The structure of phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4. Production and characterization of site-specific antibodies

Fifteen peptides corresponding in sequence to segments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the antipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demonstrated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 125I-labeled P-450 PB-4 and also in vitro-synthesized apo-P-450 PB-4, the yields of immunoprecipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibodies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5. Three antipeptide antibodies raised against hydrophilic segments located in the amino-terminal one-third of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed O-deethylation of the model substrate 7-ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 beta NF-B, the major aromatic hydrocarbon-inducible rat hepatic P-450, suggesting that certain amino acid sequences or regions of secondary structure are conserved between the major phenobarbital-induced and polycyclic-induced rat liver P-450 isoenzymes. These studies demonstrate the utility of antipeptide antibodies for evaluation of antigenic sites exposed in native P-450 PB-4, for identification of specific amino acid sequences important for the interaction of P-450 PB-4 with its substrate and/or with cytochrome P-450 reductase in a reconstituted system and for elucidation of structural and immunochemical homologies between P-450 PB-4 and other P-450 isoenzymes present in rat liver endoplasmic reticulum.     

The structure of an antigenic determinant in a protein

The immunogenic and antigenic determinants of a synthetic peptide and the corresponding antigenic determinants in the parent protein have been elucidated. Four determinants have been defined by reactivity of a large panel of antipeptide monoclonal antibodies with short, overlapping peptides (7-28 amino acids), the immunizing peptide (36 amino acids), and the intact parent protein (the influenza virus hemagglutinin, HA). The majority of the antipeptide antibodies that also react strongly with the intact protein recognize one specific nine amino acid sequence. This immunodominant peptide determinant is located in the subunit interface in the HA trimeric structure. The relative inaccessibility of this site implies that antibody binding to the protein is to a more unfolded HA conformation. This antigenic determinant differs from those previously described for the hemagglutinin and clearly demonstrates the ability of synthetic peptides to generate antibodies that interact with regions of the protein not immunogenic or generally accessible when the protein is the immunogen.     

Exposure of antigenic sites during immunization. Monoclonal antibodies to monomer of lactose repressor protein

Monoclonal antibodies specific for the lactose repressor protein have been purified from three mouse hybridoma cell lines, and ascitic fluids from five other cell lines producing repressor antibodies have been assayed for immunoglobulin subclass and antigenic specificity. The chymotryptic core region (amino acids 57-360) of the repressor reacted with all antibodies examined, while no reaction with the NH2-terminal domain (1-56) could be detected. All of the purified antibodies and ascitic fluids reacted with the carboxyl-terminal fragment (amino acids 281-360) produced by cyanylation and base-catalyzed cleavage at the cysteine residues. Although none of the purified antibodies associated with native, tetrameric lac repressor, reaction was observed with repressor which had been denatured or dissociated into monomers by treatment with low levels of sodium dodecyl sulfate. Additionally, a mutant repressor which exists as a monomer in solution reacted with the antibodies in the absence of any denaturing treatments. These data indicate the carboxyl-terminal region is inaccessible in the intact repressor tetramer and further suggest that denaturation/dissociation of a protein during the initial immunologic challenge may result in the production of monoclonal antibodies to antigenic areas of the protein which are not exposed in the native conformation.     

Engineering aggregation resistance in IgG by two independent mechanisms: lessons from comparison of Pichia pastoris and mammalian cell expression

Aggregation is an important concern for therapeutic antibodies, since it can lead to reduced bioactivity and increase the risk of immunogenicity. In our analysis of immunoglobulin G (IgG) molecules of identical amino acid sequence but produced either in mammalian cells (HEK293) or in the yeast Pichia pastoris (PP), dramatic differences in their aggregation susceptibilities were encountered. The antibodies produced in Pichia were much more resistant to aggregation under many conditions, a phenomenon found to be mainly caused by two factors. First, the mannose-rich glycan of the IgG from Pichia, while slightly thermally destabilizing the IgG, strongly inhibited its aggregation susceptibility, compared to the complex mammalian glycan. Second, on the Pichia-produced IgGs, amino acids belonging to the α-factor pre-pro sequence were left at the N-termini of both chains. These additional residues proved to considerably increase the temperature of the onset of aggregation and reduced the aggregate formation after extended incubation at elevated temperatures. The attachment of these residues to IgGs produced in cell culture confirmed their beneficial effect on the aggregation resistance. Secretion of IgGs with native N-termini in the yeast system became possible after systematic engineering of the precursor proteins and the processing site. Taken together, the present results will be useful for the successful production of full-length IgGs in Pichia, give indications on how to engineer aggregation-resistant IgGs and shed new light on potential biophysical effects of tag sequences in general.     

