Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

The heparin-binding site(s) of histidine-rich glycoprotein as suggested by sequence homology with antithrombin III

A high degree of sequence homology has been found between the N-terminal region of histidine-rich glycoprotein (HRG) and that of antithrombin III (AT III) where the putative heparin-binding site of AT III is located. The amino acid residue at the position corresponding to Arg-47 of AT III that is essential for the heparin-binding was also arginine (Arg 23 and 78) in the homologous sequences of HRG. These observations strongly suggest that the heparin-binding sites of HRG and AT III are evolutionarily related. There was no apparent sequence similarity between the remaining about 70% portions of the two proteins.     

Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides

Native type IV collagen was isolated from human placental tissue by pepsin digestion, fractional salt precipitation, reduction and alkylation, a second pepsin digestion, and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. After denaturation, 10 distinct peptides were isolated from this material by molecular sieve, ion-exchange, and high-performance liquid chromatography. All of the peptides were found to have amino acid compositions characteristic of type IV collagen. Analysis of the eight major peptides by amino-terminal amino acid sequencing and by cyanogen bromide and tryptic peptide mapping has revealed the manner in which they are derived from type IV collagen. Pepsin liberates two large peptides by attacking non-triple-helical regions, one derived from the alpha 1 (IV) chain (F2, Mr 90 000) and one derived from the alpha 2 (IV) chain (F3, Mr 75 000). The alpha 1 (IV)-derived F2 peptide is also represented in the pepsin digest by amino-terminal and carboxy-terminal subfragments [F4c (Mr 41 000) and F4a (Mr 60 000)], as is the alpha 2 (IV)-derived F3 peptide [F5 (Mr 28 000) and F4b (Mr 50 000), respectively]. These findings indicate that the molecular regions from which the larger peptides are derived in themselves contain pepsin-sensitive (non-triple-helical) domains. In addition, several of the peptides examined were found to be present in two slightly different forms, suggesting that closely adjacent pepsin-sensitive sites often exist within the type IV collagen molecules. The methods outlined here provide a reliable means by which identifiable type IV collagen peptides can be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of the carboxy-terminal cyanogen bromide peptide of the human fibrinogen beta-chain: homology with the corresponding gamma-chain peptide and presence in fragment D.

The carboxy-terminal cyanogen bromide fragment of the human fibrinogen beta-chain has been isolated and its structure determined. It is a nonapeptide with the sequence Lys-Ile-Arg-Pro-Phe-Phe-Pro-Gln-Gln and is homologous with a portion of the carboxy-terminal cyanogen bromide fragment of the gamma-chain. The peptide has also been isolated in full yield from cyanogen bromide digests of the plasmin-derived fragment D, indicating that the carboxy-terminal region of the beta-chain is resistant to plasmin digestion. In contrast, a small portion of the corresponding gamma-chain carboxy-terminal region was missing in the same fragment D.     

A 10-kDa cyanogen bromide fragment from the epidermal growth factor homology domain of rabbit thrombomodulin contains the primary thrombin binding site

We have isolated a fragment (approximately equal to 10 kDa) of thrombomodulin containing the fifth and sixth epidermal growth factor (EGF)-like regions which retains thrombin binding capacity. The amino-terminal sequence of a 50-kDa active fragment of thrombomodulin derived from elastase proteolysis begins 11 residues before the first EGF-like structure of native thrombomodulin. Subsequent digestion with cyanogen bromide yields a 10-kDa thrombin binding fragment. The amino-terminal sequence of this fragment starts at the fifth EGF-like structure (Phe407). The amino acid composition suggests that this fragment contains the fifth and sixth EGF-like structures with a total of approximately 77 residues. This fragment lacks cofactor activity, but acts as a competitive inhibitor for protein C activation (Ki = 8.6 +/- 1.4 nM). We propose that the fifth and sixth EGF-like structures contain the thrombin binding site of thrombomodulin.     

