Proteasome inhibition in glyoxal-treated fibroblasts and resistance of glycated glucose-6-phosphate dehydrogenase to 20 S proteasome degradation in vitro.

Glycation and glycoxidation protein products are formed upon binding of sugars to NH(2) groups of lysine and arginine residues and have been shown to accumulate during aging and in pathologies such as Alzheimer's disease and diabetes. Because the proteasome is the major intracellular proteolytic system involved in the removal of altered proteins, the effect of intracellular glycation on proteasome function has been analyzed in human dermal fibroblasts subjected to treatment with glyoxal that promotes the formation of N epsilon-carboxymethyl-lysine adducts on proteins. The three proteasome peptidase activities were decreased in glyoxal-treated cells as compared with control cells, and glyoxal was also found to inhibit these peptidase activities in vitro. In addition, the activity of glucose-6-phosphate dehydrogenase, a crucial enzyme for the regulation of the intracellular redox status, was dramatically reduced in glyoxal-treated cells. Further analysis was performed to determine whether glycated proteins are substrates for proteasome degradation. In contrast to the oxidized glucose-6-phosphate dehydrogenase, both N epsilon-carboxymethyl-lysine- and fluorescent-glycated enzymes were resistant to degradation by the 20 S proteasome in vitro, and this resistance was correlated with an increased conformational stability of the glycated proteins. These results provide one explanation for why glycated proteins build up both as a function of disease and aging. Finally, N epsilon-carboxymethyl-lysine-modified proteins were found to be ubiquitinated in glyoxal-treated cells suggesting a potential mechanism by which these modified proteins may be marked for degradation.

Comparison of the kinetics of S-S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme.

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and/or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.

Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed alpha/beta protein, Escherichia coli thioredoxin.

Oxidized Escherichia coli thioredoxin (Trx) is a small protein of 108 residues with one disulfide bond (C32-C35 essentially involved in the activity) and no prosthetic moieties, which folds into a structural motif containing a central twisted beta-sheet flanked by helices that is found in many larger proteins. The kinetics of refolding of Trx in vitro have been investigated using a newly developed active site titration assay and continuous or stopped-flow (SF) methods in conjunction with circular dichroism (CD) and fluorescence (Fl) spectroscopy. These studies revealed the presence of early folding intermediates with "molten globule or pre-molten globule" characteristics. Measurements of the ellipticity at 222 nm indicated that about 68% of the total change associated with refolding occurred during the dead time (4 ms) of the stopped-flow instrument, suggesting the formation of substantial secondary structure. The reconstruction of the far-UV CD spectrum of the burst intermediate using combined continuous and stopped-flow methods showed the formation of a defined secondary structure that contains more beta-structure than the native state. Kinetic measurements using SF far-UV CD and Fl over a wide range (0.087-6 M) of GuHCl concentrations at two temperatures (6 and 20 degreesC) demonstrated that the population formed during the 4 ms dead time contained multiple species that are stabilized mainly by hydrophobic interactions and undergo further folding along alternative pathways. One of these species leads directly and rapidly to the native state as demonstrated by active site titration, while the two others fold into a fourth intermediate that is slowly converted to the native protein. Double-jump experiments suggest that the heterogeneity in folding behavior results from proline isomerizations occurring in the unfolded state. Conversely, the accumulation of the burst intermediate does not depend on proline isomerizations.

Kinetics of secondary structure recovery during the refolding of reduced hen egg white lysozyme.

We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 M guanidinium chloride, 60 microM oxidized glutathione, and 20 microM reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi- or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.

The "pre-molten globule," a new intermediate in protein folding.

In vitro folding studies of several proteins revealed the formation, within 2-4 msec, of transient intermediates with a large far-UV ellipticity but no amide proton protection. To solve the contradiction between the secondary structure contents estimated by these two methods, we characterized the isolated C-terminal fragment F2 of the tryptophan synthase beta 2 subunit. In beta 2, F2 forms its tertiary interactions with the F1 N-terminal region. Hence, in the absence of F1, isolated F2 should remain at an early folding stage with no long-range interactions. We shall show that isolated F2 folds into, and remains in, a "state" called the pre-molten globule, that indeed corresponds to a 2- to 4-msec intermediate. This condensed, but not compact, "state" corresponds to an array of conformations in rapid equilibrium comprising native as well as nonnative secondary structures. It fits the "new view" on the folding process.

