Development of methotrexate alpha-peptides as prodrugs for activation by enzyme-monoclonal antibody conjugates.

Selective delivery of lethal concentrations of drugs to tumors, allowing the latter to be eradicated without damage to other tissues, continues to be a major goal in cancer chemotherapy. Prodrugs (i.e. drugs that have been derivatized to prevent uptake into cells or interaction with targets), activated by enzyme-monoclonal antibody conjugates positioned at tumor sites, offer promise for achieving this objective. Methotrexate alpha-peptides (derivatives in which an amino acid is linked to the alpha-carboxyl group of the glutamate moiety) are ideal prodrugs, since they are not transported into cells and can be converted to the parent drug by carboxypeptidases. The L,L-diastereomer of MTX-alpha-Phe, synthesized in good yield by treatment of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with Glu-alpha-Phe, was hydrolyzed readily by carboxypeptidase A (CP-A). Conjugate was prepared by derivatizing the enzyme and monoclonal antibody KS1/4 with linkers containing maleimide and sulfhydryl groups, respectively; interaction of these groups to form a stable thioether bond joined the proteins. When administered in vitro to UCLA-P3 human lung adenocarcinoma cells (ca. 5 x 10(4) antibody binding sites/cell) that had been pre-treated with the conjugate (whose antibody KS1/4 is targeted to these cells), and excess conjugate removed by extensive washing, MTX-Phe (ID50 = 6.3 x 10(-8) M) approached the toxicity of MTX (ID50 = 4.5 x 10(-8) M). In the absence of conjugate, MTX-Phe was much less toxic (ID50 = 2.2 x 10(-6) M).

Methotrexate-alpha-phenylalanine: optimization of methotrexate prodrug for activation by carboxypeptidase A-monoclonal antibody conjugate.

Methotrexate-alpha-phenylalanine (MTX-Phe), a second-generation prodrug in the MTX alpha-peptide series designed for activation to MTX by carboxypeptidase-mAb conjugates, was synthesized by reaction of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with L-glutamyl-alpha-L-phenylalanine. Production of MTX from MTX-Phe, catalyzed by bovine pancreas carboxypeptidase A (CPA), was 250-fold faster than the corresponding reaction involving methotrexate-alpha-alanine, previously the best MTX peptide substrate for the enzyme. The amount of CPA required to make MTX-Phe equitoxic with MTX, when tested against UCLA-P3 human lung adenocarcinoma cells in vitro, was more than 10-fold lower than that required to achieve the same result with MTX-alpha-alanine. When the lung tumor cells were treated with CPA conjugated to KS1/4 (a mAb targeted to these cells) and excess conjugate removed by extensive washing, the ID50 for MTX-Phe improved from 2.2 x 10(-6) M (no enzyme present) to 6.3 x 10(-8) M; the latter value was comparable to that of the parent drug MTX (4.5 x 10(-8) M). [3H]MTX-Phe was synthesized and used to investigate the mechanism by which the prodrug exerts its cytotoxic effect in the presence and absence of CPA. The present results demonstrate that, for use in conjunction with CPA-mAb conjugates, the alpha-phenylalanine derivative is the optimal prodrug form of MTX (and probably other antifols that contain the glutamate moiety).

Novel substrate analogs delineate an endocytotic mechanism for uptake of folate via the high-affinity, glycosylphosphatidylinositol-linked transport protein in L1210 mouse leukemia cells.

The mechanism of folate uptake, mediated by the high-affinity folate receptor linked via glycosylphosphatidylinositol to the plasma membrane of L1210 mouse leukemia cells, has been investigated with the use of two substrate analogs, a fluorescein derivative of folate and a gold-conjugated, streptavidin-biotin derivative of folate, in conjunction with fluorescence microscopy and electron microscopy. These substrate probes bind to receptors that are uniformly distributed rather than clustered on the membrane, and the receptor-probe complexes are internalized by an endocytotic mechanism in which vesicles containing the complexes migrate from the membrane toward the nucleus. Measurements using [3H]-folate demonstrate that receptors are recycled to the plasma membrane.

