A streptavidin mutant with altered ligand-binding specificity

The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between biotin and these biotin analogs. Free energy calculations predicted that the introduction of a negative charge at the position of S27 plus the mutation N23A should disrupt two of the three hydrogen bonds between natural streptavidin and the ureido oxygen of biotin. In contrast, the imino hydrogen of 2-iminobiotin should form a hydrogen bond with the side chain of an acidic amino acid at position 27. This should reduce the biotin-binding affinity by approximately eight orders of magnitude, while leaving the affinities for these biotin analogs virtually unaffected. In good agreement with these predictions, a streptavidin mutant with the N23A and S27D substitutions binds 2-iminobiotin with an affinity (Ka) of 1 x 10(6) M-1, two orders of magnitude higher than that for biotin (1 x 10(4) M-1). In contrast, the binding affinity of this streptavidin mutant for diaminobiotin (2.7 x 10(4) M-1) was lower than predicted (2.9 x 10(5) M-1), suggesting the position of the diaminobiotin in the biotin-binding site was not accurately determined by modeling.     

Reactions of the eco RV restriction endonuclease with fluorescent oligodeoxynucleotides: identical equilibrium constants for binding to specific and non-specific DNA

The EcoRV restriction endonuclease cleaves DNA specifically at its recognition sequence in the presence of magnesium ions, but several studies have indicated that it binds to DNA in the absence of Mg2+ without any preference for its recognition site. However, specific binding to the recognition site has also been reported. To distinguish between these reports, oligodeoxynucleotides were tagged with either dansyl or eosin fluorophores at their 5' termini and annealed to form duplexes of 12 to 16 base-pairs. For each length of duplex, one derivative had the EcoRV recognition sequence while another lacked this sequence. For the duplexes with the recognition site, the fluorophores had no effect on DNA cleavage rates by EcoRV in the presence of Mg2+. The binding of the specific and non-specific duplexes to EcoRV in the absence of Mg2+ was measured by fluorescence resonance energy transfer and by fluorescence depolarization. In both procedures, the signal from the specific complex differed from the complex with non-specific DNA, with the depolarization data indicating that non-specific DNA bound to EcoRV retains a higher rotational freedom than specific DNA. Even so, the equilibrium constant for the binding of specific DNA was identical, within error limits, to that for non-specific DNA.     

Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the C1 beta of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher Kcat/Km for 1-chlorohexane and a 2-fold higher Kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in Kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.     

Isolation of a non-classical mutant of the DNA recognition subunit of the type I restriction endonuclease R.EcoR124I

We have used deletion mutagenesis and PCR-based misincorporation mutagenesis to produce a collection of mutations in the central conserved region of the DNA binding subunit of the type IC restriction endonuclease EcoR124I. It has been proposed that this domain is involved in protein-protein interactions during the assembly of the endonuclease. While a large percentage of these mutations gave a classical Res- Mod- phenotype, one mutant was isolated with a nonclassical Res- Mod+ phenotype. The loss of restriction activity, but retention of the ability to modify indicates that this mutation cannot affect DNA binding and must alter the assembly of the endonuclease in such a way as to prevent DNA cleavage but allow methylation. This mutant resulted from a single amino acid change Trp212-->Arg. The location of the single amino acid change is at the border of the central conserved region and the second target recognition domain (TRD2) and suggests that this region is extremely important for the assembly of the methylase with the HsdR subunit into a functional restriction endonuclease.     

Site-directed mutagenesis of putative active site residues of MunI restriction endonuclease: replacement of catalytically essential carboxylate residues triggers DNA binding specificity

Mapping of the conserved sequence regions in the restriction endonucleases MunI (C/AATTG) and EcoRI (G/AATTC) to the known X-ray structure of EcoRI allowed us to identify the sequence motif 82PDX14EXK as the putative catalytic/Mg2+ ion binding site of MunI [Siksnys, V., Zareckaja, N., Vaisvila, R., Timinskas, A., Stakenas, P., Butkus, V., & Janulaitis, A. Gene (1994) 142, 1-8]. Site-directed mutagenesis was then used to test whether amino acids P82, D83, E98, and K100 were important for the catalytic activity of MunI. Mutation P82A generated only a marginal effect on the cleavage properties of the enzyme. Investigation of the cleavage properties of the D83, E98, and K100 substitution mutants, however, in vivo and in vitro, revealed either an absence of catalytic activity or markedly reduced catalytic activity. Interestingly, the deleterious effect of the E98Q replacement in vitro was partially overcome by replacement of the metal cofactor used. Though the catalytic activity of the E98Q mutant was only 0.4% of WT under standard conditions (in the presence of Mg2+ ions), the mutant exhibited 40% of WT catalytic activity in buffer supplemented with Mn2+ ions. Further, the DNA binding properties of these substitution mutants were analyzed using the gel shift assay technique. In the absence of Mg2+ ions, WT MunI bound both cognate DNA and noncognate sequences with similar low affinities. The D83A and E98A mutants, in contrast, in the absence of Mg2+ ions, exhibited significant specificity of binding to cognate DNA, suggesting that the substitutions made can simulate the effect of the Mg2+ ion in conferring specificity to the MunI restriction enzyme.     

