Long-term treatment with subcutaneous T-20, a fusion inhibitor, in HIV-infected patients: patient satisfaction and impact on activities of daily living.

T-20 is a novel antiretroviral agent that inhibits the fusion of human immunodeficiency virus (HIV) with target cell membranes. It is delivered by self-administered, twice-daily, subcutaneous injections. The impact of this mode of administration on patients' ability to conduct normal activities of daily living (ADL) and comply with a T-20 treatment regimen was assessed as part of a 48-week, phase 2 trial (T20-205). Patients' opinions on the impact of T-20 on ADL, ease of use of T-20, and choice to continue with T-20 were assessed by two questionnaires completed at baseline and week 48 (or study withdrawal). ADL were measured using a Likert-type scale based on established instruments with questions added to assess HIV-specific issues. Seventy previously treated patients received T-20 in combination with an average of five oral antiretroviral agents. Relative to other HIV/AIDS drugs, T-20 had little impact on ADL, with the majority of patients (54%-96%) agreeing (somewhat or strongly) that subcutaneous injections had not limited ADL. Patients found the injections relatively easy to perform with more than 47% of patients stating that each aspect of the injections (ease of injection, storage, reconstitution, and disposal of sharps) were very easy or easy. If medically indicated, 98% of patients stated that they would choose to continue with T-20. The most common reasons for this were the perceived effectiveness of T-20 and lack of side effects. In conclusion, the need to deliver T-20 via twice-daily subcutaneous injections was not considered an important barrier by HIV-positive patients seeking improvement or stabilization of their condition.

The CD58 (LFA-3) binding site is a localized and highly charged surface area on the AGFCC'C" face of the human CD2 adhesion domain.

Using site-directed mutagenesis in conjunction with NMR structural data on the adhesion domain of human CD2, we have defined the binding region for CD58. Previous structural studies of rat and human CD2 indicate that this adhesion domain is immunoglobulin-like. Here we report that the CD58 binding site is a well-circumscribed, charged surface area covering approximately 770 A2 on the AGFCC'C" face of the CD2 beta barrel. This site contains beta-strand residues in the carboxyl-terminal half of the F strand (including Lys-82 and Tyr-86), the top of the C strand (Asp-32 and Lys-34), and the C' strand (Gln-46), which are all solvent exposed. In addition, several exposed residues on the FG loop (Gly-90, Lys-91, Asn-92, and Val-93), the CC' loop (Lys-41 and Lys-43), and the C'C" loop (Arg-48 and Lys-51) form this site. In contrast, neither residues on the more peripheral G and C" strands of the same CD2 surface nor residues on B, E, and D strands of the opposite face are involved in CD58 binding. This CD58 binding site is predicted to lie most distal to the T-lymphocyte surface membrane, with ready access to CD58 on the surface of the opposing antigen-presenting cell.

A simple and rapid method for the purification of GroEL, an Escherichia coli homolog of the heat shock protein 60 family of molecular chaperonins.

GroEL, an Escherichia coli homolog of the heat shock protein 60 family of molecular chaperonins, has been implicated as a target of T cell-mediated immune responses in a broad spectrum of infections. In order to produce large quantities of native protein for raising and stimulating GroEL specific T cell lines, we have developed a simple and rapid two-step protocol for purifying native E. coli GroEL heat shock (or stress) protein which takes advantage of the inherent structural and functional properties of the protein. Based on a combination of gel exclusion chromatography, ATPase activity assay, isoelectric focusing, and circular dichroism analyses we conclude that our purification process yields native tetradecameric GroEL.

1H resonance assignments and secondary structure of the 13.6 kDa glycosylated adhesion domain of human CD2.

