ABSTRACT
The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Adhesion/drug effects , Lovastatin/pharmacology , Lymphocyte Function-Associated Antigen-1/metabolism , Naphthalenes/pharmacology , Pravastatin/pharmacology , T-Lymphocytes/drug effects , Allosteric Site , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Disease Models, Animal , Drug Design , Humans , Integrin alpha4beta1 , Integrins/metabolism , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Function-Associated Antigen-1/chemistry , Lymphocyte Function-Associated Antigen-1/genetics , Lymphocyte Function-Associated Antigen-1/pharmacology , Macrophage-1 Antigen/metabolism , Mice , Models, Molecular , Molecular Structure , Mutagenesis, Site-Directed , Naphthalenes/toxicity , Peritonitis/drug therapy , Protein Binding/drug effects , Receptors, Lymphocyte Homing/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/physiologyABSTRACT
The interaction between the leukocyte function-associated antigen-1 (LFA-1) and the intercellular adhesion molecule is thought to be mediated primarily via the inserted domain (I-domain) in the alpha-subunit. The activation of LFA-1 is an early step in triggering the adhesion of leukocytes to target cells decorated with intercellular adhesion molecules. There is some disagreement in the literature over the respective roles of conformational changes in the I-domain and of divalent cations (Mg(2+), Mn(2+)) in the activation of LFA-1 for intercellular adhesion molecule binding. X-ray crystallographic structures of the I-domains of LFA-1 and Mac-1 in the presence and absence of cations show structural differences in the C-terminal alpha-helix; this change was proposed to represent the active and inactive conformations of the I-domain. However, more recent X-ray results have called this proposal into question. The solution structure of the Mg(2+) complex of the I-domain of LFA-1 has been determined by NMR methods, using a model-based approach to nuclear Overhauser enhancement spectroscopy peak assignment. The protein adopts the same structure in solution as that of the published I-domain X-ray structures, but the C-terminal region, where the X-ray structures are most different from each other, is different again in the solution structures. The secondary structure of this helix is well formed, but NMR relaxation data indicate that there is considerable flexibility present, probably consisting of breathing or segmental motion of the helix. The conformational diversity seen in the various X-ray structures could be explained as a result of the inherent flexibility of this C-terminal region and as a result of crystal contacts. Our NMR data are consistent with a model where the C-terminal helix has the potential flexibility to take up alternative conformations, for example, in the presence and absence of the intercellular adhesion molecule ligand. The role of divalent cations appears from our results not to be as a direct mediator of a conformational change that alters affinity for the ligand. Rather, the presence of the cation appears to be involved in some other way in ligand binding, perhaps by acting as a bridge to the ligand and by modulation of the charge of the binding surface.
Subject(s)
Lymphocyte Function-Associated Antigen-1/chemistry , Nuclear Magnetic Resonance, Biomolecular , Amino Acid Sequence , Autoantigens/chemistry , Autoantigens/metabolism , Binding Sites/drug effects , Cations, Divalent/metabolism , Cations, Divalent/pharmacology , Cell Adhesion Molecules/metabolism , Crystallization , Deuterium/metabolism , Humans , Ligands , Lymphocyte Function-Associated Antigen-1/metabolism , Magnesium/metabolism , Magnesium/pharmacology , Models, Molecular , Molecular Sequence Data , Pliability , Protein Structure, Secondary/drug effects , Protein Structure, Tertiary/drug effects , Solutions , Structure-Activity Relationship , ThermodynamicsABSTRACT
The lymphocyte function-associated antigen (LFA-1) belongs to the family of beta2-integrins and plays an important role in T-cell activation and leukocyte migration to sites of inflammation. We report here that lovastatin, a drug clinically used for lowering cholesterol levels, inhibits the interaction of human LFA-1 with its counter-receptor intercellular adhesion molecule-1. Using nuclear magnetic resonance spectroscopy and X-ray crystallography we show that the inhibitor binds to a highly conserved domain of the LFA-1 alpha-chain called the I-domain. The first three-dimensional structure of an integrin inhibitor bound to its receptor reveals atomic details for a hitherto unknown mode of LFA-1 inhibition. It also sheds light into possible mechanisms of LFA-1 mediated signalling and will support the design of novel anti-adhesive and immunosuppressive drugs.