Programming of enzyme specificity by substrate mimetics: investigations on the Glu-specific V8 protease reveals a novel general principle of biocatalysis

BACKGROUND: The purpose of the study was to determine the accuracy and role of the sentinel node technique in patients with non-small cell lung cancer. METHODS: This study was carried out on 36 consecutive patients undergoing lung resection. Peritumoral tissue was infiltrated with isosulfan blue dye and the first lymph node to stain was identified as a sentinel node. Sensitivity and specificity of the sentinel node in predicting the status of other lymph node stations were determined. RESULTS: Seventeen patients had sentinel lymph nodes. In 9 of these 17 cases neither the sentinel node nor any other lymph node contained metastatic carcinoma. In 5 cases the sentinel node was in the mediastinum and documented unexpected N2 disease. In 19 patients no sentinel node was found. Final lymph node statuses were N0 in 13 patients, N1 in 5, and N2 in 1. CONCLUSIONS: The use of isosulfan blue for intraoperative lymphatic mapping is feasible. The specificity in our experience was good; 9 of 9 patients with negative sentinel nodes were found to be N0 on the final pathology report. Unexpected N2 disease was found in 5 patients. The accumulation of further experience will determine the role of the sentinel node technique in patients with non-small cell lung cancer.     

Characterization of an exocellular protein phosphatase with dual substrate specificity from the yeast Yarrowia lipolytica

In previous work, the major endocellular protein phosphatase activity has been identified in the secretory yeast Yarrowia lipolytica as a PP2A. The aim of the present work was to seek the presence of one protein phosphatase excreted in the exocellular medium and to study its activity during yeast growth in media supplemented or not supplemented with inorganic phosphate. Protein phosphatase was purified and activity was assayed by following the dephosphorylation of three substrates, [32P]casein, phosphotyrosine and a synthetic tyrosine-phosphorylated peptide. Phosphatase activity recovered in the medium after 25 h culture was greatly enhanced by Pi-deficiency. After several purification steps, the enzyme preparation presents an apparent electrophoretic homogeneity on SDS-PAGE with associated phosphoseryl/threonyl and phosphotyrosyl activities. The kinetic properties exclude contamination by a copurified protein and it is concluded that the two activities are carried by the same single proteic species. It was characterized by gel filtration as a 33 kDa protein with one single subunit demonstrated by SDS-PAGE. An absolute requirement for reducing-agents is observed suggesting that the enzyme contains at least one essential reactive cysteinyl residue. Optimum pH value is 6.1, apparent K(m) for phosphotyrosine was calculated to be 760 microM and Hill coefficient 3.2 indicating a rather high cooperativity. These results showed that the involvement of alkaline and/or acid phosphatase was unlikely. In conclusion, a protein phosphatase distinct from endocellular PP2A is secreted by Yarrowia lipolytica and characterized as a phosphotyrosine protein phosphatase with associated phosphoseryl/threonyl activity.     

Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB

FhuD is the periplasmic binding protein of the ferric hydroxamate transport system of Escherichia coli. FhuD was isolated and purified as a His-tag-labeled derivative on a Ni-chelate resin. The dissociation constants for ferric hydroxamates were estimated from the concentration-dependent decrease in the intrinsic fluorescence intensity of His-tag-FhuD and were found to be 0.4 microM for ferric aerobactin, 1.0 microM for ferrichrome, 0.3 microM for ferric coprogen, and 5.4 microM for the antibiotic albomycin. Ferrichrome A, ferrioxamine B, and ferrioxamine E, which are poorly taken up via the Fhu system, displayed dissociation constants of 79, 36, and 42 microM, respectively. These are the first estimated dissociation constants reported for a binding protein of a microbial iron transport system. Mutants impaired in the interaction of ferric hydroxamates with FhuD were isolated. One mutated FhuD, with a W-to-L mutation at position 68 [FhuD(W68L)], differed from wild-type FhuD in transport activity in that ferric coprogen supported promotion of growth of the mutant on iron-limited medium, while ferrichrome was nearly inactive. The dissociation constants of ferric hydroxamates were higher for FhuD(W68L) than for wild-type FhuD and lower for ferric coprogen (2.2 microM) than for ferrichrome (156 microM). Another mutated FhuD, FhuD(A150S, P175L), showed a weak response to ferrichrome and albomycin and exhibited dissociation constants two- to threefold higher than that of wild-type FhuD. Interaction of FhuD with the cytoplasmic membrane transport protein FhuB was studied by determining protection of FhuB degradation by trypsin and proteinase K and by cross-linking experiments. His-tag-FhuD and His-tag-FhuD loaded with aerobactin specifically prevented degradation of FhuB and were cross-linked to FhuB. FhuD loaded with substrate and also FhuD free of substrate were able to interact with FhuB.     

