Tumor necrosis factor-alpha-converting enzyme mediates the inducible cleavage of fractalkine.

Fractalkine (FK, CX3CL1) is a novel multidomain protein expressed on the surface of endothelial cells. As a full-length transmembrane protein, FK binds cells expressing CX3CR1, its cognate receptor, with high affinity. Proteolytic cleavage of FK releases a soluble form that is a potent chemoattractant for monocytes, T cells, and natural killer cells. Activation of protein kinase C dramatically increases the rate of this cleavage. Regulation of FK cleavage is critical for maintaining the balance between the immobilized and soluble forms, but the protease responsible has not been identified. Here we report that tumor necrosis factor-alpha-converting enzyme (TACE) is primarily responsible for the inducible cleavage of FK. After transfection into host cells, the proteolytic cleavage of FK was blocked by TACE-specific inhibitors and was not detected in cells genetically altered to remove TACE activity. In contrast, the constitutive cleavage of FK was not mediated by TACE and proceeded normally in TACE-null fibroblasts. We conclude that TACE is primarily responsible for the inducible cleavage of FK. These studies identify a potentially important link between local generation of potent cytokines and control of the balance between the cell adhesion and chemotactic properties of FK.

Human placental cytotrophoblasts attract monocytes and CD56(bright) natural killer cells via the actions of monocyte inflammatory protein 1alpha.

During human pregnancy, the specialized epithelial cells of the placenta (cytotrophoblasts) come into direct contact with immune cells in several locations. In the fetal compartment of the placenta, cytotrophoblast stem cells lie adjacent to macrophages (Hofbauer cells) that reside within the chorionic villus stroma. At sites of placental attachment to the mother, invasive cytotrophoblasts encounter specialized maternal natural killer (NK) cells (CD56(bright)), macrophages, and T cells that accumulate within the uterine wall during pregnancy. Here we tested the hypothesis that fetal cytotrophoblasts can direct the migration of these maternal immune cells. First, we assayed the chemotactic activity of cytotrophoblast conditioned medium samples, using human peripheral blood mononuclear cells as targets. The placental samples preferentially attracted NK cells (both CD56(dim) and CD56(bright)), monocytes, and T cells, suggesting that our hypothesis was correct. A screen to identify chemokine activity through the induction of a Ca(2)+ flux in cells transfected with individual chemokine receptors suggested that cytotrophoblasts secreted monocyte inflammatory protein (MIP)-1alpha. This was confirmed by localizing the corresponding mRNA and protein, both in vitro and in vivo. MIP-1alpha protein in conditioned medium was further characterized by immunoblotting and enzyme-linked immunosorbent assay. Immunodepletion of MIP-1alpha from cytotrophoblast conditioned medium showed that this chemokine was responsible for a significant portion of the induced monocyte and CD56(bright) NK cell chemotaxis. These data suggest the specific conclusion that cytotrophoblasts can attract monocytes and CD56(bright) NK cells by producing MIP-1alpha and the more general hypothesis that these cells may organize and act on leukocytes at the maternal-fetal interface.

High concentrations of D-glyceraldehyde-3-phosphate dehydrogenase stabilize the enzyme against denaturation by low concentrations of GuHCl.

It is known that denaturation of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in low concentrations of GuHCl, around 0.5 M, at 25 degrees C, leads first to a burst phase drop of activity, followed by slow unfolding with further loss of enzyme activity and aggregation. However, GAPDH at higher concentrations does not increase the aggregation in the slow phase as would be expected but decreases both the inactivation and aggregation of the enzyme instead. It seems that GAPDH at high concentrations protects the enzyme against GuHCl-denaturation. This protection is not a general effect of GuHCl binding by increased protein concentration but specific for GAPDH, as either bovine serum albumin or alpha-lactalbumin does not show any protection at similar concentrations. It is proposed that dissociation of tetrameric GAPDH into dimers in the early phase of denaturation in dilute GuHCl is reversible and further unfolding of the dimer to an aggregation prone species is irreversible and rate-limiting for the unfolding process. High concentrations of the enzyme shift the equilibrium towards the tetramer thus decrease the aggregation of GAPDH in dilute GuHCl.

Structure of active site carboxymethylated D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor.