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antigenic determinants of influenza virus hemagglutinin. V. Antigenicity of the HA2 chain

All the polypeptide fragments obtained by cyanogen bromide cleavage of the hemagglutinin from A/Memphis/102/72 influenza virus were examined for their ability to bind to IgG raised against purified virus. Within the hemagglutinin heavy chain the only fragment displaying antigenicity is HA1CN1, which comprises the amino-terminal 168 amino acid residues. By the use of a sensitive radioimmunoassay in which the antigen is unlabeled, it was shown that the light chain is also antigenic. Inhibition studies have localized the activity to the HA2CN1 region, which comprises the carboxy-terminal 90 amino acids. The determinant on HA2 is shown to be subtype specific.     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

The structural organization of photosystem II proteins has been investigated by use of the zero-length protein cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem II membranes were treated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl2 washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganese-stabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal 8-kDa peptide of the manganese-stabilizing protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The molecular basis of the requirement for antigen processing of pigeon cytochrome c prior to T cell activation

Antigen-induced activation of T lymphocytes that co-recognize Ia molecules has been shown to require an antigen-processing step by the presenting cell before T cell stimulation can occur. In this report, we demonstrate that antigen presentation of pigeon cytochrome c to an E kappa beta:E kappa alpha-restricted T cell hybridoma, 2C2, is inhibited by pretreatment of the antigen-presenting cells (APC) either with chloroquine or with fixation by paraformaldehyde. The chloroquine effect was partially reversible after 22 hr; the paraformaldehyde effect was not. In contrast, these treatments had little or no effect on the presentation of the carboxy-terminal cyanogen bromide cleavage fragment of pigeon cytochrome c, residues 81 to 104. There was at least a 50-fold greater potency of the fragment, as compared to that of the intact molecule, when paraformaldehyde-fixed APC were used. In addition, the fixed cells did not present synthetic fragments of the cytochrome c that were nonstimulatory when presented by unfixed cells. This observation showed that the loss of potency, demonstrated previously for analogs of pigeon cytochrome c with single amino acid substitutions at positions such as 99, was not a consequence of an alteration in the rate of antigen-processing. This result is consistent with our earlier hypothesis that these residues are contact amino acids with the antigen-specific T cell receptor or the Ia molecule. The major goal of these experiments was to define the molecular transition that occurred as a result of antigen processing. To achieve this end, we tested a variety of pigeon cytochrome c molecules and fragments for their ability to be presented by paraformaldehyde-fixed APC. Apocytochrome c, the denatured form of the molecule with the heme removed, could not be presented by the fixed cells, nor could the fragment 60-104, derived by acid cleavage of the tryptophan at position 59. Both molecules stimulated an IL 2 response from the T cell hybridoma when unfixed APC were utilized, demonstrating that the conditions used to prepare these two molecules did not destroy their antigenic determinant. In contrast, carboxy-terminal fragments, both native and synthetic, ranging in size from 16 to 39 amino acids, were capable of stimulating in the presence of paraformaldehyde-fixed APC. In particular, the partial-digest cyanogen bromide fragment, residues 66 to 104, was only twofold less potent than the pigeon fragment 81-104.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.     

Amino acid sequence of cyanogen bromide fragments of potato phosphorylase

Amino acid sequence analysis of the cyanogen bromide peptides of potato alpha-glucan phosphorylase was undertaken for comparison with rabbit muscle glycogen phosphorylase and for elucidation of the structural bases for the differences in the catalytic and regulatory properties between the animal and plant enzymes. The potato enzyme was carboxymethylated and cleaved with cyanogen bromide. The 17 distinct fragments produced were isolated by a combination of gel filtration, sulfopropyl ion exchange chromatography, and high performance liquid chromatography. The molecular weights of these fragments are distributed in a range of 300 to 30,000. Fragment CI has a blocked amino terminus, and has the same amino acid sequence as CII, which has been assigned as the amino-terminal fragment of potato phosphorylase. The blocking group was deduced to be an acetyl group from the results of fast atom bombardment mass spectrometry of an amino-terminal pentapeptide. This paper describes the sequence determination of all the cyanogen bromide fragments of potato phosphorylase. The complete structure is presented in the following paper (Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236).     