Tumor targeting: activation of prodrugs by enzyme-monoclonal antibody conjugates.

Selective delivery of lethal levels of drugs to tumors, without concomitant damage to normal tissues, is a major challenge in cancer chemotherapy. Prodrugs used in conjunction with enzyme-monoclonal antibody conjugates that can target tumors and convert prodrugs to their active drug forms in situ, offer exceptional promise in achieving this objective. Synthesis of prodrugs, acquisition of appropriate enzymes and monoclonal antibodies, and manufacture of conjugates afford considerable flexibility in experimental design.

The methotrexate story: a paradigm for development of cancer chemotherapeutic agents.

Methotrexate (MTX), one of the earliest cancer chemotherapy agents, continues to be used extensively in the treatment of leukemia and a variety of other tumors. The efficacy of this drug results from its facile uptake by cells, rapid polyglutamylation and virtually stoichiometric inhibition of dihydrofolate reductase (DHFR), a key enzyme in cell replication. From the work of a multitude of biochemists, molecular biologists, organic chemists and pharmacologists, much is known about the mode of action of MTX and the mechanisms by which tumors exhibit inherent or acquired resistance to this drug. MTX enters cells primarily by a carrier-mediated active transport system whose principal substrate is 5-methyltetrahydrofolate, and additional glutamates are added to the gamma-position of the parent glutamate moiety. The tight binding of MTX to DHFR is defined from NMR and X-ray crystallographic studies of the enzyme and its drug or substrate complexes, supplemented by site-directed mutagenesis to confirm specific interactions. Resistance to the drug, encountered in cell culture model systems or in cancer patients, can result from an increased level of DHFR (due to gene amplification), mutant DHFR with reduced affinity for MTX, or decreased uptake or polyglutamylation of the drug. Although DHFR is an extremely well-studied enzyme, there is still some uncertainty about its kinetics, mechanism for reduction of folate, multiple forms, and activation by a diverse group of agents. Prodrug forms of MTX, e.g., MTX alpha-phenylalanine, which can be activated by carboxypeptidase A-monoclonal antibody conjugates, offer promise for improved efficacy of the drug by selective targeting to tumors. The large body of information summarized above has aided in the development of other folate antagonists, provides a paradigm for assessing the status of other cancer chemotherapeutic agents in current use, and offers a platform from which to speculate about the future of the field.

Membrane transport of folate compounds.

All eukaryotic cells and some prokaryotes that are unable to synthesize folic acid utilize membrane-associated transport systems for acquisition of the pre-formed vitamin or its coenzyme forms from external sources. These transport systems, in addition to providing folates essential for cell replication, are also important because of their role in the internalization of antifolates such as Methotrexate (MTX) that are used extensively in cancer chemotherapy. Information about the components and mechanism of folate transport systems has been derived, in large part, from studies with Lactobacillus casei and L1210 mouse leukemia cells, which serve as convenient models for prokaryotes and eukaryotes, respectively. L. casei contain a single folate transport system whose Kt value (i.e., concentration for half-maximum rate of uptake) for the preferred substrate folate is in the nanomolar range. The hydrophobic membrane-associated folate transport protein (18 kDa) has been purified to homogeneity and characterized. Expression of this transporter is repressed in cells grown on high concentrations (microM) of folate. L1210 cells contain two separate transport systems for folate compounds: (1) the low affinity system (Kt values for the preferred substrates 5-methyl- and 5-formyltetrahydrofolate and MTX in the microM range); and (2) the high affinity system (Kt for folate in the nM range). Fluorescein and biotin derivatives of MTX and folate, after conversion to N-hydroxysuccinimide esters, can be attached covalently to the transporters. These probes have been used for visualizing the transporters by fluorescence and electron microscopy and for their purification to homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple folate transport systems in L1210 cells.