Cleavage of IgGs by proteases associated with invasive diseases

The effective functioning of immunoglobulins and IgG mAbs in removing pathological cells requires that the antigen binding regions and the Fc (effector) domain act in concert. The hinge region that connects these domains itself presents motifs that engage Fc receptors on immune effector cells to achieve cell lysis. In addition, sequences in the lower hinge/CH2 and further down the CH2 region are involved in C1q binding and complement-mediated cell killing. Proteolytic enzymes of little relevance to human physiology were successfully used for decades to generate fragments of IgGs for reagent and therapeutic use. It was subsequently noted that tumor-related and microbial proteases also cleaved human IgG specifically in the hinge region. We have shown previously that the “nick” of just one of the lower hinge heavy chains of IgG unexpectedly prevented many effector functions without impacting antigen binding. Of interest, related single-cleaved IgG breakdown products were detected in breast carcinoma extracts. This suggested a pathway by which tumors might avoid host immune surveillance under a cloak of proteolytically-generated, dysfunctional antibodies that block competent IgG binding. The host immune system cannot be blind to this pathway since there exists a widespread, low-titer incidence of anti-hinge (cleavage-site) antibodies in the healthy population. The prevalence of anti-hinge reactivity may reflect an ongoing immune recognition of normal IgG catabolism. Tumor growth and bacterial infections potentially generate hostile proteolytic environments that may pose harsh challenges to host immunity. Recent findings involving physiologically-relevant proteases suggest that the potential loss of key effector functions of host IgGs may result from subtle and limited proteolytic cleavage of IgGs, and that such events may facilitate the incursion of invasive cells in local proteolytic settings.     

Quantitative comparison of the efficiency of antibodies against S1 and S2 subunit of SARS coronavirus spike protein in virus neutralization and blocking of receptor binding: implications for the functional roles of S2 subunit

Neutralizing effects of antibodies targeting the C-terminal stalk (S2) subunit of the spike protein of severe acute respiratory syndrome coronavirus have previously been reported, although its mechanism remained elusive. In this study, high titered mouse antisera against the N-terminal globular (S1) and S2 subunits of the S protein were generated and total immunoglobulin G (IgG) was purified from these antisera. The efficiency of these purified IgGs in virus neutralization and blocking of receptor binding were compared quantitatively using virus neutralization assay and a previously developed cell-based receptor binding assay, respectively. We demonstrated that anti-S1 IgG neutralizes the virus and binds to the membrane associated S protein more efficiently than anti-S2 IgG does. Moreover, both anti-S1 and anti-S2 IgGs were able to abolish the binding between S protein and its cellular receptor(s), although anti-S1 IgG showed a significantly higher blocking efficiency. The unexpected blocking ability of anti-S2 IgG towards the receptor binding implied a possible role of the S2 subunit in virus docking process and argues against the current hypothesis of viral entry. On the other hand, the functional roles of the previously reported neutralizing epitopes within S2 subunit were investigated using an antigen specific antibody depletion assay. Depletion of antibodies against these regions significantly diminished, though not completely abolished, the neutralizing effects of anti-S2 IgG. It suggests the absence of a major neutralizing domain on S2 protein. The possible ways of anti-S2 IgGs to abolish the receptor binding and the factors restricting anti-S2 IgGs to neutralize the virus are discussed.     

