Binding of pyridoxal 5'-phosphate to bovine serum albumin and albumin fragments obtained after proteolytic hydrolysis. Localization and nature of the primary PLP binding site.

The binding of pyridoxal 5'-phosphate (PLP) to bovine serum albumin (BSA), and to large BSA fragments obtained after proteolytic hydrolysis, was investigated in order to study the structure of these fragments in relation to the albumin structure itself, and to get information about the PLP binding sites on albumin. From absorbance and circular dichroism spectra, combined with peptide mapping of the tryptic digests of the reduced PLP-protein complexes, it could be concluded that the primary binding site is localized with the NH2-terminal part of the albumin molecule. The COOH-terminal part contains one or more secondary sites. It appeared that in albumin and in the largest NH2-terminal fragment, the environment of the primary binding site is rather apolar in character. However, in the smallest NH2-terminal fragment this site is more exposed to the solvent. This suggests that the part of the peptide chain which is not common in both fragments has a stabilizing effect on the structure around the primary binding site.     

The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin

In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.     

Fragments of bovine serum albumin produced by limited proteolysis. Conformation and ligand binding.

Twelve fragments of bovine serum albumin, isolated following limited tryptic or peptic hydrolysis, have been studied to define secondary structure and locate ligand-binding sites. Based on circular dichroism, the conformational pattern of albumin (68% alpha helix and 18% beta structure) is substantially retained by individual fragments, indicating that secondary configuration is locally determined and is not destroyed during the cleavage process nor during fragment purification. The strong bilirubin-binding site of bovine serum albumin is present in 3 of the 12 fragments. Residues 186-238 are common to the three fragments and absent from those fragments which do not bind bilirubin; consequently the strong bilirubin-binding site is suggested to involve this region. By similar reasoning, the presence of palmitate-binding sites in some fragments and not in others indicates that the three strongest sites for the binding of palmitate are located in the carboxyl-terminal two-thirds of the molecule. The first site (KA approximately 2 X 10(7) M-1) is suggested as residues 377-503; the second site (KA approximately 8 X 10(6) M-1), residues 239-306; the third site (KA approximately 2 X 10(6) M-1), residues 307-377. Bromocresol Green, a reagent used in the assay of ablumin, was bound by fragments rougly in proportion to their size but showed particular affinity for the region of the strong bilirubin-binding site. The fluorescent probe, 8-anilino-1-naphthalensulfonate, was in general bound by large fragments, supporting the concept that this ligand is held principally in clefts between domains of the macromolecule.     

Definition of IgG- and albumin-binding regions of streptococcal protein G

Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., J?rnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.     

Physical and binding properties of large fragments of human serum albumin.

Three large fragments of human serum albumin were produced by peptic digestion of the native protein [Geisow & Beaven (1977) Biochem. J. 161, 619-625]. Fragment P44 represents residues 1-386 and fragments P29 and P31 represent residues 49-307 and residues 308-584 respectively of the albumin molecule. The large N-terminal fragment P44 has a similar percentage of alpha-helix to stored defatted albumin, although the alpha-helix content of all the fragments is significantly less than that of freshly prepared albumin. The fragment P44 appears to account for all the binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonate to albumin. N-Acetyl-L-tryptophan binds to this fragment and displaces one of the bound molecules of 8-anilinonaphthalene-1-sulphonate. Bilirubin binds to fragments P44 and P29, and the complexes show similar circular-dichroism spectra to that of the complex between bilirubin and whole albumin. These results are in agreement with affinity-labeling work on albumin with reactive ligands where substitution occurs in the N-terminal region of the molecule. The sharp conformational transitional transition in albumin which is observed between pH4 and 3.5 was absent from the fragments. This isomerization, usually called the N-F transition, probably occurs in intact albumin as a result of the unfolding or separation of the C-terminal third of the protein from the remainder of the molecule.     