Human CD2, a glycosylated transmembrane receptor found on all T-lymphocytes, plays a key role in facilitating cellular adhesion between T-cells and target cells or antigen-presenting cells by binding to its counter receptor CD58 (LFA-3) present on the surface of those cells. All CD2 adhesion functions are localized within the amino-terminal 105-residue domain, which contains a single high mannose N-glycan required for maintaining both the conformational stability and CD58 binding properties of the glycoprotein. In order to better understand the structural basis for CD2-CD58-mediated adhesion and the critical role of the carbohydrate moiety in maintaining the functional stability of the molecule, we have determined the secondary structure of the N-glycosylated adhesion domain of human CD2 (hu-sCD2(105)) using NMR spectroscopy. Most of the 1H resonance assignments have been obtained from 1H-1H homonuclear 2D NMR spectra, which were further extended by applying 1H-15N heteronuclear 2D experiments on a hu-sCD2(105) sample selectively labeled with [15N]lysine. Thus, 98% of all backbone 1H resonances and over 80% of all side chain 1H resonances have been assigned. An overall topology characteristic of an immunoglobulin variable domain is observed, which consists of two beta-sheets comprised of three (residues 16-20, 67-71, and 60-63) and five (residues 94-103, 80-86, 32-37, 45-47, and 53-55) antiparallel beta-strands, respectively, with a hydrophobic core sandwiched between them. A ninth beta-strand (residues 7-12) makes parallel contacts to the carboxy-terminal beta-strand. NOEs between the N-linked glycan and the protein have tentatively been identified.

Structure of the glycosylated adhesion domain of human T lymphocyte glycoprotein CD2.

BACKGROUND: CD2, a T-cell specific surface glycoprotein, is critically important for mediating adherence of T cells to antigen-presenting cells or target cells. Domain 1 of human CD2 is responsible for cell adhesion, binding to CD58 (LFA-3) expressed on the cell to which the T cell binds. Human CD2 domain 1 requires N-linked carbohydrate to maintain its native conformation and ability to bind CD58. In contrast, rat CD2 does not require N-linked carbohydrate, and binds to a different ligand, CD48. RESULTS: The three-dimensional structure of the glycosylated form of domain 1 of human CD2 has been determined by NMR spectroscopy. The overall structure resembles the typical beta-barrel of an immunoglobulin variable domain. Nuclear Overhauser enhancement contacts between the protein and the N-linked glycan have been tentatively identified. CONCLUSION: Based on our results, we propose a model showing how the N-linked glycan might be positioned in the human CD2 domain 1 structure. The model provides an explanation for the observed instability of deglycosylated human CD2, and allows residues that are important for CD58 binding to be differentiated from those affecting conformational stability via interactions with the glycan.

Inhibition of T cell activation and adhesion functions by soluble CD2 protein.

The CD2 (T11) molecule belongs to a family of cell-surface glycoproteins that function as adhesion molecules in the immune system. Human CD2 is found exclusively on cells of the T lineage: peripheral T lymphocytes, NK cells, and thymocytes. CD2 binds specifically to the surface glycoprotein LFA-3. CD2/LFA-3 adhesion is the basis for the formation of rosettes between T cells and sheep erythrocytes (SRBC) which bear the sheep homologue of LFA-3. More importantly, CD2/LFA-3 adhesion functions in the immune system to augment T cell activation; it initiates conjugate formation between participating T cells and antigen-presenting cells (APC). We investigated the effects of soluble forms of CD2 (sCD2), produced in either baculovirus or CHO expression systems, on the rosetting of T cells with SRBC and on the activation of T cells by antigen plus major histocompatibility complex (MHC) molecules. Rosette formation between T cells and SRBC was completely inhibited by as little as 1 microM sCD2. Furthermore, sCD2 effectively inhibited (at micromolar concentrations) the T cell proliferative response to recall antigens including rubella, tetanus toxoid, and herpes simplex virus (HSV-1), as well as alloantigens in a mixed lymphocyte culture. These findings are consistent with the notion that the CD2/LFA-3 interaction augments antigen-specific T cell functions. The use of a CD2 "decoy" molecule rather than anti-CD2 or anti-LFA-3 antibodies to block the CD2/LFA-3 interaction rules out secondary antibody effects, via the Fc portion, as the basis for inhibition of T cell activation and directly stresses the importance of this adhesion interaction in T cell responses.

Detection of a glycosylation-dependent ligand for the T lymphocyte cell adhesion molecule CD2 using a novel multimeric recombinant CD2-binding assay.