Computer prediction of potential immunogenic determinants from protein amino acid sequence

In proteins, immunogenic determinants that can induce protein-reactive antipeptide antibodies reside mostly in those parts of the molecule that have a high tendency to form beta-turns. A program for an IBM personal computer which predicts protein immunogenic determinants is described. The program predicts potential immunogenic determinants from protein amino acid sequences according to a Chou-Fasman-based probability of a beta-turn occurrence, p greater than 1.5 X 10(-4)(P. Y. Chou and G. D. Fasman, 1978, Adv. Enzymol. 47, 46-148). Oncopeptides (whose efficacy in generating protein-reactive antipeptide antibodies has been described) with a beta-turn probability of p greater than 1.5 X 10(-4) elicited antipeptide antibodies that reacted with the parent oncoprotein at a rate of 96%, thus showing a surprisingly good correlation between the tendency to form a beta-turn and the protein reactivity of antipeptide antibodies. Potential immunogenic determinants were predicted on myohemerythrin and myoglobin.     

Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients

To examine the identity of binding sites for thyrotropin (TSH) and thyroid stimulating antibodies (TSAbs) associated with Graves' disease, we constructed eight human TSH receptor/rat LH-CG receptor chimeras. Substitution of amino acid residues 8-165 of the TSH receptor with the corresponding LH-CG receptor segment (Mc1 + 2) results in a chimera which retains high affinity TSH binding and the cAMP response to TSH but loses both the cAMP response to Graves' IgG and Graves' IgG inhibition of TSH binding. Two of three IgGs from idiopathic myxedema patients which contain thyroid stimulation blocking antibodies (TSBAbs) still, however, react with this chimera. Chimeras which substitute residues 90-165 (Mc2) and 261-370 (Mc4) retain the ability to interact with TSH, Graves' IgG, and idiopathic myxedema IgG. The data thus suggest that residues 8-165 contain an epitope specific for TSAbs and that TSH receptor determinants important for the activities of TSAbs and TSH are not identical. Further, binding sites for TSBAbs in idiopathic myxedema may be different from receptor binding sites for both Graves' IgG TSAb as well as TSH and may be different in individual patients.     

Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen

Three mouse hybridomas secreting antibodies against the undecapeptide Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr, corresponding to the carboxy terminus of simian virus 40 large T antigen, were isolated and cloned. A sensitive enzyme-linked immunosorbent assay was used to characterize the properties of the monoclonal antibodies. All three hybridomas, designated KT1, KT3, and KT4, produced antibodies that immunoprecipitated large T. The antibodies differed in their affinities for the peptide and for the native protein. Antibodies from KT3 precipitated large T better than those from KT1 or KT4. KT3 antibodies also had the highest affinity for the free peptide (5.2 X 10(6) M-1) as determined by radioimmunoassay; KT1 and KT4 antibodies had ca. 5- and 1,000-fold lower affinities, respectively. Inhibition studies with shorter peptides, overlapping the undecapeptide, revealed the approximate regions recognized by the different monoclonal antibodies. KT3 antibodies bound to a region within the carboxy-terminal six amino acids of large T. Antibodies from KT1 and KT4 reacted with sequences located further towards the amino terminus of the undecapeptide. Surprising results were obtained with KT4 antibodies. Their binding to the undecapeptide was completely inhibited by the undecapeptide itself or the carboxy-terminal hexapeptide. The carboxy-terminal pentamer, on the other hand, slightly enhanced binding, and the carboxy-terminal tetramer, Glu-Pro-Glu-Thr, was strongly stimulatory. A model for this effect is proposed. Using the enzyme-linked immunosorbent assay, we confirmed previous studies (W. Deppert and G. Walter, Virology 122:56-70, 1982) which found that antiserum against sodium dodecyl sulfate-denatured large T reacts strongly with the carboxy terminus of large T. By inhibition studies, we identified the approximate region within the undecapeptide recognized by anti-sodium dodecyl sulfate-denatured large T and compared this region with the region identified by antipeptide serum.     

Monoclonal antipeptide antibodies to the glucocorticoid receptor.

The generation of monoclonal antibodies to synthetic peptides of the glucocorticoid receptor is described. Two antibodies to sequences from the DNA binding region are IgMs. Two other antibodies to sequences in the steroid binding region and the C-terminus belong to the IgG class. The specificity of the IgG binding to the receptor in an ELISA assay is demonstrated by competition with the relevant peptides. Both IgGs are able to recognize the receptor in Western blots, but do not form stable complexes in sucrose gradients. Steroid binding to the receptor is not influenced by preincubation with antibodies. This indicates that denaturation or distortion of the receptor is necessary for the accessibility of these antibodies to their epitopes. Both antibodies can be used to stain the glucocorticoid receptor in neoplastic cells of patients suffering from chronic lymphatic leukemia.     