Subject(s)
CD11 Antigens/metabolism , Lovastatin/pharmacology , Lymphocyte Function-Associated Antigen-1/metabolism , Amino Acid Sequence , Binding Sites , CD11 Antigens/chemistry , Cell Adhesion/drug effects , Crystallography, X-Ray , Humans , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Molecular Structure , Protein Binding/drug effects , Protein Structure, Secondary , Tumor Cells, CulturedABSTRACT
The molecular docking computer program SANDOCK was used to screen small molecule three-dimensional databases in the hunt for novel FKBP inhibitors. Spectroscopic measurements confirmed binding of over 20 compounds to the target protein, some with dissociation constants in the low micromolar range. The discovery that FK506 binding protein is a steroid binding protein may be of wider biological significance. Two-dimensional NMR was used to determine the steroid binding mode and confirmed the interactions predicted by the docking program.
Subject(s)
Chemistry, Pharmaceutical/methods , Enzyme Inhibitors/chemistry , Immunophilins/antagonists & inhibitors , Software , Steroids/chemistry , Binding Sites , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hydrogen Bonding , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Spectrometry, Fluorescence/methods , Steroids/metabolism , Steroids/pharmacology , Tacrolimus Binding ProteinsABSTRACT
A novel variant of the 13C/15N ω2 half-filter experiment is reported for studying the hydration of an unlabelled ligand bound to a 15N and 13C uniformly labelled biological macromolecule. This doubly tuned filter experiment represents a powerful tool for obtaining resonance assignments, structure determination and hydration properties of a ligand. Its application to the binary complex formed by the inserted-domain (I-domain) of the leukocyte function-associated antigen-1 (LFA-1) with a ligand reveals the presence of H2O molecules at the binding interface.
ABSTRACT
A novel X-filter experiment based on 1H PFG DQ spectroscopy is described. Excellent suppression of proton bound to 13C and 15N is achieved. Successful application of the method to a protein-ligand complex is demonstrated.
Subject(s)
Lymphocyte Function-Associated Antigen-1/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Binding , Algorithms , Carbon Isotopes , Electron Spin Resonance Spectroscopy , Glucose/chemistry , Hydrogen , Image Enhancement/methods , Ligands , Nitrogen Isotopes , Phenylalanine/chemistry , ProtonsABSTRACT
The three-dimensional structure of cyclopeptolide HUN-7293, a naturally-occurring inhibitor of cell adhesion molecule expression, has been determined from nuclear magnetic resonance data recorded in solution and from X-ray diffraction analysis of single crystals. The backbone conformation of HUN-7293 is characterized by two cis-peptide bonds in both the solution and crystalline state. Differences between the solution and crystal structure are visible for the orientation of some side chains and the strength of two transannular hydrogen bonds. Such structural information helps to provide insight into the molecular architecture of HUN-7293 on the atomic level and opens the way for structure-based modifications of this novel inhibitor of cell adhesion molecule expression.
Subject(s)
Cell Adhesion Molecules/drug effects , Fungal Proteins/chemistry , Fungal Proteins/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Amino Acid Sequence , Crystallography, X-Ray , Humans , Hydrogen Bonding , In Vitro Techniques , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Molecular Structure , Protein Conformation , SolutionsABSTRACT
Src homology 2 domains (SH2) are protein molecules found within a wide variety of cytoplasmic signalling molecules that bind with high affinity to phosphotyrosyl (pY)-containing protein sequences. We report here for crystal structure of the SH2 domain from the adaptor protein SHC (Shc), which has been refined by restrained least-squares methods to an R-factor of 17.3% to 2.7 A. The overall Shc architecture is essentially similar to that determined in other SH2 domains but it shows significant differences in a number of loops, thus providing a molecular surface with no obvious secondary pocket. Based on the knowledge of the crystal structure of the protein a model for a low affinity Shc-bound peptide has been generated from nuclear magnetic resonance data in solution using transferred nuclear Overhauser enhancements as intramolecular distance restraints. The model shows that the tyrosine moiety binds Shc in a rather similar way to that observed for other SH2-peptide complexes, but that the residue in position +3 does not seem to make specific contact with the protein. An intermolecular crystallographic interaction occurs between the pY-binding site and the C-terminal residues of a symmetry-related molecule. This crystal packing interaction suggests how inhibitory regulation could play a role in SHC activity.
Subject(s)
Models, Molecular , Phosphotyrosine/metabolism , Protein Conformation , Proteins/chemistry , src Homology Domains , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Proteins/metabolismABSTRACT
Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.