Ribonuclease P substrate specificity: cleavage of a bacteriophage phi80-induced RNA

RNase P can cleave in vitro a bacteriophage phi80-induced RNA which is 62 nucleotides long [M3 RNA, G. Pieczenik et al. (1972) Arch. Biochem. Biophys. 152, 152-165] to yield two specific fragments 25 and 37 nucleotides long. As is the case for another substrate of RNase P; the precursor to Escherichia coli 4.5S RNA, the cleavage site in M3 RNA is at the end of a long double-stranded region immediately adjacent to a single-stranded segment. Similar nucleotide sequences span the cleavage site in both substrates. These and other features of the reaction of RNase P with M3 and 4.5S precursor RNA are different from some aspects of the reaction of this enzyme with tRNA precursor molecules. A qualitative scheme is presented that is directed towards the understanding of the differences in RNase P cleavage site specificity for these substrates.     

A mutant antigen-presenting cell defective in antigen presentation expresses class II MHC molecules with an altered conformation

Ag presentation by APC to class II MHC-restricted T cells involves a sequence of events: 1) intracellular processing of protein Ag into immunogenic peptides, 2) specific binding of peptides to class II MHC molecules, and then 3) transport of the MHC-peptide complexes to the plasma membrane. The critical event in the activation of T cells by APC is the recognition of MHC-associated antigenic determinants by the TCR/CD3 complex. In this report we describe the isolation and characterization of a mutant APC with a defect in an intracellular process that results in its inability to form MHC-peptide complexes for recognition by T cells. The mutant APC cannot present many different protein Ag with both I-A and I-E molecules but is able to present processing-independent peptides. The functional defect in the mutant APC is not caused by either a decrease in expression or a structural mutation in class II MHC molecules. Further, there is no mutation in the invariant chain (li) and it displays a normal kinetics of association and dissociation from the class II MHC molecules during biosynthesis. Although the mutation is not in the genes encoding for the class II MHC molecules or li, the mutant APC expresses class II MHC molecules with distinct serological epitopes suggestive of an altered conformation. Pulse-chase experiments suggest that a conformational difference between I-Ad molecules of wild-type and mutant cells occurs after the class II molecules exit from the endoplasmic reticulum but while they are still associated with li. The mutant cell produces few compact (SDS-resistant) class II heterodimers. This mutant APC provides a tool for studying the cell biology of Ag processing and presentation.     

Crystal structures of the inactive D30N mutant of feline immunodeficiency virus protease complexed with a substrate and an inhibitor

Crystal structures of complexes of a D30N mutant of feline immunodeficiency virus protease (FIV PR) complexed with a statine-based inhibitor (LP-149), as well as with a substrate based on a modification of this inhibitor (LP-149S), have been solved and refined at resolutions of 2.0 and 1.85 A, respectively. Both the inhibitor and the substrate are bound in the active site of the mutant protease in a similar mode, which also resembles the mode of binding of LP-149 to the native protease. The carbonyl oxygen of the scissile bond in the substrate is not hydrated and is located within the distance of a hydrogen bond to an amido nitrogen atom from one of the two asparagines in the active site of the enzyme. The nitrogen atom of the scissile bond is 3.25 A from the conserved water molecule (Wat301). A model of a tetrahedral intermediate bound to the active site of the native enzyme was built by considering the interactions observed in all three crystal structures of FIV PR. Molecular dynamics simulations of this model bound to native wild-type FIV PR were carried out, to investigate the final stages of the catalytic mechanism of aspartic proteases.     