Stabilization of pancreatic ribonuclease A by immobilization on Sepharose-linked antibodies that recognize the labile region of the enzyme.

The stabilizing potential of the antibodies recognizing the labile region of pancreatic ribonuclease A (RNase) has been investigated. The dodecapeptide SRNLTKDRAKPV corresponding to the labile region 32--43 on RNase was synthesized by the solid-phase method. Antiserum raised against the dodecapeptide-bovine serum albumin conjugate showed good cross-reactivity with the peptide and native RNase. RNase immobilized on Sepharose support precoupled either with the antipeptide immunoglobulin (IgG) or anti-RNase IgG proved to be more resistant to thermal inactivation than the soluble enzyme. Besides, stability against inactivation by trypsin at 55 degrees C was markedly high when enzyme was immobilized on the antipeptide IgG support, as compared to the soluble and other immobilized preparations. These results suggest that matrices bearing antibodies recognizing specific labile regions of enzyme may be useful in selectively improving their stability against specific forms of inactivation.     

Aggregation-Prone Motifs in Human Immunoglobulin G

Therapeutic antibodies of many different IgG subclasses (IgG1, IgG2 and IgG4) are used in the treatment of various cancers, rheumatoid arthritis and other inflammatory and infectious diseases. These antibodies are stored for long durations under high concentrations as required in the disease treatment. Unfortunately, these antibodies aggregate under these storage conditions, leading to a decrease in antibody activity and raising concerns about causing an immunological response. Thus, there is a tremendous need to identify the aggregation-prone regions in different classes of antibodies. We use the SAP (spatial-aggregation-propensity) technology based on molecular simulations to determine the aggregation-prone motifs in the constant regions of IgG1 classes of antibodies. Mutations engineered on these aggregation-prone motif regions led to antibodies of enhanced stability. Fourteen aggregation-prone motifs are identified, with each motif containing one to seven residues. While some of these motifs contain residues that are neighbors in primary sequence, others contain residues that are far apart in primary sequence but are close together in the tertiary structure. Comparison of the IgG1 sequence with those of other subclasses (IgG2, IgG3 and IgG4) showed that these aggregation-prone motifs are largely preserved among all IgG subclasses. Other broader classes of antibodies (IgA1, IgD, IgE and IgM), however, differed in these motif regions. The aggregation-prone motifs identified were therefore common to all IgG subclasses, but differ from those of non-IgG classes. Moreover, since the motifs identified are in the constant regions, they are applicable for all antibodies within the IgG class irrespective of the variable region. Thus, the motif regions identified could be modified on all IgGs to yield antibodies of enhanced stability.     

Antipeptide antibodies to the porcine lactate dehydrogenase isoenzyme M4 active center fragment as a probe for the structural analysis of active centers of human lactate dehydrogenase isoforms

Antipeptide antibodies (AB) to the fragment of the active center of porcine lactate dehydrogenase M4 isoform were used for the analysis of antigenic properties and structural comparison of active centers of human lactate dehydrogenase isoforms. Selective precipitation of the M-subunit-containing isoforms using an immunoadsorbent based on antipeptide AB as well as selective inhibition of the enzymic activity of the M4 isoform by antipeptide AB testify to the specific binding of isoforms to antipeptide AB. The experimental results confirm the literary data on conformational changes in the structure of the active centers of corresponding human lactate dehydrogenase isoforms. The specific interaction of antipeptide AB with human lactate dehydrogenase isoforms suggests that the site of the amino acid sequence (residues 180-214) in both human and porcine M4 isoenzymes is immunochemically identical. The data obtained suggest that antipeptide AB are convenient probes for detecting differences (including minor ones) in the primary and spatial structure of enzymes.     