The CD2 molecule plays an important role in T cell adhesion by interacting with the ligands CD58 (LFA-3) and CD59. In order to detect additional ligands for CD2, potentially of low binding affinity, we have prepared a highly fluorescent, multimeric form of rCD2 whose binding to cells can be quantified by flow cytometry. Initial studies demonstrated that binding of multimeric rCD2 to cells was CD2-specific, concentration and time dependent, and saturable. The negative charge on cells was also found to play a critical role in the efficiency of multimeric rCD2 binding. Analysis of binding of multimeric rCD2 to 17 CD58+ cell types revealed that only 8 of the cells exhibited binding. Failure of multimeric rCD2 to interact with the other cells could not be explained by differences in CD58 expression, suggesting that, in terms of CD2 binding, there are qualitative differences in CD58 on different cell types. Binding of multimeric rCD2 to six of the seven reactive cells was virtually totally inhibited by CD58 mAb pretreatment, whereas binding to the erythroleukemic line K562 was only partially blocked, suggesting the existence of another CD2 ligand. Subsequent studies demonstrated that the putative new ligand is not CD59, and that it interacts with a different region of the CD2 molecule than CD58, probably a site located between the T11(1) and T11(2) epitopes. The binding affinity of CD2 for the new ligand is 10-fold lower than for CD58 and, based on studies with truncated rCD2, the binding site for the new ligand is located within the amino-terminal 105 amino acids of the CD2 polypeptide. Unlike CD58, the new ligand is tunicamycin sensitive suggesting that it contains a N-linked carbohydrate structure that is essential for functional activity.

A soluble multimeric recombinant CD2 protein identifies CD48 as a low affinity ligand for human CD2: divergence of CD2 ligands during the evolution of humans and mice.

To search for possible ligands of CD2 distinct from CD58 (lymphocyte function-associated antigen 3), we have produced a soluble pentameric CD2-immunoglobulin (Ig) fusion protein (spCD2) linking the 182-amino acid human CD2 extracellular segment with CH2-CH3-CH4 domains of human IgM heavy chain, thus enhancing the micromolar affinity of the CD2 monomer through multimeric interaction. Using quantitative immunofluorescence and standard stringency wash conditions, we observed that the binding of spCD2 to human B lymphoblastoid JY cells and red blood cells is virtually inhibited by anti-CD58 TS2/9 monoclonal antibody, even though these cells express levels of CD48 and CD59 comparable to CD58. Consistent with these results, spCD2 did not show any binding to Chinese hamster ovary (CHO) cells transfected with human CD48 or CD59. However, binding studies on CD48-, CD58-, or CD59-transfected CHO cells with spCD2 under low stringency wash conditions revealed that human CD48 is a low affinity ligand of human CD2 compared with CD58 (Kd approximately 10(-4) vs. approximately 10(-6) M, respectively). The findings are noteworthy given that in the murine system CD48 is the major ligand for CD2. No detectable binding was observed to CD59-transfected CHO cells despite a report suggesting that CD59 may bind to the human CD2 adhesion domain. Importantly, in cell-cell adhesion assays between CD2+ Jurkat T cells and CD48- or CD59-transfected CHO cells, there was no conjugate formation, whereas binding of Jurkat T cells to CD58-transfected CHO cells was readily detected. Collectively, our findings provide evidence for a conservation of the CD2-CD48 interaction in the human species that may be of limited, if any, functional significance. Given the importance of the CD2-CD48 interaction in the murine system and CD2-CD58 interaction in humans, it would appear that there has been a divergence of functional CD2 ligands during the evolution of humans and mice.

N-glycosylation is required for human CD2 immunoadhesion functions.

The T-lymphocyte glycoprotein receptor, CD2, mediates cell-cell adhesion by binding to the surface molecule CD58 (LFA-3) on many cell types including antigen presenting cells. Two domains comprise the CD2 extracellular segment, with all adhesion functions localized to the amino-terminal domain that contains a single N-glycosylation site at Asn65. We have defined an important role for the N-linked glycans attached to Asn65 of this domain in mediating CD2-CD58 interactions and also characterize its N-glycotype structure. Analysis of deglycosylated soluble recombinant CD2 as well as a mutant transmembrane CD2 molecule containing a single Asn65-Gln65 substitution demonstrates that neither deglycosylated CD2 nor the mutant CD2 transmembrane receptor binds CD58 or monoclonal antibodies directed at native CD2 adhesion domain epitopes. Electrospray ionization-mass spectrometry demonstrates that high mannose oligosaccharides ((Man)nGlcNAc2, n = 5-9) are the only N-glycotypes occupying Asn65 when soluble CD2 is expressed in Chinese hamster ovary cells. Based on a model of human CD2 secondary structure, we propose that N-glycosylation is required for stabilizing domain 1 in the human receptor. Thus, N-glycosylation is essential for human CD2 adhesion functions.

Rapid, two-step purification process for the preparation of pyrogen-free murine immunoglobulin G1 monoclonal antibodies.