Kinetic mechanism of luciferase subunit folding and assembly.

The kinetic mechanism in vitro of the folding and assembly of the heterodimeric flavin monooxygenase bacterial luciferase has been defined by a unique set of rate constants which describe both the productive refolding pathway and competing off-pathway reactions in 50 mM phosphate, pH 7.0 at 18 degrees C. The individual alpha and beta subunits fold independently to form heterodimerization-competent species, alpha i and beta i. The alpha i beta i species can interact to form an inactive heterodimeric intermediate, [alpha beta ]i, which isomerizes to form the active alpha beta structure; the structure of the enzyme has been determined to 1.5 A resolution [Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., & Rayment, I. (1996) J. Biol. Chem. 271, 21956-21968]. In the absence of alpha i, beta i can form a kinetically trapped homodimer, beta 2, with a second-order rate constant of about 180 M-1 s-1 [Sinclair, J. F., Ziegler, M. M., & Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]; the structure of beta 2 has recently been reported [Thoden. J. B., Holden, H. M., Fisher, A. J., Sinclair. J. F., Wesenberg, G., Baldwin, T.O., & Rayment, I. (1997) Protein Sci. 6, 13-23]. The beta i species, or some other form that precedes beta i on the refolding pathway, can also undergo a first-order conversion into a form (designated beta x) that cannot associate with alpha i to form the native enzyme. The rate constant for this process, assigned here, accounts well for the previously observed dependence of final yield on concentration of refolding species [Ziegler, M.M., Goldberg, M.E., Chaffotte, A. F., & Baldwin, T. O. (1993) J. Biol. Chem. 268, 10760-10765]. In simulations of the refolding reaction, all processes associated with the refolding of the individual subunits were combined into single first-order rate constants for each subunit which were consistent with the rate constants determined from stopped-flow circular dichroism studies. The first-order rate constant for the folding of the alpha subunit, estimated from the concentration-independent lag preceding the appearance of active enzyme, and the second-order rate constant for assembly of alpha i and beta i into the heterodimer, estimated from the concentration-dependent rate of appearance of active enzyme, were consistent with the rates of first- and second-order processes monitored by changes in fluorescence of an extrinsic probe [the product of modification with N-(4-anilino-1-naphthyl)maleimide] on the alpha subunit during refolding. The rate constant for the isomerization of [alpha beta]i to form the active heterodimer was estimated from the kinetic data of a secondary dilution experiment and from fluorescence measurements of protein diluted 20-fold from 2.1 M urea-containing buffer. The rate constants reported here for the kinetic mechanism of refolding permitted simulation of the time courses and yields for activity recovery during the refolding of luciferase from about 1 to 25 micrograms/mL which are in excellent agreement with our previously reported data.

Recognition between disordered states: kinetics of the self-assembly of thioredoxin fragments.