Biotin derivatives of methotrexate (biotin-SS-MTX) and folate (biotin-SS-folate), in which the functional components are joined by a dissociable disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by HPLC, elemental analysis and mass spectrometry. These compounds provide a convenient means for the single-step purification of the folate transporters from L1210 cells. Parental L1210 murine leukemia cells, which contain only the microM transporter (the reduced folate/MTX transport protein) were treated with the N-hydroxysulfosuccinimide ester of biotin-SS-MTX, and a detergent extract of the plasma membranes was exposed to streptavidin-agarose beads to adsorb the labeled protein. Dithiothreitol cleavage of the disulfide linkage released the transporter, which migrated as a well-defined component (43 kDa) on SDS-PAGE gels; no other proteins were present. An L1210 subline (JF), obtained by adapting cells to grow on nanomolar concentrations of folate, contains both the microM transporter and the nM transporter (high-affinity folate binding protein). When these cells were treated with the N-hydroxysulfosuccimide ester of biotin-SS-folate and processed as described above, analysis on SDS-PAGE gels revealed the presence of two proteins, the microM transporter (43 kDa) and the nM transporter (39 kDa). Both transporters were characterized with respect to amino acid content; blocked N-termini precluded Edman sequencing. Treatment of the nM transporter with peptide:N-glycosidase F produced a smaller component (32 kDa); the microM transporter, conversely, was unchanged by this procedure. When the microM transporter in parental L1210 cells was labeled with fluorescein-MTX and then treated with phosphoinositol-specific phospholipase C (PI-PLC), no change in fluorescence was detected. Alternatively, when the nM transporter in the JF subline was labeled with fluorescein-folate and then treated with PI-PLC, complete loss of fluorescence was observed. These results indicate that the L1210 microM transporter is a non-glycosylated, integral membrane protein, while its nM counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.

Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate.

Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted enzyme and antibody. On the basis of SDS-PAGE analysis and measurement of catalytic activity, the preparation contained approximately equal amounts of 1:1 and 2:1 (enzyme:antibody) conjugates; binding activity of the conjugate (1.8 x 10(5) molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies with UCLA-P3 cells demonstrated the ability of cell-bound conjugate to convert the prodrug methotrexate-alpha-alanine (MTX-Ala) to methotrexate (MTX). In the absence of conjugate, ID50 values for MTX-Ala and MTX were 8.9 x 10(-6) and 5.2 x 10(-8) M, respectively. ID50 for the prodrug improved to 1.5 x 10(-6) M with cells containing bound conjugate. This potentiation of MTX-Ala cytotoxicity by conjugate-bound CP-A, which was at least 30-fold greater than that produced by a comparable amount of free enzyme, is attributed to enhanced effectiveness of MTX generated at the cell surface as opposed to the surrounding medium. Examination of the time course of cytotoxicity over a 96-h period showed that the conjugate-prodrug combination (at 2.5 x 10(-6) M) was nearly as effective as MTX in preventing cell replication. These results demonstrate the chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs.

Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells.

Biotin derivatives of methotrexate and folate (2-(biotinamido)ethyl-1,3'-dithiopropionyldiaminopentyl methotrexate and/or folate), in which carboxyl groups of the functional components are joined by a disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by high pressure liquid chromatography and mass spectrometry. These bifunctional, dissociable probes were utilized for the single-step purification to homogeneity of two folate transport proteins (43 and 39 kDa) from L1210 cells. Treatment of the 39-kDa protein with peptide N-glycosidase F produced a smaller component (32 kDa); the 43-kDa protein, conversely, was unchanged by this procedure. When the 39-kDa transporter in intact cells was labeled with a fluorescein derivative of folate and then treated with phosphoinositol-specific phospholipase C, complete loss of fluorescence was observed. Alternatively, there was no change in fluorescence when the 43-kDa transporter was labeled with a fluorescein derivative of methotrexate and treated with the enzyme. These results indicate that the 43-kDa transporter is a nonglycosylated, integral membrane protein, whereas the 39-kDa counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.

Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate.

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and chemotherapeutic potential of carboxypeptidase-A/monoclonal antibody conjugate.

Carboxypeptidase-A and a monoclonal antibody (KS1/4) directed against a human lung carcinoma cell line (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. Admixture of these entities produced a stable conjugate containing 4 to 5 enzyme molecules per molecule of antibody. The conjugate (Mr approximately equal to 300 kDa) was purified to homogeneity by HPLC gel filtration and HPLC ion-exchange chromatography. Neither the catalytic activity of the enzyme nor the antigen-binding capacity of the monoclonal antibody was impaired in the conjugate. UCLA-P3 cells that had been exposed to the conjugate and then washed thoroughly were extremely sensitive to methotrexate alpha-alanine (MTX-Ala), a prodrug form of MTX. At 10(-5) M, MTX-Ala was almost as effective as free MTX in blocking the replication of conjugate-treated cells. These results demonstrate the chemotherapeutic potential of enzyme-monoclonal antibody conjugates used in conjunction with prodrugs.

Occurrence and significance of diastereomers of methotrexate alpha-peptides.

The L,L diastereomer of methotrexate-alpha-alanine (L,L-MTX-Ala) was synthesized by reaction of alpha-L-glutamyl-L-alanine di-tert-butyl ester with 4-amino-4-deoxy-10-methylpteroic acid, followed by removal of the blocking groups. It was identified by HPLC (C18 reversed-phase silica gel; acetic acid/CH3OH) as the slower of two closely spaced components in DL,L-MTX-Ala prepared previously by a different route [Kuefner et al. (1989) Biochemistry 28, 2288-2297]. The L,L diastereomer was hydrolyzed by pancreatic carboxypeptidase A (to yield MTX and Ala) twice as rapidly as the DL,L mixture. Analysis of the latter by HPLC established that the slower component was hydrolyzed to MTX and that the unreactive, faster component was D,L-MTX-Ala. DL,L-MTX-Arg was resolved by HPLC (NH4OAc/CH3CN) into two closely spaced components, and the diastereomers were partially separated by chromatography on DEAE-Trisacryl (H2O----2% NH4HCO3). Serum carboxypeptidase N hydrolyzed only the slower HPLC component (to yield MTX and Arg), thereby identifying it as the L,L diastereomer. When tested for cytotoxicity against L1210 cells, L,L-MTX-Arg (ID50 = 1.6 X 10(-8) M) was more effective than the D,L diastereomer (ID50 = 2.2 X 10(-7) M). Treatment of MTX with dicyclohexylcarbodiimide and N-hydroxysuccinimide (NHS), followed by hydrolysis of the NHS ester, led to racemization in the L-glutamate moiety of MTX as shown by the fact that the product was hydrolyzed by carboxypeptidase G2 (at the pteroate-Glu bond) only to the extent of ca. 50% compared to the untreated control. These observations have a broad significance, since coupling agents are employed extensively in the derivatization of MTX for attachment to affinity supports and monoclonal antibodies.

Visualization of folate transport proteins by covalent labeling with fluorescein methotrexate.

Fluorescein-methotrexate (F-MTX) has been synthesized by an improved procedure and separated via chromatography on DEAE-Trisacryl into the alpha- and gamma-isomers. Purity of each isomer was verified by TLC, HPLC, and absorbance spectra. Identity of the alpha- and gamma-isomers was established by the following biological criteria: the gamma-isomer inhibited dihydrofolate reductase and was hydrolyzed by carboxypeptidase G2 (at the pteroate-glutamate linkage). The alpha-isomer, conversely, was unreactive in both systems, which is consistent with the specificity of these enzymes. Based upon these results, the gamma-isomer was selected for covalent labeling of proteins. Fluorescent bands were observed when the 22 kDa human dihydrofolate reductase and the 18 kDa folate transporter from Lactobacillus casei were treated with gamma-F-MTX (activated by N-hydroxysuccinimide) and subjected to SDS-PAGE. The probe was also useful for visualizing in situ the micromolar folate transport protein in L1210 cells.