Complete amino acid sequence of flavocytochrome b2 from baker's yeast

Each subunit of baker's yeast flavocytochrome b2 can be selectively cleaved by proteases into two fragments, amino-terminal fragment alpha and carboxy-terminal fragment beta. The primary structure of the former has been reported before [Ghrir, B., Becam, A. M. & Lederer, F. (1984) Eur. J. Biochem. 139, 59-74]. The amino acid sequence of the 197-residue fragment beta has now been established. The fragment was cleaved with cyanogen bromide; the three peptides thus obtained were submitted to digestions with Staphylococcus aureus V8 protease, chymotrypsin and trypsin, sometimes after succinylation. The complete fragment was also submitted to tryptic cleavage after citraconylation. Peptides were separated by thin-layer finger-printing or high-pressure liquid chromatography. They were mostly sequenced in a liquid-phase sequenator. The 511-residue amino acid sequence of the mature protein is thus completely established. Secondary structure predictions indicate an alternation of helical and extended structure, with a higher percentage of the former. Comparisons with other flavoproteins do not detect any significant sequence similarity.     

Determination of peptide contact points in the human angiotensin II type I receptor (AT1) with photosensitive analogs of angiotensin II

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166-199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of [Sar1, Bpa8]AngII interacts with the second extracellular loop of the AT1 receptor.     

The primary structure of skeletal muscle myosin heavy chain: I. Sequence of the amino-terminal 23 kDa fragment

Subfragment-1 was prepared from adult chicken pectoralis myosin by limited digestion with alpha-chymotrypsin, and an amino-terminal 23 kDa fragment of the heavy chain was obtained by digesting the subfragment-1 with trypsin. The 205-residue sequence of the fragment was determined by sequencing its cyanogen bromide, tryptic, and chymotryptic peptides. The amino-terminal alpha-amino group of the fragment was acetylated, and two methylated lysines; epsilon-N-monomethyllysine and epsilon-N-trimethyllysine were recognized at the 35th and 130th positions, respectively, as in rabbit skeletal myosin. Comparing the 205-residue sequence of the skeletal myosin with those of cardiac, and gizzard myosins from chicken, considerable differences are recognized, especially in the amino-terminal region, but strong homologies are observed around the reactive lysine residue, around the epsilon-N-trimethyllysine residue, and around the consensus sequence of GXXGXGKT for nucleotide-binding proteins. On the other hand, only 12 amino acid substitutions are recognized between adult and embryonic skeletal myosins, allowing for the post-translational methylation.     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

Protein phosphatase type 1 catalytic subunit forms nondissociable dimers

Protein phosphatase type 1 is the major enzyme in skeletal muscle and liver for the dephosphorylation of Ser(P) and Thr(P) phosphoproteins. The cDNA for the catalytic subunit encodes a polypeptide of Mr 35,400 kDa, consistent with the Mr of 36,000-38,000 of the active protein purified in various laboratories. However, several investigators have found a Mr 70,000 protein for phosphatase type 1. In this report proteins of Mr 38,000 and 70,000 were resolved by Mono Q chromatography after extensive copurification from rabbit skeletal muscle. Antibodies affinity-purified against a type 1 phosphatase catalytic fragment reacted with both proteins in Western immunoblotting. Fractions from each peak were cleaved with cyanogen bromide and the major peptides were the same size by electrophoresis in gradient polyacrylamide gels. Cyanogen bromide peptides of the individual bands also were mapped by reversed-phase high-performance liquid chromatography. The purified Mr 38,000 and 70,000 proteins had identical HPLC peptide maps and also gave the same amino acid compositions after acid hydrolysis. Purified Mr 38,000 phosphatase catalytic subunit spontaneously formed a Mr 70,000 dimer that resisted usual dissociation conditions, i.e., boiling dodecyl sulfate plus 2-mercaptoethanol, but could be cleaved to about half size by various proteases, indicating that monomers were bound together near their amino or carboxy termini. Physiological changes in protein phosphatase type 1 are reflected in the amount of nondissociable dimers detected in tissue extracts.     

Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland

The complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase (thioesterase II) from rat mammary gland is presented. Most of the sequence was derived by analysis of peptide fragments produced by cleavage at methionyl, glutamyl, lysyl, arginyl, and tryptophanyl residues. A small section of the sequence was deduced from a previously analyzed cDNA clone. The protein consists of 260 residues and has a blocked amino-terminal methionine and calculated Mr of 29,212. The carboxy-terminal sequence, verified by Edman degradation of the carboxy-terminal cyanogen bromide fragment and carboxypeptidase Y digestion of the intact thioesterase II, terminates with a serine residue and lacks three additional residues predicted by the cDNA sequence. The native enzyme contains three cysteine residues but no disulfide bridges. The active site serine residue is located at position 101. The rat mammary gland thioesterase II exhibits approximately 40% homology with a thioesterase from mallard uropygial gland, the sequence of which was recently determined by cDNA analysis [Poulose, A.J., Rogers, L., Cheesbrough, T. M., & Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 15953-15958]. Thus the two enzymes may share similar structural features and a common evolutionary origin. The location of the active site in these thioesterases differs from that of other serine active site esterases; indeed, the enzymes do not exhibit any significant homology with other serine esterases, suggesting that they may constitute a separate new family of serine active site enzymes.     

Primary structure of the elongation factor G from Escherichia coli. VI. Structure of peptides of cyanogen bromide cleavage of the G-factor molecule

Peptides obtained as a result of cyanogen bromide cleavage of the G-factor have been studied. All 12 peptides embracing the whole structure of fragment T4 have been isolated. For their amino acid sequence determination, cyanogen bromide peptides have been further cleaved with trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease and BNPS-skatole. The complete primary structure of 9 from 12 cyanogen bromide peptides has been determined.     

Evidence for a polypeptide segment at the carboxyl terminus of recombinant human gamma interferon involved in expression of biological activity

A panel of 18 murine monoclonal antibodies was raised in BALB/c mice to the full-length, 146 amino acid residue recombinant human gamma interferon (rHuIFN gamma-A). Two monoclonal antibodies, designated 47N3-6 and 30N47-1, were purified from ascites tumors and further characterized. Antibody 47N3-6 neutralized both the antiviral and antiproliferative activities of rHuIFN gamma-A. Both Western blotting and enzyme-linked immunosorbent assays indicated that antibody 47N3-6 could bind to rHuIFN gamma-A as well as to a genetically engineered truncated form lacking the first three amino-terminal residues (rHuIFN gamma-D) but did not recognize a genetically engineered variant terminating at residue 131 (rHuIFN gamma-B). This antibody also demonstrated binding to a 15 amino acid residue oligopeptide, designated F-1, corresponding to residues 132-146 at the carboxyl terminus of rHuIFN gamma-A. Chemical cleavage of peptide F-1 with cyanogen bromide produced two fragments that were separated by reversed-phase high-pressure liquid chromatography. Dot-blot analysis indicated that antibody 47N3-6 could bind to a fragment, KRKRSQHse, derived from residues 132-137 of rHuIFN gamma-A, but could bind only weakly to the cyanogen bromide fragment corresponding to residues 138-146. It was consistent with these results that antibody 47N3-6 demonstrated binding to a form lacking the five carboxyl-terminal amino acids (rHuIFN gamma-D') but did not bind to a synthetic polypeptide corresponding to residues 138-146. Peptide F-1 exhibited neither antiviral nor antiproliferative activity, and it did not antagonize the antiviral activity of rHuIFN gamma-A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of helper and suppressor T cells by nonoverlapping determinants on the large protein antigen, beta-galactosidase

The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppressor (Ts)-inducing determinants was examined with cyanogen bromide and tryptic peptides of Escherichia coli beta-galactosidase (GZ), a large tetrameric protein (monomer molecular weight = 116 kDa). Immunization with cyanogen bromide fragment 2 [CB-2, amino acids (a.a.) 3-92] induced both specific Th and Ts cells. Study of the induction of these functionally opposite T cell subpopulations with tryptic peptides of CB-2 indicated that Th and Ts were activated by separate, nonoverlapping determinants. Th-inducing activity resided in a nonapeptide, T6 (a.a. 44-52), whereas T4 (a.a. 27-37) induced Ts cells. The presence of distinct helper and suppressor determinants suggests that the specificity repertoire in these T cell subpopulations may differ, perhaps owing to the expression of antigen-recognizing receptors that are coded by unique gene families. Alternatively, antigen presentation structures may be physicochemically quite different, and bind to distinct parts of the peptide antigen.