The disordered N- (1-73) and C- (74-108) fragments of oxidized Escherichia colithioredoxin (Trx) reconstitute the native structure upon association [Tasayco, M. L., & Chao, K. (1995) Proteins: Struct., Funct., Genet. 22, 41-44]. Kinetic measurements of the formation of the complex (1-73/74-108) at 20 degrees C under apparent pseudo-first-order conditions using stopped-flow far-UV CD and fluorescence spectroscopies indicate association coupled to folding, an apparent rate constant of association [kon = (1330 +/- 54) M-1 s-1], and two apparent unimolecular rate constants [k1 = (0. 037 +/- 0.007) s-1 and k2 = (0.0020 +/- 0.0005) s-1]. The refolding kinetics of the GuHCl denatured Trx shows the same two slowest rate constants. An excess of N- over C-fragment decreases the kon, and the slowest phase disappears when a P76A variant is used. Stopped-flow fluorescence measurements at 20 degrees C indicate a GuHCl-dependent biphasic dissociation/unfolding process of the complex, where the slowest phase corresponds to 90% of the total. Their rate constants, extrapolated to zero denaturant, k-1 = (9 +/- 3) x 10(-5) s-1 and k-2 = (3.4 +/- 1.2) x 10(-5) s-1, show m# values of (4.0 +/- 0.4) kcal mol-1 M-1 and (3.5 +/- 0.1) kcal mol-1 M-1, respectively. Our results indicate that: (i) a compact intermediate with trans P76 and defined tertiary structure seems to participate in both the folding and unfolding processes; (ii) not all the N-fragment is competent to associate with the C-fragment; (iii) conversion to an association competent form occurs apparently on the time scale of P76 isomerization; and (iv) the P76A variation does not alter the association competency of the C-fragment, but it permits its association with "noncompetent" forms of the N-fragment.

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.

Structural analysis of the predicted coiled-coil rod domain of the cytoplasmic bullous pemphigoid antigen (BPAG1). Empirical localization of the N-terminal globular domain-rod boundary.

The bullous pemphigoid antigen BPAG1 is required for keratin filament linkage to the hemidesmosome, an adhesion complex in epithelial basal cells. BPAG1 structural organization is similar to the intermediate filament-associated proteins desmoplakin I (DPI) and plectin. All three proteins have predicted dumbbell-like structure with central alpha-helical coiled-coil rod and regions of N- and C-terminal homology. To characterize the size of the N-terminal globular domain in BPAG1, two polypeptides spanning possible boundaries with the coiled-coil rod domain of BPAG1 were expressed in Escherichia coli. BP-1 (Mr = 111,000), containing amino acids 663-1581 of BPAG1 (Sawamura, D., Li, K., Chu, M.-L., and Uitto, J. (1991) J. Biol. Chem. 266, 17784-17790), and BP-1A, with a 186 amino acid N-terminal deletion, were purified. BP-1 and BP-1A behave as highly asymmetric dimers in aqueous solution according to velocity sedimentation and gel filtration. Both have globular heads with rod-like tails of roughly equal length, 55-60 nm, upon rotary shadowing. BP-1A content of alpha-helix, determined by circular dichroism, is approximately 90%, consistent with alpha-helical coiled-coil formation in the rod-like tails. The estimated rod length, 383 +/- 57 amino acids (0.15 nm/amino acid), implies that globular folding in the BPAG1 N-terminal extends to the end of N-terminal homology with DPI and plectin. These findings support the existence of a common domain structure in the N-terminal regions of the BPAG1/DPI/plectin family.

Protein folding intermediates with rapidly exchangeable amide protons contain authentic hydrogen-bonded secondary structures.

Recent studies on protein folding intermediates by pulsed amide proton exchange and by far-ultraviolet circular dichroism have shown important discrepancies between the secondary structure contents estimated by these two methods at early folding stages. To solve these apparent discrepancies, structural studies have been performed on the isolated, 101 residue long, C-terminal proteolytic domain (F2) of the Escherichia coli tryptophan synthase beta chain, which had previously been reported to behave as an early folding intermediate [Chaffotte, A. F., Cadieux, C., Guillou, Y., & Goldberg, M. E. (1992) Biochemistry 31, 4303-4308]. The secondary structure of F2 has been investigated by far-UV circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and NMR. The CD and FTIR spectra clearly indicate that isolated F2 has about 30-45% of its residues involved in secondary structures stabilized by conventional hydrogen bonds. The characteristics of the NMR spectrum (line broadening, absence of structure-induced chemical shifts, absence of nuclear Overhauser effects in the amide region, few dipolar interactions between the side-chain protons) suggest that isolated F2 is oscillating between several conformations in rapid equilibrium. The rate of amide proton exchange has been studied by one-dimensional NMR, which indicates a significant extent of proton protection, with, however, protection factors that can be estimated to be at most 60 and more probably closer to 10. Thus, F2 appears to exist as a molten globule that exhibits very low amide proton protection and yet contains a large fraction of its residues involved in authentic secondary structures stabilized by hydrogen bonds. Such a state is likely to correspond to the earliest structured folding intermediates thus far characterized.