Carboxypeptidase-mediated release of methotrexate from methotrexate alpha-peptides.

Methotrexate (MTX) alpha-peptides containing representative neutral (alanine), acidic (aspartic acid), and basic (arginine) amino acids were synthesized by a regiospecific route. Purity and authenticity of MTX-Ala, MTX-Asp, and MTX-Arg were established by TLC, HPLC, elemental analysis, and NMR and absorbance spectra. These peptides were hydrolyzed by carboxypeptidases to yield MTX and the amino acids. Reactions were monitored by using a ninhydrin assay for the amino acids and HPLC and spectrophotometric assays for MTX. Pancreatic carboxypeptidase A (CP-A) hydrolyzed MTX-Ala and, at a much slower rate, MTX-Asp and MTX-Arg. MTX-Ala was also a substrate for pancreatic carboxypeptidase B (CP-B); marginal activity was observed with this enzyme and MTX-Arg. Human serum hydrolyzed only MTX-Arg; biphasic inhibition of this activity by 2-(mercaptomethyl)-3-(guanidinoethyl)thiopropionate was consistent with the known presence of two types of endogenous carboxypeptidase (CP-N). Cytotoxicity of the MTX peptides toward L1210 cells in culture was enhanced considerably in the presence of the appropriate carboxypeptidases. MTX-Ala was much less toxic than MTX (ID50 values of 2.0 X 10(-6) M and 2.4 x 10(-8) M, respectively), but in the presence of CP-A the ID50 of the peptide improved to 8.5 X 10(-8) M. Similar results were obtained with MTX-Asp/CP-A and MTX-Ala/CP-B combinations. MTX-Arg showed good cytotoxicity (ID50 of 5.0 X 10(-8) M), due to CP-N activity in the fetal bovine serum of the culture medium; inclusion of CP-B lowered the ID50 to that of MTX. Possible clinical uses of MTX peptides are discussed.

Visualization of membrane-associated folate transport proteins.

Transport of Methotrexate (MTX) into cells, via the "reduced folate" transport system, is a critical factor in the effectiveness of the drug in cancer chemotherapy, and defective transport is one of the principal types of resistance to MTX. Probes capable of detecting membrane-associated folate transport proteins (ftp's) in individual cells are potentially useful for identifying structural and functional domains and for investigating mechanisms of substrate translocation. Polyclonal antibody to highly purified ftp from Lactobacillus casei, in conjunction with a second, gold-labeled antibody, has been used to visualize, via electron microscopy, the protein in Triton-treated membrane fragments and in the membrane and cytoplasm of spheroplasts. To visualize ftp in L1210 cells, the substrate-binding site was first labeled covalently with activated fluorescein-Methotrexate, and the cells were then treated with anti-fluorescein antibody and the gold-labeled antibody.

Overview: rational basis for development of fluoropyrimidine/5-formyltetrahydrofolate combination chemotherapy.

Fluorodeoxyuridylate (FdUMP) and thymidylate synthase (TS) are one of the better understood systems of drug-target interaction in cancer chemotherapy. Isolation and characterization of TS (initially from Lactobacillus casei and later from a variety of other sources), cloning and sequencing of the gene, determination of the 3-D structure of the enzyme by X-ray diffraction, and elucidation of the structure of both the catalytic intermediate and the enzyme-inhibitor complex have revealed critical parameters of the target at the molecular level. Potentiation of FdUMP binding by 5,10-methylenetetrahydrofolate (CH2-FH4), discovered at the enzymatic level, has been exploited to increase the clinical effectiveness of fluoropyrimidines. CH2-FH4 can be generated from folate, 5-methyltetrahydrofolate, or 5-formyltetrahydrofolate (citrovorum factor, CF); the latter is the compound of choice for therapeutic regimens. Transformation of CF to CH2-FH4 can occur via two pathways: (a) CF----5,10-methyltetrahydrofolate----CH2-FH4; or (b) CF----tetrahydrofolate----CH2-FH4. The relative importance of these pathways in various cells is not yet clear. The role of CH2-FH4 in FdUMP toxicity, and its central position in folate coenzyme-dependent C1 metabolism, emphasize the need for development of methods to quantitate intracellular levels of this compound.