Subject(s)
Aldose-Ketose Isomerases , Carbohydrate Epimerases/metabolism , Escherichia coli/enzymology , Indole-3-Glycerol-Phosphate Synthase/metabolism , Multienzyme Complexes/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Carbohydrate Epimerases/isolation & purification , Chromatography, Gel , Chromatography, Ion Exchange , Enzyme Stability , Genes, Bacterial , Indole-3-Glycerol-Phosphate Synthase/isolation & purification , Kinetics , Molecular Sequence Data , Multienzyme Complexes/isolation & purification , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Operon , Protein Engineering , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrophotometry , Substrate SpecificityABSTRACT
Data from steady state and transient kinetics show that the functional phosphoribosyl anthranilate isomerase domain of the naturally bifunctional enzyme from Escherichia coli has properties similar to those of its artificially excised domain. The naturally monofunctional enzyme from Saccharomyces cerevisiae has significantly higher values of both kcat and kcat/KMPRA. The primary product of a single turnover of phosphoribosylanthranilate is fluorescent, but it slowly isomerizes to the nonfluorescent stable product. The latter is the competent substrate of indoleglycerol phosphate synthase, which catalyzes the subsequent step of tryptophan biosynthesis. The isomerization is characterized by a monoexponential decay independent of phosphoribosyl anthranilate isomerase. Due to a tentative assignment of the fluorescent, primary product and the nonfluorescent, stable product to an enol and a keto compound, respectively, tryptophan biosynthesis appears to be rate-limited by an uncatalyzed enol/keto tautomerization. A formal kinetic mechanism of the reaction catalyzed by phosphoribosyl anthranilate isomerase is proposed that is consistent with the combined enzymic and ligand binding properties of the three variants of phosphoribosyl anthranilate isomerase.
Subject(s)
Aldose-Ketose Isomerases , Carbohydrate Epimerases/metabolism , Escherichia coli/enzymology , Saccharomyces cerevisiae/enzymology , Kinetics , Mathematics , Models, Theoretical , Molecular Structure , Ribosemonophosphates/chemical synthesis , Ribosemonophosphates/metabolism , Spectrometry, Fluorescence , Time Factors , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/metabolismABSTRACT
New H(2)O-selective homonuclear and heteronuclear 2D NMR experiments have been designed for the observation of protein hydration (PHOGSY, Protein Hydration Observed by Gradient Spectroscop Y). These experiments utilize selective excitation of the H(2)O resonance and pulsed field gradients for coherence selection and efficient H(2)O suppression. The method allows for a rapid and sensitive detection of H(2)O molecules in labelled and unlabelled proteins. In addition it opens a way to measure the residence time of water bound to proteins. Its application to uniformly (15)N-labelled FKBP-12 (FK-506 binding protein) is demonstrated.
ABSTRACT
Intracellular protein phosphorylation by protein kinase C (PKC) plays a major role in the translation of extracellular signals into cellular events. Speculations on the structural basis for PKC activation are based on sequence homology between their cysteine-rich domains (CRD) and the DNA-binding 'zinc-fingers'. We produced a fragment comprising the second CRD (CRD2) of rat PKC-alpha and determined its three-dimensional structure in solution by NMR spectroscopy. This revealed that CRD2 adopts a globular fold allowing two non-consecutive sets of zinc-binding residues to form two separate metal-binding sites. The fold is different to those previously proposed and allows insight into the molecular topology of a family of homologous proteins.