Refolding of luciferase subunits from urea and assembly of the active heterodimer. Evidence for folding intermediates that precede and follow the dimerization step on the pathway to the active form of the enzyme.

Conditions have been established that allow reversible refolding of luciferase from 5 M urea. The kinetics of formation of the active enzyme showed a concentration-independent lag, suggesting the existence of intermediate structures on the pathway of refolding. The rate of approach to the final level of activity was strongly concentration-dependent at protein concentrations below 10 micrograms/ml, but at concentrations above about 20 micrograms/ml, the rate of approach to the final activity value did not change with concentration. The concentration dependence presumably reflects the second-order step yielding the heterodimeric structure. The finding that at concentrations above 20 micrograms/ml, the rate becomes insensitive to concentration suggests that under these conditions, some step subsequent to dimerization become rate-limiting. When the refolding reaction was initiated by dilution out of 5 M urea at 50 micrograms/ml followed at various times by a secondary dilution to a final concentration of 5 micrograms/ml, it was found that the increase in activity continued at the rate characteristic of the higher protein concentration for a period of about 1-2 min following the dilution before slowing to the rate expected for the lower protein concentration. These observations indicate that there are inactive heterodimeric species that form from assembly of the individual subunits and that these species must undergo further folding to yield the active heterodimeric species. At protein concentrations of 5-50 micrograms/ml, the final yield of active enzyme was about 65-85%, decreasing at higher and lower concentrations. At higher concentrations, aggregation probably accounts for the limit in recovery, whereas at lower concentrations, it appears that the reduced yield of activity is due to the competing process of the folding of one or both individual subunits into some form incompetent to interact with each other. These experiments demonstrate the existence of slow steps in the refolding of luciferase subunits from urea and the formation of the active heterodimeric structure, both preceding and following the dimerization. Furthermore, the failure of protein at low concentrations to efficiently reassemble into the active heterodimer is consistent with the prior finding that luciferase subunits produced independently in Escherichia coli fold into conformations that cannot interact to form the active heterodimer upin mixing (Waddle, J. J., Johnston, T. C., and Baldwin, T. O. (1987) Biochemistry 26, 4917-4921).

Contribution of folding steps involving the individual subunits of bacterial luciferase to the assembly of the active heterodimeric enzyme.

Bacterial luciferase is an alpha beta heterodimer with a single active center in which the reaction of reduced FMN, O2, and an aliphatic aldehyde yields a photon of blue-green light. We have shown that refolding of the luciferase subunits from 5 M urea occurs via the intermediacy of several species, one of which is an inactive heterodimeric structure, resulting from the dimerization of alpha and beta, which isomerizes to the active alpha beta structure in a first-order reaction (Ziegler, M. M., Goldberg, M. E., Chaffotte, A. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10760-10765). We have also demonstrated the existence of an inactive heterodimeric species that is well populated at equilibrium in the presence of 1.6-2.8 M urea (Clark, A. C., Sinclair, J. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10773-10779). We have separated the alpha and beta subunits by ion exchange chromatography and investigated the effects on reformation of active luciferase of allowing the individual subunits to refold separately prior to mixing. These investigations show that the lag in formation of active luciferase is due to slow steps in folding of the individual subunits. The beta subunit appears to fold faster than the alpha subunit, but folding of the beta subunit also shows a distinct lag. When the alpha and beta subunits were allowed to refold from urea for periods of several hours or more prior to mixing, the yield of active heterodimeric luciferase was compromised, which is consistent with the finding that individual subunits produced in vivo fold into structures incompetent to interact with each other to form the active heterodimer (Waddle, J. J., Johnston, T. C., and Baldwin, T. O. (1987) Biochemistry 26, 4917-4921). It appeared that the rate with which the beta subunit assumed the heterodimerization-incompetent structure was faster than the rate with which the alpha subunit became heterodimerization-incompetent. These observations support a model for folding and assembly of the subunits of luciferase in which the two subunits fold into assembly-competent structures that associate to form the heterodimer. In a slow competing process, the subunits undergo a conformational rearrangement to form stable structures incompetent to form heterodimers. It appears that the association of the luciferase subunits might constitute an example of one polypeptide modifying the folding pathway of another, a model that is consistent with the suggestion that the formation of the heterodimeric structure of luciferase is a kinetic trap on the folding pathway of the individual subunits (Sugihara, J., and Baldwin, T. O. (1988) Biochemistry 27, 2872-2880).