The structure of active site carboxymethylated D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor was determined in the presence of coenzyme NAD+ at 1.88 A resolution with a final R-factor of 0.175. The structure refinement was carried out on the basis of the structure of holo-GAPDH at 2.0 A resolution using the program XPLOR. The carboxymethyl group connected to Cys149 is stabilized by a hydrogen bond between its OZ1 and Cys149N, and charge interaction between the carboxyl group and the nicotinamide moiety. The modification of Cys149 induced conformational changes in the active site, in particular, the site of sulphate ion 501 (the proposed attacking inorganic phosphate ion in catalysis), and segment 208-218 nearby. Extensive hydrogen-bonding interactions occur in the active site, which contribute to the higher stability of the modified enzyme. The modification of the active site did not affect the conformation of GAPDH elsewhere, including the subunit interfaces. The structures of the green and red subunits in the asymmetric unit are nearly identical, suggesting that the half-site reactivity of this enzyme is from ligand-induced rather than pre-existing asymmetry. It is proposed that the carboxymethyl group takes the place of the acyl group of the reaction intermediate, and the catalytic mechanism of this enzyme is discussed in the light of a comparison of the structures of the native and the carboxymethylated GAPDH.

Metal regulation of metallothionein participation in redox reactions.

Like glutathione or dithiothreitol, metallothionein effects the formation of pancreatic ribonuclease A from its S-sulfonated derivative catalyzed by protein disulfide isomerase. EDTA increases the yield of ribonuclease A activity recovery with metallothionein but does not affect the reaction with glutathione or dithiothreitol. EDTA also increases the reactivity of thiol groups in metallothionein with 5,5'-dithiobis-(2-nitrobenzoic acid) by chelation of zinc ions. It is suggested that the thiol groups in metallothionein form a part of the pool of cellular thiols in the regulation of cellular redox reactions and their availability is modulated by zinc chelation.

Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies.

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.

The inactivation kinetics of papain by guanidine hydrochloride: a re-analysis.

The kinetic theory of the substrate reaction during modification of enzyme activity has been applied to study the inactivation kinetics of enzymes by denaturant. However, an important problem related to the determination of the inactivation rate constants has not been considered in a previous publication (Xiao, et al., Biochim. Biophys. Acta, 1164 (1993) 54-60). In most denaturation experiments, the high concentrations of denaturants may greatly affect the kinetic behavior of the system to preclude the use of the kinetic parameters determined in the absence of denaturant. In the present study, the kinetic equation of substrate reaction in presence of denaturant has been derived. A re-examination of the effect of high concentrations of guanidine hydrochloride on the inactivation of papain, taking into consideration the effect of high concentrations of guanidine hydrochloride on the Michaelis constant, showed that, for papain, the substrate gives no protection on its inactivation. It is the purpose of the present communication to stress the importance of observing the effect of the denaturant on the kinetic parameters for kinetic analysis of enzyme inactivation by denaturants generally.

Identification of C-C chemokine receptor 1 (CCR1) as the monocyte hemofiltrate C-C chemokine (HCC)-1 receptor.

Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1alpha. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes. In this paper, we have investigated the chemokine receptor usage and downstream signaling pathways of HCC-1. Cross-desensitization experiments using THP-1 cells suggested that HCC-1 and MIP-1alpha activated the same receptor. Experiments using a panel of cloned chemokine receptors revealed that HCC-1 specifically activated C-C chemokine receptor (CCR)1, but not closely related receptors, including CCR5. HCC-1 competed with MIP-1alpha for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1alpha). Similarly, HCC-1 was less potent than MIP-1alpha in inducing inhibition of adenylyl cyclase in CCR1-transfected cells. HCC-1 induced chemotaxis of freshly isolated human monocytes, THP-1 cells, and CCR1-transfected cells, and the optimal concentration for cell migration (100 nM) was approximately 100-fold lower than that of MIP-1alpha (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes.

The role of active site flexibility in enzyme catalysis.