Transmembrane topography and evolutionary conservation of synaptophysin

Synaptophysin is the major integral membrane protein of small synaptic vesicles. Its primary structure deduced from rat and human complementary DNA sequences predicts that synaptophysin contains four transmembrane regions and a carboxyl-terminal domain having a novel repetitive structure. To elucidate the transmembrane organization of this protein in the synaptic vesicle, five antipeptide antibodies were raised. The site-specific antibodies were used to map the cognate sequences to the cytoplasmic or intravesicular side of the synaptic vesicle membrane by determining the susceptibility of the epitopes to proteolysis. The results confirm a topographic model for synaptophysin in which the protein spans the vesicle membrane four times, with both the amino and carboxyl terminus being cytoplasmic. In addition, the evolutionary conservation of the synaptophysin domains was addressed as a function of their membrane localization. To this end the primary structure of bovine synaptophysin was determined. Sequence comparisons between bovine, rat, and human synaptophysin revealed that only the intravesicular loops showed a significant number of amino acid substitutions (22%), while the transmembrane regions and cytoplasmic sequences were highly conserved (3% substitutions). These results depict synaptophysin as a protein with multiple membrane spanning regions whose functional site is likely to reside in highly conserved intramembranous and cytoplasmic sequences.     

Design, expression and characterization of a soluble single-chain functional human neonatal Fc receptor

The neonatal Fc receptor (FcRn) is responsible for transporting maternal IgGs to fetus/newborns and maintaining the homeostasis of IgGs in adults. FcRn resembles class I major histocompatibility complex in structure, and is composed of a transmembrane heavy chain and an invariant beta 2 microglobulin. Changes in the affinity of IgGs to FcRn lead to changes in the half-life of engineered IgGs and Fc fusion proteins. Longer half-life of therapeutic antibodies means lower dose and longer interval between administering. For some diagnostic agents including imaging or radio-labeled agents a shorter half life in circulation results in lower non-specific binding and decreased side effects. Therefore, studying the interaction of FcRn and therapeutic antibodies has direct clinical implications. A reliable method to prepare soluble and functional FcRn protein is essential for such studies. In this study, we describe a new method to express in mammalian cells soluble human FcRn (sFcRn) as a single-chain soluble fusion protein. The highly hydrophilic beta 2 microglobulin was joined with the hydrophobic heavy chain via a 15 amino acid linker. The single-chain fusion protein format not only improved the expression level of the heavy chain but also simplified the purification process. The sFcRn maintained its pH-dependent binding to IgG. This method typically yielded ～1 mg/100ml culture without optimization, and is easy to scale up for production of large quantities.     

Recognition of HLA class I molecules by antisera directed to synthetic peptides corresponding to different regions of the HLA-B7 heavy chain

Antisera have been prepared in rabbits and in mice against different peptides corresponding to four hydrophilic and variable regions of HLA-B7 heavy chain (65-82, 99-118, 138-157, and 164-187). Specific antipeptide sera have been obtained with all synthetic peptides; for three of them which were more than 20 amino acids long, highly potent sera were elicited by injection of the free peptide. Three overlapping peptides included in region 138-157 have been used, and two different antigenic sites were detected in this region. HLA molecules solubilized in nonionic detergent were precipitated by antipeptide sera directed against regions 65-82, 138-157, and 164-187, but not by antipeptide serum directed against the less hydrophilic region 99-118. Analysis by two-dimensional electrophoresis of the isolated molecules confirmed the anti-HLA specificity of the antipeptide 65-82 and 138-157 sera. Variable numbers of HLA-related spots were found according to the antisera used. Antipeptide 138-157 serum precipitated numerous HLA molecules and therefore probably reacted with monomorphic determinants whereas antipeptide 65-82 appeared specific for a more limited number of HLA antigens. Such reagents directed against well-defined regions of the HLA class I heavy chain are of considerable interest, notably for the mapping of antigenic epitopes on the molecule and for the study of relationships between structure and function.     

The structural basis of rabbit VH allotypes: serologic studies on a1 H chains with defined amino acid sequence.