A cost-efficient process was specifically designed for the preparation of gram amounts of highly pure murine immunoglobulin (Ig) G1 monoclonal antibodies (mAbs). This rapid, simple and scalable purification process employs a unique binding and elution protocol for IgG1 mAbs on a silica-based, mixed-mode ion-exchange resin followed by conventional anion-exchange chromatography. mAbs are bound to BakerBond ABx medium at pH 5.6 directly from serum-supplemented hybridoma culture supernatants. Contaminating proteins and nucleic acids are removed by an intermediate wash at pH'6.5, followed by the specific elution of IgG1 mAbs with 100 mM Tris-HCl (pH 8.5). The mAb eluate is then loaded directly on to QAE-Sepharose Fast Flow medium and eluted with 10 mM sodium phosphate buffer (pH 7.4), containing 150 mM sodium chloride. The resulting IgG1 mAbs are greater than 98% pure, free from measurable endotoxin, formulated in a physiological buffer and suitable for in vivo applications.

A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties.

A strategy for the production of small, soluble, single-chain T-cell receptor (scTCR) fragments that carry an intact TCR antigen-combining site is presented. The rationale is based on structural similarity between TCR and antibody molecules and use of computer modeling methods to derive a model structure of a human scTCR variable (V)-domain dimer. A gene encoding the RFL3.8 TCR protein, specific for the hapten fluorescein in the context of major histocompatibility complex class II and composed of one V alpha and one V beta domain joined via a flexible peptide linker, was assembled in an Escherichia coli plasmid. Subsequently, the protein was produced in a bacterial expression system, purified, refolded, and found to be poorly soluble at neutral pH in aqueous buffers. An inspection of the computer-generated V alpha-V beta domain model showed several surface exposed hydrophobic residues. When these were replaced by water-soluble side chains via site-directed mutagenesis of the corresponding gene, a soluble protein resulted and was shown to have antigen-binding properties equivalent to those of the intact TCR of the RFL3.8 T-cell clone. These results demonstrate the feasibility of obtaining TCR fragments endowed with antigen-combining properties by protein engineering in E. coli.

Complementary roles for CD2 and LFA-1 adhesion pathways during T cell activation.

The influence of T cell receptor (TcR) triggering on T cell adhesion function has been systematically investigated in the present studies; we show that the adhesion function of LFA-1 is minimal in non-activated T cells but is augmented within minutes following TcR-mediated activation. In contrast, CD2 function is essentially optimal in non-activated T cells and undergoes no detectable modification within 12 h of TcR stimulation. Protein kinase C activation augments LFA-1 but not CD2 adhesion function and cyclic AMP reduces LFA-1 adhesion without affecting CD2-LFA-3 interactions. Up-regulation of the LFA-1 pathway occurs in the absence of any detectable surface redistribution of this molecule, suggesting an activation dependent modification leading to a high-affinity ICAM-1 binding state. The TcR independence of CD2 adhesion function implies a critical role of the CD2 pathway in initiating cell-cell interactions prior to TcR engagement and LFA-1-ICAM-1 binding and underscores the complementary nature of the CD2 and LFA-1 adhesion pathways during the immune response.

Structural and functional characterization of the CD2 immunoadhesion domain. Evidence for inclusion of CD2 in an alpha-beta protein folding class.

The T-lymphocyte transmembrane glycoprotein CD2 plays an important physiological role in facilitating adhesion between T-lymphocytes and their cognate cellular partners. This interaction is mediated by binding of CD2 to the broadly distributed surface polypeptide LFA-3 and augments the recognition function of the CD3-Ti antigen-major histocompatibility complex receptor via stabilization of conjugate formation between cells. To define better the structural components of the CD2 extracellular region which are important in contact-mediated cellular adhesion, a single-domain CD2 immunoadhesion protein has been prepared from papain digestion of a soluble two-domain CD2 molecule. This amino-terminal domain fragment binds to LFA-3 on human B-cells with a dissociation constant of 0.4 microM, possesses functional immunoadhesion epitopes as defined by the binding of monoclonal antibodies raised to native CD2, and retains the ability to inhibit sheep erythrocyte rosette formation with human T-cells. Thus, all of the immunoadhesion functions ascribed to CD2 reside within the amino-terminal domain. Circular dichroism analysis of the isolated CD2 adhesion domain suggests the presence of substantial alpha-helical character (22%), consistent with earlier computer modeling analyses that predicted a pattern of alternating alpha-helices and beta-sheets within the extracellular region of CD2. Despite the existence of short stretches of sequence homology between CD2 and immunoglobulin superfamily members, the circular dichroism data provide supporting biophysical evidence for classification of CD2 in an alpha-beta (either alpha/beta or alpha + beta) protein folding class.