Characterization of a 59 kDa gelatin-binding fragment of buffalo plasma fibronectin

Limited proteolysis of buffalo plasma fibronectin (FN) by thermolysin yielded four gelatin-binding fragments of which, the major 59 kDa fragment, GBF1, was isolated by gelatin-Sepharose and heparin-Sepharose affinity columns. GBF1 appeared during early phase of thermolysin digestion and remained intact even after 4 hr of digestion. GBF1 may be similar to 56 kDa gelatin-binding fragment of FNs from human and hamster plasma. But, it is more resistant to thermolysin cleavage. The fragment binds to heparin with low affinity. On the basis of the structure of human plasma FN, the modular structure of GBF1 may be given as: 6Fn1 1Fn2 2Fn2 7Fn1 8Fn1 9Fn1 1Fn3. Biophysical properties of GBF1 suggest an expanded native conformation. The interaction of the fragment with gelatin is pH-dependent and independent of NaCl concentration.     

Protease resistance of the beta subunit of the hamster fibronectin receptor. Evidence for differential cleavage of membrane-bound and soluble receptor

The structural stability of the hamster fibronectin receptor has been studied using limited proteolytic digestion and anti-(fibronectin receptor) monoclonal antibodies of known specificity. Treatment of the solubilized intact receptor or of the dissociated alpha and beta chains with any one of several proteases generated large protease-resistant fragments (92-110 kDa). Western blot analysis of tryptic digests using subunit-specific monoclonal antibodies revealed the large trypsin-generated fragment to be of beta-subunit origin. No products from the alpha subunit were detected. The protease-resistant fragment is lost upon exposure to reducing conditions; thus, the highly disulfide-bonded region of the beta subunit is important in the maintenance of the tertiary structure of the entire subunit. In contrast to solubilized fibronectin receptor, membrane-bound receptor is much more stable to proteolysis, and tryptic cleavage results in two large immunoreactive fragments of approximately 100 kDa and 95 kDa. This suggests a difference in the conformation and/or oligomeric organization of the membrane-bound receptor as compared with the solubilized heterodimeric receptor.     

Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H.

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.     

Characterization of proteolytic fragments of the laminin-nidogen complex and their activity in ligand-binding assays

Some 12 new nidogen and laminin fragments were purified from elastase, thrombin and trypsin digests and characterized by their sizes (22 kDa to greater than 300 kDa), subunit patterns on electrophoresis, partial amino acid sequences, content of specific epitopes and their binding to laminin or nidogen structures in radioligand assays. This permitted the various fragments to be ordered along the dumbbell-shaped structure of nidogen and to compare them with previously described nidogen fragments arising by endogenous proteolysis. Two nidogen fragments (E-50, E-90; 50 kDa and 90 kDa) remain associated with a large laminin fragment in elastase digests of the complex and could be dissociated with 2 M guanidine.HCl. Recombination studies demonstrated Kd = 10-20 nM for this interaction. Nidogen fragments devoid of binding activity included the tryptic peptide T-40 (40 kDa) corresponding to the rod-like domain and several larger fragments extending more to the N-terminus of nidogen. An N-terminal thrombin fragment of about 50 kDa was also inactive. Together the data show a lack of laminin binding to the N-terminal globule and rod of nidogen and provide indirect evidence that this activity is located within or close to its C-terminal globular domain. Nidogen-binding structures of laminin were obtained as two large fragments (greater than 300 kDa), P1X and E1X. They correspond to the short arm structure of laminin with one (E1X) or two (P1X) arms decreased in size to the inner rod-like segment. Shortening in E1X is mainly due to the B1 chain segment including the central globular domain which was identified as a new laminin fragment E10. Binding of E1X and P1X to nidogen was comparable to that of laminin while much lower activity was found for other laminin fragments. A 10-fold lower binding potential was also observed for the laminin-nidogen complex whose structure can now be defined in more precise molecular terms.     