Subject(s)
Cell Cycle Proteins , Models, Molecular , Protein Conformation , Protein Kinase C/chemistry , Amino Acid Sequence , Animals , Binding Sites , Chimerin 1 , Consensus Sequence , Cysteine , Diacylglycerol Kinase , Drosophila melanogaster/metabolism , Enzyme Activation , Fungal Proteins/chemistry , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-raf , Proto-Oncogene Proteins c-vav , Rats , Recombinant Fusion Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Solutions , Swine , Zinc FingersABSTRACT
The solution structure of the 53 amino acid peptide hormone, human epidermal growth factor (hEGF), has been determined to high resolution from nuclear magnetic resonance (n.m.r.) data. A large number of internuclear distance and dihedral restraints was obtained, including data from uniformly 15N-labelled hEGF. Dynamical simulated annealing methods using the program XPLOR were used for structure calculation. An improved protocol was developed combining efficient conformational searching at a reduced computational cost. The general fold of the calculated structures compared well with that of a derivative of the carboxy-terminally truncated hEGF determined previously. A group of 44 structures were calculated with no violations greater than 0.3 A and 3 degrees for distance and dihedral restraints, respectively. The average pairwise root mean square (r.m.s.) deviation of all backbone atoms for these structures was 2.25 A for all 53 residues, 0.92 A for the bulk of the protein, and 0.23 A for the functionally important carboxy-terminal domain. Two new helical segments containing highly conserved amino acids have been identified; one between cysteines 6 and 14 and a second at the end of the carboxy-terminal domain. New insight into the molecular architecture of the site of putative receptor binding was provided by comparing the structure of hEGF with its biologically equipotent analogue, human transforming growth factor alpha. This comparison revealed a close structural relationship between the two growth factors and provides an improved understanding of the structure/function relationships in EGF.
Subject(s)
Epidermal Growth Factor/chemistry , Transforming Growth Factor alpha/genetics , Amino Acid Sequence , Humans , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protein Conformation , Recombinant Proteins , SolutionsABSTRACT
In order to elucidate the mechanism of interaction between human epidermal growth factor (EGF) and its receptor, selected variants of EGF, differing by single amino acid substitutions, have been made by site-directed mutagenesis. The receptor affinity of these mutants was determined by a receptor binding competition assay, and the effects of the substitution on the structure of the protein were assessed by 1H nuclear magnetic resonance techniques. Various substitutions of Arg-41 resulted in substantial reduction in receptor affinity of EGF whereas change of Tyr-13 did not affect binding to the receptor. The 1H resonances of all nonexchangeable protons of the Tyr-13----Leu, Arg-41----His, and Leu-47----Glu variants were assigned and compared in order to assess the structural integrity of these mutants, which possess very different spectral and biological properties. In the case of the Leu-47----Glu mutant, only minor localized spectral changes were observed, confirming that the tertiary structure of the protein is preserved upon mutation. In contrast, for both the Arg-41----His and Tyr-13----Leu variants, significant and strikingly similar spectra changes were observed for many residues located far away from the mutated residues. This implies that similar structural alterations have taken place in both proteins, an idea further supported by hydrogen-exchange experiments where the exchange rates of hydrogen-bonded amide protons for both the Tyr-13----Leu and the Arg-41----His mutants were found to be about 4 times faster than in the wild-type protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Epidermal Growth Factor/genetics , Mutagenesis, Site-Directed , Animals , Arginine/genetics , Epidermal Growth Factor/chemistry , Epidermal Growth Factor/physiology , ErbB Receptors/metabolism , Genetic Variation , Haplorhini , Humans , Magnetic Resonance Spectroscopy , Mice , Protein Conformation , Radioligand Assay , Structure-Activity Relationship , Tyrosine/genetics , Vero CellsABSTRACT
Anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae has been purified to homogeneity from an overproducing strain. Analytical ultracentrifugation demonstrated that the enzyme is a dimer of Mr = 83,000 +/- 4,000 (S20.w = 4.7 S). Moreover, as shown by active enzyme sedimentation, the enzyme remains dimeric even at low concentrations. The presence of yeast phosphoribosylanthranilate isomerase in the gradient does not lead to complex formation between the two enzymes as might be expected if phosphoribosyl anthranilate, the very labile product of the anthranilate phosphoribosyltransferase, were channelled to phosphoribosylanthranilate isomerase in vivo. The steady-state-kinetic behaviour of the enzyme suggests that catalysis involves a ternary enzyme-substrate complex, with KANTm = 1.6 microM, and KPRib-PPm = 22.4 microM. The enzyme has been used to generate phosphoribosylanthranilate in situ for kinetic studies of phosphoribosylanthranilate isomerase from Escherichia coli: KPRAm = 5 microM, kcat = 40 s-1.