Role of interaction of CD2 molecules with lymphocyte function-associated antigen 3 in T-cell recognition of nominal antigen.

The role of the interaction of CD2 molecules with lymphocyte function-associated antigen 3 (LFA-3) in facilitating nominal antigen recognition by T lymphocytes was studied by utilizing an HLA-DR4-restricted CD4+ cytotoxic human T-cell clone specific for human immunodeficiency virus envelope glycoprotein gp120 as a responder and murine fibroblasts transfected with human class II major histocompatibility complex (MHC) and/or human LFA-3 molecules as antigen-presenting cells (APC). Although expression of the DR4 restriction element in fibroblasts is sufficient for T-cell recognition of a gp120 peptide as judged by induction of proliferation coexpression of human LFA-3 on DR4+ APC decreases the molar requirement of nominal antigen by greater than one order of magnitude. Both LFA-3 and the relevant class II MHC molecules are necessary for antigen-independent conjugate formation, but the binding is further enhanced by specific nominal antigen. CD2-LFA-3 interaction is independent of T-cell receptor-MHC interaction and contributes directly to the stabilized conjugate between the T cell and LFA-3-bearing APC; soluble CD2 and monoclonal antibodies to LFA-3 and CD2 reduce T-cell-APC binding to the level mediated by nominal antigen and MHC. During conjugate formation, CD2 but not CD3 molecules are reorganized into the cell-cell interaction site in an antigen-independent manner. Thus, reorganization and/or coassociation of CD2 with CD3 molecules is not essential for T-cell activation.

Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin.

Recombinant human erythropoietin (rhEPO) has been purified to apparent homogeneity from a Chinese hamster ovary cell line expressing a cDNA clone of the human gene. NH2-terminal sequencing of the recombinant hormone indicates that the 27-residue leader peptide is correctly and consistently cleaved during secretion of the recombinant protein into conditioned medium, yielding the mature NH2 terminus (Ala-Pro-Pro-Arg...). Analysis of the COOH terminus of rhEPO by peptide mapping and fast atom bombardment mass spectrometry (FABMS) demonstrates that the arginyl residue predicted to be at the COOH terminus (based on confirmation of both genomic and cDNA sequences) is completely missing from the purified protein. The truncated form of the recombinant hormone, designated des-Arg166 rhEPO, displays an in vivo specific activity of greater than 200,000 units/mg protein. Structural characterization of natural human urinary EPO (uEPO) by peptide mapping and FABMS reveals that the urinary hormone is also missing the COOH-terminal Arg166 amino acid residue, a modification that remained undetected until now. There is no evidence of further proteolytic processing at the COOH terminus beyond specific removal of the Arg166 amino acid residue in either rhEPO or uEPO. On the basis of the FABMS data, we propose that the physiologically active form of the hormone circulating in plasma and interacting with target cells in vivo is des-Arg166 EPO.

Identification of the high-affinity lipid binding site in Escherichia coli pyruvate oxidase.

Pyruvate oxidase from Escherichia coli is a peripheral membrane associated enzyme which is activated by lipids. We have investigated the high-affinity lipid binding site associated with lipid activation of pyruvate oxidase by covalent attachment of [14C]lauric acid to the enzyme. Lauric acid is bound stoichiometrically (1 mol/mol of active sites), and the enzyme is essentially irreversibly activated. Mild tryptic digestion of the modified enzyme shows that the lauric acid is bound within the last 100 residues of the 572-residue monomer. Digestion with thermolysin releases two closely related peptides, A and B, in approximately equal amounts. Comparison of the amino acid composition of peptide A with the entire sequence of the protein shows that peptide A corresponds to the sequence from Ala-543 to Ile-554. The analysis of peptide B is very similar to that of A. Limited sequence analysis of peptide B shows that residue 1 is Ala and residue 2 is labeled. These results support the assignment of residue 1 in peptide B as Ala-543 and indicate that lauric acid is bound to Lys-544. Previous work in this laboratory has shown that pyruvate oxidase may be activated independently of lipids by mild protease digestion. Proteolytic activation is accompanied by the release of a small peptide (residues 550-572) from the carboxyl terminus of the protein. The present work locates the lipid binding site very close to this peptide. The significance of these results for the mechanism of activation of pyruvate oxidase and other lipid-activated systems is discussed.