Kinetic resolution of peptide bond and side chain far-UV circular dichroism during the folding of hen egg white lysozyme.

The kinetics of regain of the native ellipticity in the far- and near-UV spectra have been investigated during the refolding at pH 7.8 and 20 degrees C of guanidine-unfolded, nonreduced hen egg white lysozyme. Stopped-flow studies showed that the ellipticities at 260 and 289.5 nm exhibit biphasic kinetics with rate constants of about 50 s-1 and 2.5 s-1 for the rapid and slow phase, respectively. The ellipticity in the far-UV obeyed triphasic kinetics. In addition to a rapid and a slow phase with rate constants similar to those observed in the near-UV, a "burst" of ellipticity was shown to occur in the dead time of the experiments. The effects of low pH and of concentrations of guanidine ranging from 0.075 to 1.5 M on the rapid and slow rate constants were studied. Under all conditions investigated, the rate constants observed in the far- and near-UV for a given phase were the same, thus suggesting that the molecular events observed in the two regions of the UV spectrum are either identical or strongly coupled. Continuous-flow experiments at different wavelengths between 214 and 240 nm under conditions where the dead time for the observation was only 4 ms, followed by a detailed analysis of the kinetics of ellipticity change at each wavelength, provided the spectrum of the molecular species formed at the end of the burst phase. This spectrum was found to closely fit that predicted from the secondary structure of native lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Early steps in cytochrome c folding probed by time-resolved circular dichroism and fluorescence spectroscopy.

The kinetics of protein folding for horse ferricytochrome c was investigated by stopped-flow methods, using far-UV circular dichroism (CD), near-UV CD, and tryptophan fluorescence to probe the formation of secondary structure and tertiary interactions. In the far-UV region of the CD spectrum (222 nm), 44% of the total change associated with refolding occurs within the dead time of the stopped-flow experiment, indicating that a significant amount of helical secondary structure is formed in less than 4 ms. The remaining changes in the ellipticity at 222 nm occur in two kinetic phases with time constants of about 40 ms and 0.7 s, respectively. In contrast, there is no evidence for rapid changes in the ellipticity at 289 nm: an aromatic CD band, which is indicative of the formation of a tightly packed core, only begins to appear in a 400-ms step and is completed in a final 10-s phase. The fluorescence of a single tryptophan at position 59, which becomes quenched upon folding via nonradiative energy transfer to the heme group, provides complementary information on the condensation of the polypeptide chain during refolding. The fluorescence-detected stopped-flow folding kinetics of ferricytochrome c exhibits a 35% decrease in fluorescence during the dead time, suggesting that a substantial decrease in the average tryptophan-heme distance occurs on a submillisecond time scale. The subsequent fluorescence changes exhibit two prominent phases with time constants of about 20 and 300 ms, followed by a minor 5-s phase.(ABSTRACT TRUNCATED AT 250 WORDS)

A possible initial folding intermediate: the C-terminal proteolytic domain of tryptophan synthase beta chains folds in less than 4 milliseconds into a condensed state with non-native-like secondary structure.