Chemotherapeutic potential of methotrexate peptides.

MTX peptides in which the amino acid was linked to the alpha-carboxyl group have been prepared and examined for cytotoxicity before and after treatment with proteolytic enzymes. The alanine, aspartic acid and arginine derivatives (MTX-ala, MTX-asp and MTX-arg) were synthesized by a regio-specific route, following the general procedures of Rosowsky and Montgomery. Each compound was obtained in good yield, and purity was established by TLC, HPLC, absorbance spectra and elemental analyses. The MTX peptides were not hydrolyzed by a variety of proteolytic enzymes (e.g., trypsin, plasmin, urokinase, aminopeptidase). Pancreatic carboxypeptidase A, however, hydrolyzed MTX-ala readily, MTX-asp slowly and MTX-arg not at all. The MTX-ala and, to a lesser extent, MTX-arg were substrates for pancreatic carboxypeptidase B. MTX-arg was also hydrolyzed by the endogenous carboxypeptidase N in human serum. The cytotoxicity of these MTX peptides toward L1210 cells was measured in a microculture assay system using a tetrazolium dye. MTX-ala was weakly cytotoxic (ID50 = 2.0 x 10(-6)M) compared to MTX (ID50 = 2.4 x 10(-8)M). When MTX-ala was tested in the presence of carboxypeptidase A, the ID50 value improved to 8.5 x 10(-8)M. MTX-arg gave an ID50 of 5.0 x 10(-8)M, which was not unexpected in view of its susceptibility to hydrolysis by the carboxypeptidase activity present in the fetal calf serum of the culture medium. Inclusion of carboxypeptidase B lowered the ID50 value to 2.5 x 10(-8)M. Possible clinical uses of MTX peptides are discussed.

L1210 dihydrofolate reductase. Kinetics and mechanism of activation by various agents.

Dihydrofolate reductase from a methotrexate-resistant subline (R6) of L1210 mouse leukemia cells is activated (i.e. has its catalytic activity increased severalfold) by treatment with (a) sulfhydryl-modifying agents (p-chloromercuribenzoate (pCMB) or 5,5'-dithiobis(2-nitrobenzoic acid], (b) salts (KCl or NaCl), or (c) chaotropes (urea or guanidinium hydrochloride). With b or c activation is rapid (less than 10 s), but with a the process is much slower; at 25 degrees C, pseudo first-order rate constants for activation by excess pCMB or 5,5'-dithiobis(2-nitrobenzoic acid) are 0.45 and 0.08 min-1, respectively. Activation can also be monitored by conformational changes in the protein as indicated by enhanced fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate or by increased intrinsic fluorescence of tryptophan residues in the enzyme. Pseudo first-order rate constants for the pCMB-induced conformational change, measured by these fluorimetric procedures (0.45 min-1 and about 0.4 min-1, respectively), are in good agreement with the value obtained from the increase in catalytic activity. The rate of modification of the single cysteine residue in the enzyme by excess 14C-labeled pCMB, however, is faster than the rate of activation, indicating that the conformational change follows derivatization and is the rate-limiting step in the overall process. Activated forms of the enzyme are more labile to thermal denaturation or proteolysis than the untreated enzyme; the former process, however, is retarded by the presence of bovine serum albumin. Activation by the various agents is considered to involve a common mechanism in which interaction of the enzyme with the agents is followed by conformational changes in the enzyme, producing a series of forms that differ in microstructure, catalytic activity, and lability.