Cloning, expression, and nucleotide sequence of the formate dehydrogenase genes from Methanobacterium formicicum.

The genes for the two subunits of the formate dehydrogenase from Methanobacterium formicicum were cloned and their sequences determined. When expressed in Escherichia coli, two proteins were produced which had the appropriate mobility on an SDS gel for the two subunits of formate dehydrogenase and cross-reacted with antibodies raised to purified formate dehydrogenase. The genes for the two formate dehydrogenase subunits overlap by 1 base pair and are preceded by DNA sequences similar to both eubacterial and archaebacterial promoters and ribosome-binding sites. The amino acid sequences deduced from the DNA sequence were analyzed, and the arrangement of putative iron-sulfur centers is discussed.

Isolation and characterization of cDNA clones for the 35-kDa pulmonary surfactant-associated protein.

A group of 35,000-dalton sialoglycoproteins is the major non-serum protein component of pulmonary surfactant. Tryptic fragments of these proteins were sequenced, and oligonucleotide probes were synthesized based on the amino acid sequences. A human lung cDNA library was then screened using the oligonucleotide probes, and clones coding for these proteins were identified and characterized. By in vitro transcription-translation experiments we have associated individual clones with particular proteins. The data suggest that co-translational modifications of two primary translation products account for many of the isoforms observed by two-dimensional gel electrophoresis in the precursors of 35,000-dalton sialoglycoproteins.

Characterization of the alpha-peptide released upon protease activation of pyruvate oxidase.

The pyruvate oxidase of Escherichia coli is a homo-tetrameric enzyme which can be activated greater than 500-fold (kcat/Km) by limited proteolytic digestion with alpha-chymotrypsin in the presence of pyruvate and thiamine pyrophosphate. The cleavage produces an Mr 2000 peptide (the alpha-peptide) from each subunit and mimics the physiologically important activation of the enzyme by phospholipids. Moreover, the proteolytic cleavage results in the loss of the high affinity lipid-binding site of the enzyme. We now report the isolation and characterization of the alpha-peptide fragment which is cleaved from the carboxyl terminus of each subunit by protease activation. Both the site of cleavage and the sequence of the alpha-peptide have been determined by a combination of Edman degradation of the purified peptide and DNA sequence analysis of the gene encoding the oxidase. The cleavage site lies within a sequence of hydrophobic amino acids predicted to form a beta-sheet. Another segment of the alpha-peptide is predicted to form an amphipathic alpha-helix. Quantitative assessment of the amphipathic nature of this alpha-helix (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623) gives a value very similar to the values for several helical peptides which spontaneously bind to the surface of phospholipid vesicles. From these analyses, we propose that the alpha-peptide may play a role in binding pyruvate oxidase to cell membrane phospholipids in vivo.

Isolation and characterization of the protease-activated form of pyruvate oxidase. Evidence for a conformational change in the environment of the flavin prosthetic group.

Pyruvate oxidase is a tetrameric enzyme consisting of four identical subunits. The specific activity of the enzyme may be increased more than 20-fold by limited proteolytic digestion by alpha-chymotrypsin in the presence of pyruvate and thiamin pyrophosphate. This "activation" phenomenon is due to the specific cleavage of an Mr = approximately 2000 peptide from each subunit. The Mr = 2000 "activation peptide" (alpha) may be readily separated from the activated enzyme by high performance liquid chromatography under nondenaturing conditions. The alpha peptide is not required to maintain the modified tetramer in the activated state. Cleavage of the alpha peptide from each monomer is directly correlated with a substantial change in the visible spectrum of the flavin, characteristic of a shift from a hydrophobic to a more hydrophilic environment. Proteolytic cleavage by alpha-chymotrypsin in the absence of thiamin pyrophosphate irreversibly inactivates the enzyme by cleavage at a different site, producing an Mr = approximately 9000 "inactivation peptide" (beta). The beta peptide remains noncovalently associated with the inactivated tetramer. Cleavage of the beta peptide does not alter the spectrum of the flavin, even though the beta peptide contains the alpha peptide sequence. These results suggest that cleavage and release of the alpha peptide opens up the flavin active site and may be directly responsible for the observed stimulation of enzymatic activity.