Comparative studies on the substrate specificity of lecithin:cholesterol acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates

We have studied, in a prospective blinded fashion, the effects of regular and extended-release gemfibrozil on plasma lipoprotein and apolipoprotein (apo) levels in hypercholesterolemic subjects with decreased high density lipoprotein (HDL) cholesterol (C) levels. Study participants were men and women 19 to 80 years of age with baseline plasma low density lipoprotein (LDL) C levels > or = 4.5 mmol/l (175 mg/dl), HDL-C levels < or = 1.2 mmol/l (45 mg/dl), and triglyceride levels < or = 3.4 mmol/L (300 mg/dl). All subjects were stabilized on a diet for eight weeks prior to entry into two different protocols. In the first protocol 229 subjects were randomized to placebo or extended-release gemfibrozil (1200 mg/day) for 3 months (placebo trial). In the second protocol 655 subjects were randomized to regular or extended-release gemfibrozil (1200 mg/day) for 6 months (equivalency trial). Changes in lipids and apos were stratified by baseline HDL-C levels (< 0.9 mmol/l, and 0.9-12.2 mmol/l). In both studies, treatment with gemfibrozil, either regular or extended-release, was associated with significant (P < 0.05) decreases in plasma very low density lipoprotein (VLDL) C and triglyceride levels of 42-45% and 33-37%, respectively, in subjects with HDL-C level < 0.9 mmol/l, and of 38-47% and 32-39%, respectively, in patients with HDL-C levels of 0.9-1.2 mmol/l. Modest reductions from baseline in directly measured LDL-C levels were observed in both groups (3-6% and 8-9%, respectively). These reductions were less than those observed for calculated LDL-C (7-10% and 11%, respectively). For apo B, reductions were 11-14% and 16-17% in the two groups. HDL-C, apo A-I, and apo A-II levels increased by 15-16%, 5-6%, and 21-25%, respectively, in patients with HDL-C < 0.9 mmol/l, and by 6-7%, 2-3%, and 19-22%, respectively, in patients with HDL-C of 0.9-1.2 mmol/l. These differences in HDL-C levels reached statistical significance in the equivalency trial (P < 0.0001) and were independent of baseline triglyceride levels. Our data indicate that gemfibrozil, either regular or extended-release, is highly effective in lowering plasma triglyceride levels and increases HDL-C levels by approximately 15% in hypercholesterolemic patients with low HDL-C levels (< 0.9 mmol/l). Moreover, this agent lowers VLDL-C somewhat more than triglyceride, resulting in an underestimation of calculated VLDL-C reductions and in an overestimation of calculated LDL-C reductions. This agent also raises apo A-II levels much more than apo A-I levels.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.     

Mannolipid donor specificity of glycosylphosphatidylinositol mannosyltransferase-I (GPIMT-I) determined with an assay system utilizing mutant CHO-K1 cells

The microsomal enzyme glycosylphosphatidylinositol mannosyltransferase I (GPIMT-I) catalyses the transfer of a mannosyl residue from beta-mannosylphosphoryldolichol (beta-Man-P-Dol) to glucosamine-alpha(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Man alpha(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis. While the transfer of [3H]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [3H]Man-P-Dol, GPIMT-I activity could be characterized with an in vitro enzyme assay system employing membrane fractions from Lec15 or Lec35 cells. These CHO cell mutants apparently contain elevated levels of endogenous GlcN-aPI due to the inability to synthesize (Lec15) or utilize (Lec35) beta-Man-P-Dol in vivo. The presence of a saturated alpha-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since beta-mannosylphosphorylpolyprenol (beta-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as beta-[3H]Man-P-Dol as a mannosyl donor. When beta-[3H]-Man-P-Dol and alpha-[3H]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the beta-mannosyl-phosphoryl linkage. beta-[3H]Man-P-dolichols containing 11 or 19 isoprenyl units were equally effective substrates for GPIMT-I. Membrane fractions from Lec 9, a CHO mutant that apparently lacks polyprenol reductase activity and synthesizes very little beta-Man-P-Dol, but accumulates beta-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with beta-[3H]Man-P-Dol in vitro. This indirect assay suggests that GlcN-aPI does not accumulate in Lec 9 cells, possibly because it is mannosylated via beta-Man-P-Poly, or perhaps the small amount of Man-P-Dol formed by the mutant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of an amino acid insertion into the omega loop region of a class C beta-lactamase on its substrate specificity

