regulation of transformation frequency by exogenous and endogenous growth factors in rat tracheal epithelial cells.

The purpose of our studies was to re-evaluate the rat tracheal epithelial (RTE) transformation system and to identify critical variables that affect the development of enhanced growth variants (EGV). The enhanced growth variant colony, which is a preneoplastic cell variant, is the quantifiable transformation endpoint in RTE cultures. Using a standard protocol the frequency of EGV colony formation was shown to be inversely related to the number of clonogenic cells (CFU) seeded per dish in control cultures as well as in cultures treated with the transforming agent 6-nitrochrysene (6-NC). Experiments showed that the major mechanisms that underlie the CFU density-dependent inhibition of EGV colony formation are depletion of growth factors from and accumulation of autocrine TGF-beta in the media. Thus the cells themselves are creating the selection environment, which allows only the EGVs to survive. The effects of agents such as 6-NC, which increase the frequency of EGV colony formation, are to induce a cellular phenotype that is less susceptible to the selection environment. We showed that TGF-beta-neutralizing antibodies added to the selection media significantly increased EGV colony formation in control cultures but not in 6-NC-exposed cultures. In addition we demonstrated that the development of EGV colonies is much less susceptible to inhibition by (exogenous) TGF-beta in 6-NC-exposed than in control cultures. Thus spontaneous and 6-NC EGV colony formation are distinguishable based on TGF-beta sensitivity. To conduct quantitative cell transformation experiments with RTE cells it is essential that the number of surviving CFU per dish is the same in control and treated cultures. Under the conditions used in the studies described here, 350-500 CFU per culture was found to be the optimum CFU density. Besides 6-NC, agents that have been shown to increase EGV colony frequency under conditions similar to those described here are nitrosamines, NNK, nickel compounds and X-rays.

Fluvoxamine. A review of its safety profile in world-wide studies.

Fluvoxamine, a selective serotonin reuptake inhibitor, was studied extensively in 34,587 predominantly depressed patients in 66 studies conducted world-wide. These studies were largely uncontrolled trials representing the use of fluvoxamine by psychiatric and general practice physicians in everyday conditions. The safety data were analyzed according to standardized medical review and data management policies. Approximately 70% of the fluvoxamine population were female and 44% were aged 31-51 years. The modal total daily dose was 100 mg. Safety findings revealed a pharmacological adverse event profile similar to that seen with other serotonin reuptake inhibitors. Nausea was found to be the only common symptom, with an incidence rate of 16%. Approximately 2% of the fluvoxamine population reported at least one serious adverse event (per FDA criteria). Overall suicidality rates of fluvoxamine were found to be low (0.7%). No cases of zimelidine syndrome, bleeding syndrome or Guillain-Barré syndrome were identified. Overall, fluvoxamine was found to be safe and well tolerated suggesting a favorable alternative in the treatment of depression.

Characterization of a third transforming growth factor beta 1 transcript in rat tracheal epithelial cells.

It has been previously reported (R. W. Steigerwalt et al., Mol. Carcinog., 5:32-40, 1992) that primary cultures of rat tracheal epithelial (RTE) cells and immortalized RTE cell lines produce three mRNA transcripts [2.5, 1.9, 1.4 kilobases (kb)] which hybridize to a murine transforming growth factor beta 1 (TGF-beta 1) complementary DNA probe. In this report, we show that the 1.9- and 1.4-kb transcripts are not detectable by Northern analysis of resting adult trachea but are induced in regenerating tracheal grafts and tumors formed from transformed RTE cells. Northern analysis of the TGF-beta 1 transcripts with subclones of the murine complementary DNA demonstrate that the 1.4-kb transcript lacks much of the 5' untranslated region (UTR). RNase protection analysis was used to map the transcriptional start site of the 1.4-kb transcript to within 30-40 base pairs of the first ATG codon. No differences in the coding or 3' UTR were detected between the 1.4-kb and the 2.5-kb transcripts. Although RTE cells produce a 1.9-kb TGF-beta 1 mRNA, we were unable to detect a previously reported unique 3' UTR, which we found to be almost identical to a rat mitochondrial ATPase sequence. Because the 1.4-kb transcript is missing most of the long GC-rich 5' UTR, it may be translated at a different rate than the 2.5- and 1.9-kb transcripts, or it may code for an intracellular form of TGF-beta 1. The 1.4-kb transcript has been observed under several conditions of injury or stress and, therefore, may be an important component of the TGF-beta 1 response to these conditions.

Enhanced growth potential of cultured rabbit tracheal epithelial cells following exposure to N-methyl-N'-nitro-N-nitrosoguanidine.