Subject(s)
Aldose-Ketose Isomerases , Anthranilate Phosphoribosyltransferase/isolation & purification , Pentosyltransferases/isolation & purification , Saccharomyces cerevisiae/enzymology , Anthranilate Phosphoribosyltransferase/antagonists & inhibitors , Carbohydrate Epimerases/isolation & purification , Carbohydrate Epimerases/metabolism , Catalysis , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Kinetics , Magnesium/pharmacologyABSTRACT
An important question in protein folding is whether the natural amino and carboxyl termini and the given order of secondary structure segments are critical to the stability and to the folding pathway of proteins. Here it is shown that two circularly permuted versions of the gene of a single-domain beta alpha barrel enzyme can be expressed in Escherichia coli. The variants are enzymically active and are practically indistinguishable from the original enzyme by several structural and spectroscopic criteria, despite the creation of new termini and the cleavage of a surface loop. This novel genetic approach should be useful for protein folding studies both in vitro and in vivo.
Subject(s)
Aldose-Ketose Isomerases , Carbohydrate Epimerases/genetics , Cloning, Molecular , Escherichia coli/enzymology , Genes , Protein Conformation , Amino Acid Sequence , Base Sequence , Carbohydrate Epimerases/metabolism , Circular Dichroism , Enzyme Stability , Escherichia coli/genetics , Genetic Variation , Kinetics , Molecular Sequence Data , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
By means of 522 singles in breech presentation the pH of umbilical artery (pH u. a.) and Apgar score in comparison to the mode of delivery, duration of pregnancy and weight of neonates have been analysed. The frequency of deliveries by cesarean section in breech presentation was 28.3%, the rate of preterms in these cases amounts to 14.6% and the perinatal mortality to 42.1%. The average value of pH u.a. after vaginal delivery (7.25) was significantly different to the value after cesarean section (7.28). 25% of the pH-values were below 7.20, 10% below 7.10. Mixed acidoses dominated, metabolic ones after vaginal and abdominal deliveries equalled each other. Vaginal deliveries were frequently followed by respiratory acidoses. In 81% of one-minute-Apgar and in 93% of five-minute-Apgar, the score was above 7. According to our experiences the only determination of the pH u.a. after breech deliveries is not sufficient, because of being at risk for the fetus it demands the measurement of the complete acid-base-status. A critical comparison of these data with the clinical status of the newborn is necessary.
Subject(s)
Apgar Score , Breech Presentation , Fetal Blood/metabolism , Infant, Premature, Diseases/blood , Acidosis/blood , Cesarean Section/mortality , Female , Humans , Hydrogen-Ion Concentration , Infant, Newborn , PregnancyABSTRACT
By means of 149 fetal microblood samplings (FBA) from the breech in deliveries in breech presentation and by means of 64 cardiotocograms the validity of biochemical parameters and biophysical ones of fetal monitoring will be investigated. It takes an increased number of suspect or pathologic CTG into account in cases of breech presentation. By means of FBA it is possible to exclude or to confirm the supposed acidoses. By this the rate of operations during delivery can be reduced or the operation will be indicated respectively. A normal fetal pH resulting from the general biochemical monitoring does enlarge the obstetricians license when the fetus presents himself in advanced parturition in pelvic floor (hold back of breech during assisted spontaneous delivery). A pH less than 7.15 demands an immediate delivery. A prognosis of fetal outcome by means of FBA and CTG is not possible due to difficulties in calculation of the final stage of breech presentation. The difference between fetal pH measured in pelvic floor and pH in umbilical artery must be kept as small as possible and it might be regarded as criterion of the management of the delivery in breech presentation.
Subject(s)
Breech Presentation , Cardiotocography , Fetal Blood/metabolism , Acidosis/blood , Acidosis/diagnosis , Apgar Score , Blood Gas Analysis , Female , Humans , Hydrogen-Ion Concentration , Infant, Newborn , Obstetric Labor Complications/blood , PregnancyABSTRACT
The binding properties of a monoclonal antibody to an early activation marker of human lymphocytes are described. The monoclonal antibody BL-Ac/p26 binds to an antigen which is expressed on activated lymphocytes. This antigen is not detectable on resting lymphocytes or other blood cells. The surface radioiodinated antigen of activated T cells isolated by the monoclonal antibody BL-Ac/p26 is separated by SDS-PAGE in a major 26k Da component and a minor band (32 kDa) under reducing conditions. These polypeptide chains form disulphide linked dimers on the cell surface (Mr 55-77 kDa). This 26 kDa antigen is a very early activation marker of T lymphocytes. Human T lymphocytes stimulated by mitogens (PHA, ConA or PWM), anti-TCR/CD3 monoclonal antibodies or allogeneic leucocytes express this 26 kDa antigen after 2-4 hours.