Procedure for the isolation of a large peptic fragment of human serum albumin

For a thorough investigation of the drug-binding behaviour and other physicochemical properties of human serum albumin, one needs large amounts of specific fragments of albumin. Such fragments were obtained by careful proteolysis of the native protein with pepsin at pH 3.70. The fast protein liquid chromatographic technique was used to find the optimum experimental conditions for the separation of the fragments. By means of anion-exchange chromatography, chromatofocusing and gel permeation, it was possible to obtain a large fragment with a relative molecular mass of 46 000. The fragment could be assigned to segment 1–387 and therefore consists of domains 1 and 2 of the albumin structure, A 1-g amount of albumin produced 50 mg of a fragment that was 98% homogeneous.     

Phosphorylation of the human erythrocyte glucose transporter by protein kinase C: localization of the site of in vivo and in vitro phosphorylation

1. The human erythrocyte glucose transporter was phosphorylated in vitro by protein kinase C. 2. Tryptic cleavage of phosphorylated native transporter produced two major unphosphorylated membrane-embedded fragments weighing 23 and 19 kDa and released numerous water-soluble peptides. 3. Ion-exchange FPLC of the soluble tryptic peptides resolved the mixture into two phosphopeptide peaks. 4. Tryptic digestion of glucose transporter that was phosphorylated in vivo in response to phorbol esters produced soluble phosphopeptides that eluted at identical salt concentrations. 5. Proteolytic digestion and peptide mapping of the transporter revealed that the site(s) of phosphorylation lie within the large cytoplasmic domain that bisects the molecule.     

Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen.

We generated fragments of simian virus 40 large tumor antigen (T antigen) by tryptic digestion and assayed them for helicase activity and helicase substrate (mostly single-stranded DNA)-binding activity in order to map the domain sites on the protein. The N-terminal 130 amino acids were not required for either activity, since a 76-kilodalton (kDa) fragment (amino acids 131 to 708) was just as active as intact T antigen. To map the helicase domain further, smaller tryptic fragments were generated. A 66-kDa fragment (131 to about 616) retained some activity, whereas a slightly smaller 62-kDa fragment (137 or 155 to 616) had none. This suggests that the minimal helicase domain maps from residue 131 to approximately residue 616. To map the helicase substrate-binding domain, we tested various fragments in a substrate-binding assay. The smallest fragment for which we could clearly demonstrate activity was a 46-kDa fragment (131 to 517). To determine the relationship between the helicase substrate domain and the origin-binding domain (131 to 257, minimal core region; 131 to 371, optimal region), we performed binding experiments with competitor DNAs present. We found that origin-containing double-stranded DNA was an excellent competitor of the binding of the helicase substrate to T antigen, suggesting that the two domains overlap. Therefore, full helicase activity requires at least a partial origin-binding domain as well as an active ATPase domain. Additionally, we found that the helicase substrate was a poor competitor of origin-binding activity, indicating that T antigen has a much higher affinity to origin sequences than to the helicase substrate.     

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.     

Requirement of the hinge domain for dimerization of Ca2+-ATPase large cytoplasmic portion expressed in bacteria

The large cytoplasmic domain of rabbit sarcoplasmic reticulum Ca2+-ATPase was overexpressed in Escherichia coli as a 48 kDa fusion protein, designated p48, containing an N-terminal hexa-His tag. Purification conditions were optimized, thus conferring long-term stability to p48. Circular dichroism spectroscopy and the pattern of limited trypsinolysis confirmed the proper folding of the domain. p48 retained 0.5 +/- 0.1 mol of high affinity 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) binding sites per mol of polypeptide chain with an apparent dissociation constant of about 8 microM. Size-exclusion FPLC using protein concentrations in the range 0.03 5 mg/ml showed that p48 was essentially monodisperse with apparent molecular mass and Stokes radius (Rs) values compatible with a dimer (100 kDa and 40 A, respectively). Analysis of p48 by small-angle X-ray scattering provided an independent second proof for a dimeric p48 particle with a radius of gyration (Rg) of 39 A, suggesting that the dimer was not spherical (Rs/Rg = 1.026). When digested by proteinase K, p48 was converted to a 30 kDa fragment, designated p30, which was very resistant to further proteolysis. p30 retained high affinity TNP-ATP binding (Kd = 8 microM) and eluted as a monomer (35 kDa) in size-exclusion FPLC. As opposed to p48, the p30 fragment did not react with monoclonal antibody A52 [Clarke et al., J. Biol. Chem. 264 (1989) 11246-11251] which recognizes region E657-R672 located upstream of the hinge domain of the Ca2+-ATPase. These results indicate a requirement of the hinge domain (670-728) region for self-association of the p48 large hydrophilic domain as a dimer. We propose that this behavior points to a possible role of the hinge domain in dimerization of sarcoplasmic reticulum Ca2+-ATPase in the native membrane.     