It has been shown in this and other laboratories that during the unfolding of a number of enzymes inactivation generally precedes global unfolding of the enzyme molecule, leading to the suggestion that enzyme active sites are usually more "fragile" and more easily "perturbed" than the molecule as a whole and are therefore conformationally more flexible than the rest of the molecule. However, the role of active site flexibility in enzyme catalysis still remains to be explored. In the induced fit hypothesis originally proposed by Koshland, the presence of the substrate induces a conformational change at the active site so as to fit with the structure of the substrate. By X-ray crystallographic structural analysis of E. coli dihydrofolate reductase liganded with cofactors and substrates, Sawaya and Kraut showed the enzyme in different conformational states indeed while complexed with different ligands, suggesting that the enzyme molecule passes through different conformational states through the whole process of catalysis. Muscle lactate dehydrogenase can be stabilized either in concentrated ammonium sulfate or by cross-linking with glutaraldehyde together with a decrease in enzyme activity which can be restored to the original level in dilute guanidine hydrochloride possibly by increased flexibility at the active site. It is known that a number of enzymes can be activated by chaotropic agents such as urea or guanidine hydrochloride. The activation of dihydrofolate reductase by either urea or guanidine hydrochloride is accompanied by an increase in susceptibility to proteolysis. Isolation of the tryptic peptides of the activated enzyme and sequence analysis allowed identification of the sites of proteolysis to be at or near the active site of the enzyme, indicating an opening up of the active site conformation in the activated state. All the above indicate that active site flexibility plays an important role in enzyme catalysis. It is possible that during the catalytic cycle, the enzyme molecule passes through different stages and each stage requires the molecule to be in a different conformation, especially at the active site. Rapid transition between the different conformational states, and hence the flexibility of the active site, is therefore mandatory for the maximal expression of enzyme activity.

Binding of a burst-phase intermediate formed in the folding of denatured D-glyceraldehyde-3-phosphate dehydrogenase by chaperonin 60 and 8-anilino-1-naphthalenesulphonic acid.

Upon dilution, D-glyceraldehyde-3-phosphate dehydrogenase (GADPH) that has been fully inactivated, but only partially unfolded, in dilute guanidine hydrochloride (GuHCl) recovers activity completely. The fully unfolded enzyme, however, is re-activated only to a limited extent after dilution, and refolds rapidly in a burst phase to a partially folded intermediate characterized by increases in both the emission intensity of intrinsic fluorescence and binding to 8-anilino-1-naphthalenesulphonic acid (ANS). This intermediate aggregates with a time lag of a few minutes, and the aggregation can be suppressed completely by chaperonin 60 (GroEL). Stoichiometric analysis of the suppression of GAPDH re-activation by GroEL suggests that the tetradecameric GroEL binds to a dimeric GAPDH folding intermediate. This intermediate can be re-activated by ATP or ATP/chaperonin 10 (GroES) to an extent considerably greater than that obtained on spontaneous re-activation of the fully denatured enzyme upon dilution. Probing with a fluorescent derivative of NAD+ shows that this folding intermediate does not have a native conformation at the active site. The similar profiles of the effects of GroEL and ANS on the re-activation of GAPDH denatured by different concentrations of GuHCl suggest that GroEL and ANS recognize and bind to the same folding intermediate, which is similar to the relatively stable, partially unfolded, state of the enzyme denatured in 0.5-1.0 MGuHCl. However, the complexes of the intermediate with GroEL or ANS appear to be different, in that GroEL, but not ANS, suppresses aggregation and assists folding in the presence of ATP.

Enzymes as chaperones and chaperones as enzymes.

Chaperones and foldases are two groups of accessory proteins which assist maturation of nascent peptides into functional proteins in cells. Protein disulfide isomerase, a foldase, and ATP-dependent proteases, responsible for degradation of misfolded proteins in cells, both have intrinsic chaperone activities. Trigger factor and DnaJ, well known Escherichia coli chaperones, show peptidyl prolyl isomerase and protein disulfide isomerase activities respectively. It is suggested that the combination of chaperone and enzyme activities in one molecule is the result of evolution to increase molecular efficiency.

Active site flexibility in enzyme catalysis.

The inactivation of a number of enzymes during denaturation by physical and chemical factors precedes detectable global conformational changes of the molecules as monitored by conventional methods. It was suggested that the enzyme active site is more flexible and more sensitive to denaturation than the molecule as a whole. The well-known "induced fit" hypothesis by Koshland implies multiconformational states of enzymes in equilibrium with one another easily perturbed by ligands. Each intermediary step during the entire catalytic process may require the molecule to be in a particular conformation state; rapid interconversion between the different conformation states may well be involved in the catalytic process. As a relative fragile and consequently flexible active site has now been envisaged, it appears that a rapid cycling of the different active site conformation states is essential for the full expression of enzyme activity.