To establish a standardized model for the transformation of rabbit airway epithelial cells, we attempted to transform rabbit tracheal epithelial (RbTE) cells in culture with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). RbTE cells, harvested by enzymatic digestion from male New Zealand white rabbits, were plated onto feeder layers of irradiated 3T3 cells. Control cells proliferated exponentially during the 2nd week of culture and reached the plateau phase by the 3rd week. Cells exposed to MNNG (0.1 microgram/ml) proliferated in a fashion similar to the control cells, except that there was some delay before proliferation began. The clonogenic activity of RbTE cells rapidly decreased in parallel with the increase in cell population equally in the control and MNNG groups. During the late plateau phase, cells exposed to MNNG regained clonogenic activity, and this compartment size expanded with time, whereas the clonogenic activity in control cultures remained below the detectable level. In RbTE cell cultures exposed three times to 0.1 microgram/ml MNNG, large, persistent and proliferating colonies emerged at a frequency of 1-3 x 10(-2) among the surviving clones, whereas all the control cultures eventually became senescent. The MNNG-induced alteration in the growth potential of RbTE cells, i.e., the extended lifespan, and the maintenance and even expansion of clonogenic activity, was similar to that of transformed rat tracheal epithelial cells. However, no immortal cell line could be established from these growth-altered RbTE cells. We therefore concluded that the growth-altered RbTE cells were partially transformed.

Refolding of barnase mutants and pro-barnase in the presence and absence of GroEL.

Three mutants of barnase and a pro-barnase variant, which have a variety of different physical properties but the same overall protein structure, were analysed for their folding in the presence of the molecular chaperone GroEL. Mutants were chosen on the basis that changes in their refolding rate constants in solution are not correlated with the changes in their stability. All barnase variants fold considerably more slowly when bound to GroEL. However, barnase refolding on GroEL parallels that in solution: there is a linear relationship between the refolding rate constants, obtained for wild-type and all mutants of barnase, in the presence and absence of GroEL. Barnase is synthesized in vivo with a 13 amino acid pro-sequence attached to the N-terminus. The pro-sequence of pro-barnase is shown by NMR spectroscopy to be devoid of defined structure. The presence of this pro-sequence has no effect on the overall refolding rate constant or the activity of barnase. In the presence of GroEL, the refolding of pro-barnase is retarded relatively more strongly than that of wild-type and the mutant barnase proteins, suggesting that the pro-sequence provides additional binding sites for the chaperone.

Refolding of barnase in the presence of GroE.

The refolding of barnase in the presence of GroEL has been monitored on the millisecond to seconds time scale using stopped-flow kinetics. GroEL binds rapidly and tightly to the denatured enzyme with a second-order rate constant of greater than 1.3 x 10(8) s-1 M-1 and slows down greatly the rate of barnase refolding. However, addition of ever increasing concentrations of GroEL does not prevent barnase refolding completely, as would be expected from mass action if folding of barnase could proceed only in free solution. At saturating concentrations of GroEL, barnase refolds with a half-life of 30 s, compared with 50 ms for refolding of free enzyme. The rate-determining step in the refolding of free barnase is the reaction of a "late" folding intermediate. A mutant of barnase that fold more slowly (Ser-->Ala91), refolds at a correspondingly lower rate when bound to GroEL, suggesting that formation of the fully folded state may be rate limiting for folding on GroEL. For the slow-folding Ser-->Ala91 mutant, the rate-determining refolding step has a half-life of 180 ms. In sequential mixing experiments, a delay was introduced to allow the Ser-->Ala91 mutant to refold for 30 ms before being mixed with GroEL. This reduces by 50% the amount of mutant barnase initially bound by GroEL. As only 11% of this mutant barnase is fully refolded from the late intermediate in 30 ms, there is preferential binding of an earlier refolding state to GroEL. We show by single mixing experiments that binding, not hydrolysis, of ATP reduces the lag in regain of barnase activity seen with GroEL alone. In the presence of high concentrations of ATP and GroEL the rate constant for refolding of barnase approaches that found in their absence, probably because ATP reduces the affinity of GroEL for refolding barnase, such that bound barnase is released and refolds unhindered. The addition of exceedingly small quantities of GroES in the presence of excess GroEL and a moderate amount of ATP also has a marked effect on the barnase refolding rate constant, suggesting that GroES may have higher affinity for the barnase: GroEL complex than for GroEL.

Safety database on fluvoxamine: analysis and report.

A review was conducted of the safety and tolerability of fluvoxamine in 54 worldwide marketing studies that enrolled 24,624 patients, the majority of whom were treated with fluvoxamine in uncontrolled studies in depression. In accordance with the general epidemiologic distribution of depressive disorder, female patients and patients aged between 30 and 50 years predominated. The majority of patients were treated for 6 weeks, the most frequent, or modal, total daily dose being 100 mg. Overall, 57.4% of the patients exposed to fluvoxamine did not have any adverse experiences. The greatest proportion of adverse experiences, as defined using COSTART body systems, affected the digestive system (24.1%), the nervous system (23.7%), and the body as a whole (15.3%). The only adverse experience with an incidence greater than 10% was nausea (15.7%); somnolence (6.9%) and asthenia (6.2%) were the next most frequent adverse experiences. Notably, the rates of agitation and anxiety were only 1.4% and 1.3%, respectively. The incidences of adverse experiences generally increased with age and were slightly higher in females than in males. In total, 15.1% of patients discontinued treatment prematurely as a result of adverse experiences, principally nausea, dizziness, vomiting, somnolence, abdominal pain, and headache. The overall incidence of serious adverse events in association with fluvoxamine treatment was 2.5% when U.S. Food and Drug Administration criteria and the most conservative approach, without causality judgments, were used.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of sodium selenite on cell proliferation and transformation of primary rat tracheal epithelial cells.