Reactivation of 3-phosphoglycerate kinase from its unfolded proteolytic fragments

Limited trypsinolysis of pig muscle 3-phosphoglycerate kinase yielded a nicked enzyme without loss of catalytic activity [Jiang, S. X. & Vas, M. (1988) FEBS Lett. 231, 151-154]. The reactivation rate of the nicked enzyme after denaturation does not differ substantially from the reactivation rate of the denatured intact enzyme: t 1/2 varies between 70-110 s at 25 degrees C, pH 7.0 in both cases. Thus, the absence of a covalent linkage between the two proteolytic fragments of the enzyme molecule apparently does not affect the refolding. The two proteolytic fragments can be separated by FPLC under denaturing conditions. Fluorescence spectra of the isolated fragments may indicate that the tryptic cleavage site is within the N-terminal domain. Thus, the larger fragment (molecular mass about 30 kDa) probably contains the whole nucleotide-binding C-terminal domain plus a small part of the N-terminal domain. The inactive isolated fragments were used in renaturation experiments to study the reassembly of active 3-phosphoglycerate kinase. Kinetic measurements revealed the presence of a bimolecular rate-limiting step of reactivation. Separate preincubation of the fragments under renaturing conditions did not cause substantial acceleration of reactivation. This implies that assembly of the separate structural units (possibly domains) may limit the reactivation of the intact enzyme.     

Stepwise phosphorylation of the NH2-terminal region of the simian virus 40 large T antigen

The simian virus 40 (SV40) large T antigen was immunoprecipitated from extracts of infected monkey cells and cleaved with trypsin under conditions of mild proteolysis. The digestion generated fragments from the NH2-terminal region of T antigen which were released from the immunoprecipitates. Pulse-chase experiments showed that most of the newly made T antigen (form A) generated an NH2-terminal fragment of 17 kDa in size, whereas most of the T antigen that had aged in the cell (form C) generated a fragment of 20 kDa. An intermediate form of T antigen (form B), which generated an 18.5- kDa NH2-terminal fragment, was produced in part from form A and was converted to form C during the chase. Phosphate-labeling experiments showed that form C was the species of T antigen that incorporated the most 32P radioactivity at the NH2-terminal region, although some label was also incorporated into forms A and B. In vitro dephosphorylation of gel-purified 18.5- and 20-kDa fragments labeled with [35S]methionine increased the electrophoretic mobility of the fragments to that of 17 kDa. This signified that phosphorylation of the NH2-terminal fragments was directly responsible for their aberrant behavior in acrylamide gels. Although peptide maps of the methionine-labeled tryptic peptides of the 17-, 18.5-, and 20-kDa fragments were very similar to one another, maps of the 32P-labeled tryptic Pronase E peptides of these fragments contained qualitative and quantitative differences. Analysis of the labeled phosphoamino acids of various peptides from these fragments indicated that the 20-kDa fragment was highly phosphorylated at Ser 123 and Thr 124, whereas the 17- and 18.5-kDa fragments were mostly unphosphorylated at these sites. These experiments indicated that T antigen is phosphorylated at the NH2-terminal region in a specific stepwise process and, therefore, that this post-translational modification of T antigen is tightly regulated.     