Inactivation kinetics of the reduced spinach chloroplast fructose-1,6-bisphosphatase by subtilisin.

The course of inactivation of the reduced spinach chloroplast fructose-1,6-bisphosphatase by digestion with subtilisin has been followed by the progress curve method [Tsou, C. L. (1988) Adv. Enzymol. 61, 381-436] and found to follow first-order kinetics. On the basis of the hydrolysis of the substrate, fructose 1,6-bisphosphate, at different concentrations during proteolysis by subtilisin, the first-order inactivation rate constants for the free enzyme and the enzyme-substrate complex can both be determined. The ratio between the inactivation rate constants for the free enzyme and the enzyme-substrate complex indicates strong protection against subtilisin proteolysis by the substrate. It is proposed that the above ratio can be used as a quantitative measure of substrate protection for enzyme inactivation generally. As it has been found that the site of proteolysis is located in a loop region near the N-terminus and well away from the active site, the substrate protection indicates a conformation change of the enzyme away from the substrate binding site.

Dissociation of chemotaxis from agonist-induced receptor internalization in a lymphocyte cell line transfected with CCR2B. Evidence that directed migration does not require rapid modulation of signaling at the receptor level.

To investigate the role of the carboxyl-terminal region (52 amino acids) of the monocyte chemoattractant protein 1 receptor (CCR2B) in chemotaxis, we created a series of mutants and expressed them in a murine pre-B lymphocyte cell line. Truncation of the cytoplasmic carboxyl tail to 20 amino acids had little or no effect on chemotaxis or signal transduction, but further truncation resulted in marked functional defects. Upon incubation with monocyte chemoattractant protein 1, CCR2B underwent rapid and extensive internalization, and this was impaired progressively as the carboxyl tail was truncated from 52 to 8 amino acids. Mutation of all of the serine and threonine residues in the carboxyl tail to alanine also resulted in markedly impaired receptor internalization but did not affect signaling or chemotaxis. We conclude that the membrane-proximal portion of the cytoplasmic carboxyl tail of CCR2B is critically involved in chemotaxis and signal transduction, but neither phosphorylation of carboxyl serines or threonines nor internalization of the receptor is required for robust chemotaxis.

Perturbation of the antigen-binding site and staphylococcal protein A-binding site of IgG before significant changes in global conformation during denaturation: an equilibrium study.

Although conformational perturbation of the active sites of many enzymes has been reported to precede global molecular conformational changes [Tsou (1993) Science 262, 380-381], little effort has been made to compare the susceptibility of the ligand-binding site of proteins and the protein molecules as a whole to perturbation by denaturants. Immunoglobulin is chosen in this study to address this problem. It is found that the variable and constant regions (Fv and Fc) of a monoclonal antibody of an IgG subclass against adenylate kinase lose their abilities to bind antigen and staphylococcal Protein A after treatment with guanidinium chloride concentrations considerably lower than those required to change the global conformation of the antibody as a whole, as detected by fluorescence and second-derivative UV absorption spectroscopy. These results indicate that both ligand-binding sites of the antibody concerned are more fragile than the molecule as a whole and that the Fv and Fc regions of the antibody molecule unfold sequentially during denaturation.

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity.

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

Organization and differential expression of the human monocyte chemoattractant protein 1 receptor gene. Evidence for the role of the carboxyl-terminal tail in receptor trafficking.