The amino acid sequences for the VH regions of three homogeneous antibodies elicited by type III pneumococcal vaccine were determined. All three antibodies had the group a allotype a1. Two of the antibody H chains (3372, 3381) had identical amino acid sequences in all framework positions that are considered correlates of the VH allotype, whereas the third H chain (3T72) differed from these at positions 15 and 16. The a1 allotypic specificities of the three homogeneous antibodies were compared by quantitative radiobinding and inhibition assays by using both insolubilized anti-a1 antisera and allotypic antiserum fractions rendered specific for the homogeneous antibody 3374. It was found that antibodies 3374 and 3381 are allotypically indistinguishable and have in common an a1 allotypic specificity that predominates in pooled a1 IgG. The allotypic specificity of the 3T72 antibody, on the other hand, was markedly deficient to those of 3374, 3381, and the a1 IgG pool. This correlation of allotypic difference with amino acid sequence variation at position 15 and 16 of the H chain indicates the involvement of these two residues in a major a1 allotypic determinant.     

Immunofluorescence localization of Z-DNA in chromosomes: quantitation by scanning microphotometry and computer-assisted image analysis

Anti-Z-DNA polyclonal and monoclonal immunoglobulins raised against left-handed polynucleotides show various degrees of specificity for base sequence and substitution. Class 1 IgGs recognize all Z-DNA with equal affinity; class 2 IgGs show a preference for d(G-C)n sequences and class 3 IgGs for d(G-C)n sequences with substitutions at the C5 position of the pyrimidine. These antibodies served as probes for the localization of Z-DNA in polytene and metaphase chromosomes and in interphase chromatin by indirect immunofluorescence. A quantitative assessment of the binding of anti-Z-DNA IgGs to polytene chromosomes of Chironomus and Drosophila was made by scanning microphotometry and by computer-assisted image analysis of double immunofluorescence and DNA-specific dye fluorescence images. The three classes of antibodies bind to most of the bands in acid fixed polytene chromosomes of C. thummi; however, preferential binding of one class of antibody over another can be observed in certain regions. These differences can be quantitated by arithmetic division or subtraction of the normalized digital images. If a class 2 antibody is first bound at saturating concentrations the binding of class 1 antibody is reduced throughout most bands by 40-50%. However, the telomeres of the three large chromosomes bind greater than 10 times as much class 1 antibody as class 2 antibody, indicating that the Z-DNA tracts in these regions are comprised largely of alternating sequences containing the A X T basepair, e.g., A-C. High-resolution image analysis of class 1 and class 2 immunofluorescence patterns and the total DNA distribution from polytene chromosomes of D. melanogaster show that the two antibody distributions are very similar in a large majority of the bands, but they often deviate from the mean DNA distribution profile. Z-DNA sequences of both G-C and A-C type are detectable at all levels of ploidy from 2n to 2(13)n and in species as diverse as insects and man. We conclude that the vast majority of polytene chromosome bands (genes) contain one or a few DNA sequences with potential for undergoing the B----Z transition and contain both alternating purine-pyrimidine G-C and A-C tracts or mixed sequences. Highly heterochromatic bands and telomeres have more Z potential sequences than do other bands.     

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.     

Study of the structure-activity features of lactate dehydrogenase isoenzymes using antipeptide antibodies to the M4-isoform of swine lactate dehydrogenase]

The structure-function peculiarities of human, porcine, rabbit, and rat lactate dehydrogenase (LDH) isoenzymes have been studied using antipeptide antibodies (AB) against the M4-isoform of porcine LDH. Antipeptide AB were raised against the hypervariable (40% homology) N-terminal fragment (residues 1-32), and the highly conservative fragment 180-214 containing histidine in the enzyme active center. Whereas antipeptide AB against the fragment of the active center of porcine LDH M4-isoform selectively inhibited the catalytic activities of LDH isoenzymes from various sources, antipeptide AB directed against the N-end were without effect. The ability of antipeptide AB to specifically interact with various isoforms of LDH suggests that sequences 1-32 and 180-214 are immunochemically identical only in the case of human and porcine M4 isoenzymes; the relatedness of the amino acid sequence to the common antigenic determinant required the absence in the given sequence of essential amino acid substituents. Chemical modification of porcine M4-isoform by diethylpyrocarbonate and the use of specific AB revealed that histidine-195 located in the active center of LDH is not directly involved in the binding to AB.