The isolated F2-V8 peptide corresponding to the 101 C-terminal residues of Escherichia coli tryptophan synthase beta chains folds into a heat-stable, yet fluctuating, condensed state that contains a lot of secondary structure. However, this state has non-native-like secondary and supersecondary structures [Chaffotte, A., Guillou, Y., Delepierre, M., Hinz, H.-J., & Goldberg, M. E. (1991) Biochemistry 30, 8067-8074]. To characterize the rate of appearance of this state, stopped-flow studies on the far-ultraviolet circular dichroism (CD) and on the binding of 1-anilino-8-naphthalenesulfonate (ANS) have been conducted during the folding of guanidine-unfolded F2-V8. It was shown that both the CD signal at 222 nm and the ANS binding properties of folded isolated F2-V8 were regained, at 20 degrees C, within the dead time of the stopped-flow apparatus, which was 4 ms. At 12 degrees C, the binding of ANS was also completed within this dead time, but the ellipticity showed some minor later changes. After a rapid overshoot of the CD signal that occurred during the 4-ms dead time, a small readjustment of the ellipticity to the final value occurred more slowly and was completed after about 25 ms. Thus, even at 12 degrees C, the hydrophobic core and most of the secondary structure of folded F2-V8 were formed in less than 4 ms. These observations strongly suggest that the previously described condensed non-native-like state of F2-V8 results from a very rapid, nonspecific, hydrophobic collapse. It is proposed that such a state may be a general early intermediate in protein folding.

Inclusion bodies of the thermophilic endoglucanase D from Clostridium thermocellum are made of native enzyme that resists 8 M urea.

Endoglucanase D from Clostridium thermocellum was purified from inclusion bodies formed upon its overproduction in Escherichia coli, using 5 M urea as a solubilizing solution. We examined the effects of denaturing agents upon the stability of the pure soluble enzyme as a function of the temperature. At room temperature, guanidinium chloride induces an irreversible denaturation. By comparison, we observed no structural or functional effects at room temperature using high concentrations of urea as denaturing agent. The irreversible denaturation process observed with guanidinium chloride also occurs with urea but only at elevated temperature (greater than or equal to 60 degrees C); in 6 M urea, the activation energy of the denaturation reaction is decreased by a factor of only 1.8. We interpret the high resistance of this protein to urea as reflecting a reduced flexibility of its structure at normal temperatures which should be correlated to the thermophilic origin of this protein.

Kinetics of the spontaneous transient unfolding of a native protein studied with monoclonal antibodies. Monomer/dimer transition in the tryptophan-synthase beta 2 subunit.

Included in a series of monoclonal antibodies obtained after immunization with the native holo beta 2 subunit of tryptophan synthase of Escherichia coli (EC 4.2.1.20), are some that interact preferentially with a denatured state of the antigen (Friguet et al., 1984). A study of the equilibrium and kinetic characteristics of the interaction of one of these antibodies with native apo beta 2 (i.e. free of pyridoxal 5'-phosphate) and with one of its proteolytic domains is reported here. The antibody is shown to interact strongly with the isolated domain in accordance with a simple equilibrium. In the presence of native beta 2, the antibody binds exclusively to the dissociated beta-monomer. The interaction of this antibody with native apo beta 2 is used to determine the equilibrium and kinetic constants of the monomer-dimer equilibrium. The values obtained are 4.5 X 10(-8) M for the equilibrium constant and 7.9 X 10(-3) s-1 for the rate constant of the dissociation of apo beta 2 into beta-monomers.

Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay.

A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.

Does a monospecific hybridoma always secrete homogeneous immunoglobulins?

The fusion of splenocytes (from mice immunized with the beta 2 subunit of E. coli tryptophan synthase) with myeloma cells which do not produce immunoglobulins gave rise to a clone secreting immunoglobulins with two distinct isotypes : gamma 1 and gamma 2b (Djavadi-Ohaniance et al. (1984) Biochemistry, 23, 97-104). Analysis of the immunoglobulins secreted by this clone indicates that these two isotypes are carried by two distinct heavy chains which are able to randomly associate to form hybrid molecules. In addition, two classes of light chains are able to randomly and to form heterologous associations with both the gamma 1 and gamma 2b heavy chains. Only the association between the gamma 2b heavy chains with one of the two classes of light chains leads to a combining site specific for the binding of the antigen beta 2.