Isolation of large peptides derived by cyanogen bromide cleavage of thermolysin using fast protein liquid chromatography (FPLC)

The recently introduced fast protein liquid chromatography (FPLC) system of Pharmacia (Uppsala, Sweden) was employed to isolate rather large peptides derived from thermolysin by selective chemical fragmentation at methionine in positions 120 and 205 of the polypeptide chain of 316 amino acid residues. Thermolysin was cleaved under conditions of limited fragmentation in order to produce, besides fragments 1-120, 121-205 and 206-316, the overlapping fragments 1-205 and 121-316. These polypeptides were separated employing prepacked Mono Q or Mono S columns (quaternary ammonium and sulfonic acid support, respectively). The columns were equilibrated with acetate-7 M urea buffer, pH 5.0 or 6.0, and eluted with a gradient of sodium chloride or acetate. Separations were achieved in 10-20 min and were carried out also at a semi-preparative level (1-3 mg per run). All five protein fragments were isolated in homogeneous form, as judged by amino acid analysis and electrophoresis. Considering that protein fragmentation with cyanogen bromide is the most commonly used procedure to achieve selective chemical fragmentation of a polypeptide chain, these results indicate that FPLC with ionic exchangers can be usefully employed to isolate rather large protein fragments especially suitable for automatic sequence analysis with the sequenator.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

The N-terminal domain I of human lactotransferrin binds specifically to phytohemagglutinin-stimulated peripheral blood human lymphocyte receptors

Human lactotransferrin receptors have been recently characterized on mitogen-stimulated human lymphocytes [(1989) Eur. J. Biochem. 179, 481-487]. In order to define the lactotransferrin recognition site by these receptors, the binding to lymphocytes of several tryptic fragments, isolated from human lactotransferrin by mild tryptic hydrolysis [(1984) Biochim. Biophys. Acta 787, 90-96], has been investigated. The 30 kDa N-tryptic fragment (residues 4-281) and the re-associated N,C-tryptic complex bind to lactotansferrin lymphocyte receptor with a dissociation constant of 44 nM and 39 nM, respectively, similar to the value obtained for the native lactotransferrin (Kd = 46 nM). However, neither the N-terminal domain II (residues 91-257) nor the 50 kDa C-tryptic fragment (residues 282-703) are recognized. These results suggest that the binding site of human lactotransferrin by the lymphocyte receptor is located in the N-terminal lobe and more precisely in the N-terminal domain I (residues 4-90 and/or 258-281).     

Binding sites for monoclonal antibodies and for mRNPs on SV40 large T-antigen determined with a cleavage map

Immune complexes of simian virus 40 large T-antigen with monoclonal papovavirus protein antibodies PAb 416, PAb 402, or PAb 423 were bound to protein-A-Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis. PAb 402 protected a specific cleavage site, located approximately within amino acid residues 450-500, from tryptic proteolysis; PAb 423 protected another site within residues 675-699. As shown by immunoblotting, 125I-labeled PAb 416 was bound to a 17-kDa N-terminal fragment of large T-antigen (amino acid residues 1-130), and PAb 423 was bound to several overlapping fragments derived from the C terminus of large T-antigen. These monoclonal antibodies were then used as accessibility probes to study the interaction of mRNPs with cytoplasmic large T-antigen. Whereas small T-antigen and nuclear large T-antigen were fully immunoreactive, cytoplasmic large T-antigen reacted poorly with PAb 402 or polyclonal antibodies unless the mRNP moiety was removed by treatment with EDTA/RNase A. In contrast, mRNP/T-antigen complexes were fully immunoreactive with PAb 416 or PAb 423 and did not require treatment with EDTA/RNase A. The results suggest that the binding site of PAb 402 is blocked due to the interaction with mRNPs whereas the N-terminal binding site of PAb 416 and the C-terminal binding site of PAb 423 remain accessible to antibodies.