The effects of sodium selenite (Na2SeO3) on cell proliferation and the development of preneoplastic transformed variants were studied in primary cultures of rat tracheal epithelial cells. Results revealed a biphasic effect of Na2SeO3 on cell proliferation: at concentrations between 6 x 10(-8) and 6 x 10(-6) M, it stimulated and at concentrations of approximately 2 x 10(-5) and above it inhibited cell proliferation (presumably due to toxicity). Nontoxic concentrations of Na2SeO3 (6 x 10(-8) -6 x 10(-7) M) significantly reduced the spontaneous transformation frequency. Transformation induced by the tobacco-specific nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was effectively inhibited by nontoxic as well as toxic concentrations of Na2SeO3. Treatment of cultures with Na2SeO3 after cessation of NNK exposure, i.e. during the selection period, also significantly reduced the transformation frequency. These experiments show that the inhibition of transformation by Na2SeO3 is not the result of an antiproliferative effect. They further indicate that the inhibitory effect occurs even when the chemical treatment occurs during the 'postinitiation' phase. Thus the inhibition of transformation by Na2SeO3 cannot solely be explained by its effects on drug metabolism.

Review of fluvoxamine safety database.

A review of the safety and tolerability of fluvoxamine in worldwide marketing studies involving 24,624 patients, predominantly receiving fluvoxamine treatment in uncontrolled studies in depression, has been conducted. There was a marked preponderance of female patients and patients aged between 30 and 50 years. The majority of patients were treated for 6 weeks, with the most frequent modal total daily dose being 100mg. The greatest proportion of adverse experiences occurring, by COSTART body system, affected the digestive system (24.1%), the nervous system (23.7%), and the body as a whole (15.3%). The only adverse experience with an incidence greater than 10% was nausea (15.7%), with somnolence (6.9%) and asthenia (6.2%) as the next most frequent experiences. Notably, the rates of agitation and anxiety were only 1.4 and 1.3%, respectively. The incidences of adverse experiences increased with age, and were slightly higher in females than males. 15.1% of patients discontinued treatment prematurely as a result of adverse experiences, principally nausea, dizziness, vomiting, somnolence, abdominal pain, and headache. The overall incidence of serious adverse events associated with fluvoxamine treatment was 2.5%, and the incidence of overall suicidality, including suicidal ideation, overdose, and intentional overdose as well as attempted and completed acts of suicide, was remarkably low at 0.8%.

Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells.

The cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied by the assays of colony-forming efficiency (CFE), micronucleus formation (MN), and cell transformation in rat tracheal epithelial (RTE) cells both in vitro and in vivo. Liver S9, primary hepatocytes and RTE cells from normal and Aroclor-1254 induced rats were compared for bioactivation of NNK using Salmonella mutagenesis as the endpoint. Results from the in vitro experiments indicated that low concentrations of NNK (0.01-25 micrograms/ml) caused from 15% to greater than 100% increases in CFE of RTE cells. At high concentrations (100-200 micrograms/ml), NNK was significantly toxic to RTE cells. NNK treatment in vitro (50-200 micrograms/ml) increased MN frequency as much as 3-fold above background and significantly increased the transformation frequency (TF) in 4/5 (50 micrograms/ml) and 6/8 (100 micrograms/ml) experiments. The in vivo exposure of rats to NNK (150-450 mg/kg, given i.p.) resulted in a 60-85% reduction in CFE and a 3-5-fold increase in MN formation in RTE cells. In vivo treatment with cumulative doses of 150 and 300 mg/kg of NNK produced significant increases in TF of tracheal cells from 3/3 and 2/3 rats, respectively. Without activation, NNK was not mutagenic in Salmonella TA1535. The bioactivation of NNK to a mutagenic metabolite was achieved by incubation of NNK with liver S9 fraction from Aroclor-1254 induced rats or primary hepatocytes from both untreated and Aroclor-1254 pretreated rats. RTE cells did not produce sufficient quantities of mutagenic NNK metabolites to be detected by the Salmonella assay.

Cooperativity in ATP hydrolysis by GroEL is increased by GroES.

The kinetics of ATP hydrolysis by the 'molecular chaperone' GroEL and the inhibition of this hydrolysis by GroES have been studied in more detail. It is shown that the hydrolysis of ATP by GroEL is cooperative with respect to ATP with a Hill coefficient of 1.86 (+/- 0.13). In the presence of GroES, there is an increase in the degree of cooperativity with a Hill coefficient of 3.01 (+/- 0.18). The observed cooperativity is not due to dissociation of the GroEL oligomer into smaller units but more probably involves structural changes within the GroEL oligomer.

Kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-tRNA synthetase analyzed by pyrophosphate-exchange kinetics.

The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.