Two forms of the monocyte chemoattractant protein-1 receptors (the type A monocyte chemoattractant protein 1 (MCP-1) receptor CCR-2A and the type B MCP-1 receptor (CCR-2B) have been recently cloned and found to differ only in their terminal carboxyl tails. Here, we report that the two isoforms are alternatively spliced variants of a single MCP-1 receptor gene. Sequencing of the gene revealed that the 47-amino acid carboxyl tail of CCR2B was located in the same exon as the seven transmembrane domains of the receptor, and the 61-amino acid tail of CCR2A was in a downstream exon. Examination of freshly isolated human monocytes by reverse transcriptase-polymerase chain reaction revealed that CCR2B was the predominant isoform and that message levels of both CCR2A and CCR2B decreased as the monocytes differentiated into macrophages. In stably transfected cell lines, CCR2B trafficked well to the cell surface, but CCR2A was found predominantly in the cytoplasm. Equilibrium binding studies revealed that those CCR2A receptors that successfully trafficked to the cell surface bound MCP-1 with high affinity (Kd = 310 pM), similar to CCR2B. In signaling studies, both CCR2A and CCR2B mediated agonist-dependent calcium mobilization, as well as inhibition of adenylyl cyclase. Creation of chimeras between CCR2A and the human thrombin receptor revealed that the cytoplasmic retention of CCR2A was due to its terminal carboxyl tail. Progressive truncation of the carboxyl tail indicated that a cytoplasmic retention signal(s) was located between residues 316 and 349. These data indicate that the alternatively spliced form of the human MCP-1 receptor (CCR2A) binds MCP-1 with high affinity and is a functional receptor and that expression at the cell surface is controlled by amino acid sequences located in the terminal carboxyl tail.

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: evidence that directed migration is mediated by betagamma dimers released by activation of Galphai-coupled receptors.

Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to Galphaq (the m3 muscarinic receptor), Galphai (the kappa-opioid receptor), and Galphas (the beta-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to Galphai, but not to Galphaq or Galphas, migrated in response to a concentration gradient of the appropriate agonist. Overexpression of Galpha transducin, which binds to and inactivates free Gbetagamma dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of Gbetagamma dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of Galphai and the release of Gbetagamma dimers, and that Galphai-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.

Phosphorylation by a G protein-coupled kinase inhibits signaling and promotes internalization of the monocyte chemoattractant protein-1 receptor. Critical role of carboxyl-tail serines/threonines in receptor function.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family of chemotactic cytokines and signals via activation of a G protein-coupled seven-transmembrane domain receptor to mediate chemotaxis. Monocyte activation is limited by desensitization and internalization of the MCP-1R, but these mechanisms are not well understood. In this study, we show that the type B MCP-1R (MCP-1RB/CCR2B) is rapidly phosphorylated and internalized in response to nanomolar concentrations of MCP-1. Co-expression of CCR2B in Xenopus oocytes with beta-adrenergic receptor kinase 2 (beta ark2), but not beta ark1 or rhodopsin kinase, specifically blocked receptor activation by MCP-1. Mutation of serine (Ser) and threonine (Thr) residues in the terminal carboxyl-tail of the receptor, which are potential targets of beta ark-mediated phosphorylation, prevented inhibition of receptor activation by beta ark2 in microinjected oocytes. Finally, a construct in which multiple Ser and Thr residues in the carboxyl-tail were changed to alanine significantly prolonged the agonist-dependent intracellular calcium flux and inhibited receptor internalization in transfected human embryonic kidney (HEK)-293 cells. These studies demonstrate that phosphorylation of Ser and Thr residues in the carboxyl-tail of CCR2B mediates receptor desensitization and internalization and may serve to limit the chemotactic response of leukocytes to MCP-1 and related chemokines.

Sequential unfolding of adenylate kinase during denaturation by guanidine hydrochloride.

The unfolding of adenylate kinase in GuHCl of increasing concentrations has been followed by a combination of different methods. Molecular packing was measured by size-exclusion chromatography (SEC), exposure of buried Tyr residues by second- derivative spectra, loss of secondary structure by circular dichroism in the far-ultraviolet and the decrease in surface hydrophobicity by ANS binding. The conformational changes of adenylate kinase as followed by the above methods depend differently on GuHCl concentration. The concentrations of GuHCl at which 50% changes as measured by the above four methods occur are 0.3, 0.46, 0.64 and 0.64 M, respectively. SEC measurements show that with increasing GuHCl concentrations, the process of unfolding of adenylate kinase involves two slowly interconvertible intermediate stages, I1, and I2, the last is in a more advanced state of unfolding but is still more compact than the fully unfolded state, U, as indicated by their elution volumes in the SEC profile. There is also evidence to suggest that both the intermediates I1 and I2 may contain additional intermediary components in rapid equilibrium as indicated by the gradual shift of both peaks in the SEC elution profile. A sequential mechanism is suggested for the unfolding of adenylate kinase with increasing guanidine hydrochloride concentrations.