The extended-substrate specificity of Enterobacter cloacae GC1 beta-lactamase is entirely due to a three amino acid insertion after position 207. To clarify the reason for the extended-substrate specificity, Ala, Ala-Ala, Ala-Ala-Ala, and Ala-Ala-Ala-Ala were inserted after position 207 on the basis of the class C beta-lactamase from E. cloacae P99, respectively. The kcat and Km values of all the mutant enzymes for cephalothin, benzylpenicillin and ampicillin were almost the same as those of the wild-type enzyme, except for those of P99-210-4A which were decreased 4-15-fold. On the other hand, the kcat and Km values for oxyimino beta-lactams such as cefuroxime, ceftazidime, and aztreonam increased with increasing numbers of inserted alanines. The kcat values of the mutant enzymes for cefroxime increased 140-7400-fold compared with that of the wild-type. The Km values also increased with almost the same magnitude, resulting in about the same kcat/Km values as that of the wild-type. On progressive inhibition analysis of aztreonam of the mutant enzymes, two kinds of inactive acyl-enzyme with distinct stabilities were observed, and the proportion of the less stable inactive enzyme increased with increasing numbers of inserted alanines. This suggests that the extension of the substrate specificity is due to instability of the acyl-intermediate caused by an increased deacylation rate in the reaction process.     

The fatty liver dystrophy mutant mouse: microvesicular steatosis associated with altered expression levels of peroxisome proliferator-regulated proteins

Fatty liver dystrophy ( fld) is an autosomal recessive mutation in mice characterized by hypertriglyceridemia and fatty liver during neonatal development. The fatty liver in fld/fld mice spontaneously resolves between the age of 14-18 days, at which point the animals develop a neuropathy associated with abnormal myelin formation in peripheral nerve. We have investigated the morphological and biochemical alterations that occur in the fatty liver of neonatal fld/fld mice. Studies at the light and electron microscopic level demonstrated the accumulation of lipid droplets and hypertrophic parenchymal cells in fld neonates, with no apparent liver pathology after resolution of the fatty liver. To better characterize the biochemical basis for the development of fatty liver in fld mice, we compared protein expression patterns in the fatty liver of fld mice and in the liver of phenotypically normal (wild-type) littermates using quantitative two-dimensional gel electrophoresis. We detected 24 proteins with significantly altered expression levels (P < 0.001) in the fld fatty liver, 15 of which are proteins that are altered in abundance by peroxisome proliferating chemicals. As these compounds characteristically elicit changes in the expression of mitochondrial and peroxisomal enzymes involved in fatty acid oxidation, we quantitated rates of fatty acid oxidation in hepatocytes isolated from fld and wild-type mice. These studies revealed that hepatic fatty acid oxidation in fld neonates is reduced by 60% compared to wild-type littermates. In hepatocytes from adult fld mice that no longer exhibit a fatty liver, oxidation rates were similar to those in hepatocytes from age-matched wild-type mice. These findings indicate that altered expression of proteins involved in fatty acid oxidation is associated with triglyceride accumulation in the fld fatty liver.     

Peptide-induced T cell clones: specificity, MHC restriction, proliferation and cytokine pattern as a function of different stimulations

CD4+ T cell clones were generated to tetanus toxin or to two tetanus toxin-derived peptides p2 (AA 830-834) and p30 (AA 947-976). 11 of the 24 p30-specific clones reacted to shorter p30 subunits (p301 or p302), and only 14 of the p2 or p30-specific clones reacted with TT presented by EBV-transformed B cell lines (B-LCL). The p30-specific clones were HLA-DP4 restricted. In contrast to autologous B cell lines, the majority of allogeneic, but HLA-DP4-positive cell lines failed to present p30 to the specific clones. We concluded that T cell clones are highly specific and that both, small alterations of the peptide length as well as discrete differences of the HLA-molecule may abrogate recognition of the peptide HLA complex by T cells. Moreover, use of peptides as stimulators of T cells may recruit and activate T cells which fail the "original" peptide, derived from normal antigen processing. Clones could usually be maintained in culture for 4-6 months, but with the help of freezing and thawing some clones are now available for over 2 years and still specific. Comparison of different autologous antigen-presenting cells, namely B-LCL and activated MHC class II-positive T cells revealed that not all clones were able to mount a proliferative response to peptide presentation by T cells, while all clones proliferated to B cells as APC. If stimulated with peptide and B-LCL, the clone proliferating to T cells as APC (so-called T responder clones) secreted a broad spectrum of cytokines (Th0-like) and were easier to maintain in culture. In contrast, clones which were unable to proliferate to peptide presentation, so-called T-nonresponder clones, showed a more restricted cytokine pattern and elevated or very low IL4/IFN gamma ratio upon antigen specific stimulation. However, all clones secreted at least small amounts of IL2, IL4, IFN gamma and TNF alpha, if stimulated by PMA and ionomycin. Thus, both chemical and antigen-specific stimulations should be considered if T cell clones are classified as Th1 or Th2, whereby those clones, which secrete a limited cytokine pattern after antigen stimulation only, might be named Th1 or Th2 like clones, while clones which even after PMA/ionomycin do not secrete all cytokines, might represent "real" Th1 or Th2 clones.