Albumin binding as a general strategy for improving the pharmacokinetics of proteins

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH(2)) was determined to be 266 +/- 8, 320 +/- 22, and 467 +/- 47 nm for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.3 h when injected by intravenous bolus into rabbits. A related sequence, fused to the anti-tissue factor Fab of D3H44 (Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., Schoenfeld, J., Lowe, D., Lai, J., Rancatore, P., Iverson, M., Lim, A., Chisholm, V., Kelley, R. F., Riederer, M., and Kirchhofer, D. (2001) Thromb. Haemost. 85, 379-389), enabled the Fab to bind albumin with similar affinity to that of SA21 while retaining the ability of the Fab to bind tissue factor. This interaction with albumin resulted in reduced in vivo clearance of 25- and 58-fold in mice and rabbits, respectively, when compared with the wild-type D3H44 Fab. The half-life was extended 37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25-43% of the albumin half-life in these animals. These half-lives exceed those of a Fab'(2) and are comparable with those seen for polyethylene glycol-conjugated Fab molecules, immunoadhesins, and albumin fusions, suggesting a novel and generic method for improving the pharmacokinetic properties of rapidly cleared proteins.     

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I.

The troponin I peptide N alpha-acetyl TnI (104-115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S. 1981, J. Biol. Chem. 256, 2798-3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726-1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974-9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C-terminal domain of troponin C (TnC) and to calcium-saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C-terminal domain hydrophobic pocket and antiparallel beta-sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 +/- 37 microM) was found to be similar to TnIp binding to TnC (48 +/- 18 microM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674-681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C-terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C-terminal domain of TnC in structure and functional interactions.     

Bilin attachment sites in the alpha, beta, and gamma subunits of R-phycoerythrin. Structural studies on singly and doubly linked phycourobilins

Three unique bilin peptides, a beta subunit peptide bearing a doubly linked phycourobilin (PUB), and two gamma subunit peptides with singly linked PUB groups, were obtained by enzymatic degradation of Gastroclonium coulteri R-phycoerythrin. These peptides were shown to have the sequences (Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856-4863): (Formula: see text) The sequence of peptide beta-3T was identical to that previously established for a doubly linked phycoerythrobilin (PEB) peptide derived from a B-phycoerythrin (Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M. (1984) J. Biol. Chem. 259, 5472-5480). Secondary ion mass spectrometry of beta-3T yielded a protonated molecular ion of 1629 mass units, the same as that given by the doubly linked PEB peptide (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484), indicating that the doubly linked PUB and PEB tetrapyrroles were isomeric structures. High resolution 1H NMR analyses of peptides beta-3T, gamma-BV8, and gamma-DP provided unambiguous structural assignments for the singly and doubly linked PUB chromophores and indicated that the peptides in gamma-BV8 and gamma-DP were linked to ring A. The determination of which peptide fragment is linked to ring A and which to ring D in peptide beta-3T was not achieved in this study. 1H NMR analyses of three PEB-peptides from G. coulteri R-phycoerythrin--alpha-1 Cys(PEB)-Tyr-Arg, alpha-2 Leu-Cys(PEB)-Val-Pro-Arg, and beta-1 Met-Ala-Ala-Cys(PEB)-Leu-Arg--showed that they were identical to previously described corresponding chromopeptides from Porphyridium cruentum B-phycoerythrin, with the peptide linked to ring A of PEB in each instance (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5485-5489). This is the first documented report on the structure of singly or doubly linked phycourobilins.     

Amino acid sequence and post-translational modification of stem cell factor isolated from buffalo rat liver cell-conditioned medium.

Stem cell factor (SCF) isolated from culture medium conditioned by Buffalo rat liver cells was subjected to detailed structural analysis. Attempts at direct N-terminal sequencing of the factor indicated that its N terminus is blocked as pyroglutamic acid (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201). The removal of the blocking pyroglutamate by pyroglutamate aminopeptidase allowed sequencing of the polypeptide chain to position 47. Stem cell factor was also digested with CNBr, trypsin, Staphylococcus aureus protease (strain V8), and AspN peptidase to generate different sets of peptides that were then separated by reverse-phase high-performance liquid chromatography and sequenced. Sequence of an internal peptide fragment obtained by cleavage of stem cell factor at a single tryptophanyl peptide bond was also obtained. From these analyses, the complete amino acid sequence could be constructed. The factor as isolated is a single polypeptide of 164 or 165 amino acids. The sequence is confirmatory to a sequence deduced from a cDNA sequence and provides important evidence for C-terminal processing of the polypeptide encoded by cDNA. There are four potential N-linked glycosylation sites. Asn65, Asn72, Asn109, and Asn120. Sequence determination of isolated peptides suggested that Asn120 is glycosylated, Asn65 and Asn109 glycosylated in some molecules but not in others, and Asn72 not glycosylated. Amino acids at three positions, i.e. 142, 143, and 155, could not be detected during sequence analysis. Since the gene sequence codes for Ser, Thr, and Thr at these positions (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. C., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C. W., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211), they could be sites of O-linked carbohydrate attachment. The four cysteines form two intramolecular disulfide bonds, Cys4-Cys89 and Cys43-Cys138.     

Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences

Tissue plasminogen activator, separated into variants I and II (differing in Mr by 2000-3000), was reduced and [14C]carboxymethylated. Fragments from cleavages with enzymes and cyanogen bromide (CNBr) were separated by reverse-phase high-performance liquid chromatography and subjected to sequence degradations. All seven CNBr fragments were purified and found to be compatible with the cDNA-derived amino acid sequence [Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Ylverton, E., Seeburg, P. H., Heynecker, H. L., Goeddel, D. V., & Collen, D. (1983) Nature (London) 301, 214-221]. Chemical characterization of 93% of the 527 residues recovered in 50 peptides confirmed the indirectly deduced primary structure of the protein. The tryptic peptide patterns from the two variants were found to differ for one peptide (T15). Since carbohydrate was present in this peptide for variant I and since a marked difference in chromatographic behavior for T15 was observed in variant II, we conclude that carbohydrate differences in this peptide (i.e., Asn-184 in the numbering system of the cDNA-derived amino acid sequence) are the explanation for the size differences between variants I and II. Carbohydrate was also found at two other positions in the protein, corresponding to Asn-117 and Asn-448. However, a fourth potential glycosylation site, Asn-218, is apparently not utilized for carbohydrate attachment. The enzyme is inactivated by diisopropyl phosphorofluoridate, which covalently modifies the serine residue corresponding to position 478, identifying this as the active site serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallization, sequence, and preliminary crystallographic data for an antipeptide Fab 50.1 and peptide complexes with the principal neutralizing determinant of HIV-1 gp120.

X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P2(1)2(1)2(1) and I222 and from polyethylene glycol in space groups P1 and P2(1). Seeds from either the P1 and P2(1) native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into preequilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 A resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.     

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases

Two epidermal growth factor-stimulated protein kinases that correspond to ERK1 and ERK2 have been purified from human epidermoid carcinoma cells (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). A consensus primary sequence for substrates of ERK1 has been identified as -Pro-Leu-Ser/Thr-Pro- (Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). However, the structural determinants for substrate recognition are not understood. We performed a systematic analysis of the effect of point mutations in the primary sequence of peptide substrates on the rate of phosphorylation by ERK1 and ERK2. The results of this investigation demonstrate that the substrate specificities of the ERK1 and ERK2 protein kinases are very similar. We propose that the primary sequence of substrates for ERK1 and ERK2 protein kinases can be generalized as -Pro-Xaan-Ser/Thr-Pro- (where Xaa is a neutral or basic amino acid and n = 1 or 2).     

Molecular anatomy of the antibody binding site

The binding region of immunoglobulins, which includes the portion of the molecule having the most variability in its amino acid sequence, is shown to have a surprisingly constant structure that can be characterized in terms of a simple, well-defined model. The binding region is composed of the antigen combining site plus its immediate vicinity and arises by noncovalent association of the light and heavy chain variable domains (VL and VH, respectively). The antigen combining site itself consists of six polypeptide chain segments ("hypervariable loops") which comprise some 80 amino acid residues and are attached to a framework of VL and VH beta-sheet bilayers. Having analyzed refined x-ray crystallographic coordinates for three antigen-binding fragments (Fab KOL (Marquart, M., Deisenhofer, J., and Huber, R. (1980) J. Mol. Biol. 141, 369-391), MCPC 603 (Segal, D., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M., and Davies, D. R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4298-4302), and NEW (Saul, F. A., Amzel, L. M., and Poljak, R. J. (1978) J. Biol. Chem. 253, 585-597] we use the results to introduce a general model for the VL-VH interface forming the binding region. The region consists of two closely packed beta-sheets, and its geometry corresponds to a 9-stranded, cylindrical barrel of average radius 0.84 nm with an average angle of -53 degrees between its two constituent beta-sheets. The barrel forms the bottom and sides of the antigen combining site. The model demonstrates that the structural variability of the binding region is considerably less than was thought previously. Amino acid residues which are part of the domain-domain interface and appear not to be accessible to solvent or antigen contribute to antibody specificity.     

Characterization of a cDNA clone for the nonspecific cross-reacting antigen (NCA) and a comparison of NCA and carcinoembryonic antigen

NCA (nonspecific cross-reacting antigen), a glycoprotein found in normal lung and spleen, is immunologically related to carcinoembryonic antigen (CEA), which is found in over 95% of colon adenocarcinomas. From a human genomic library, we previously cloned part of an NCA gene and showed that the amino-terminal region has extensive sequence homology to CEA (Thompson, J. A., Pande, H., Paxton, R. J., Shively, L., Padma, A., Simmer, R. L., Todd, Ch. W., Riggs, A. D., and Shively, J.E. (1987) Proc. Natl. Acad. Sci. U. S.A. 84, 2965-2969). We now present the nucleotide sequence of a cDNA clone, containing the entire coding region of NCA (clone 9). The clone was obtained from a lambda gt 10 library made from the colon carcinoma cell line SW 403; the clone contains a 34-amino acid leader sequence, 310 amino acids for the mature protein, and 1.4 kilobases of 3'-untranslated region of the NCA gene. A comparison of the NCA sequence to the CEA sequence (Oikawa, S., Nakazato, H., and Kosaki, G. (1987) Biochem. Biophys. Res. Commun. 142, 511-518; Zimmerman, W., Ortlieb, B., Friedrich, R., and von Kleist, S. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2690-2694) shows that both proteins contain doublets of an immunoglobulin-like domain, of which there are one copy in NCA and three copies in CEA, a 108-amino acid amino-terminal domain with no cysteine residues, and a carboxyl-terminal hydrophobic domain of sufficient length to anchor the glycoproteins in the cell membrane. Overall, the corresponding coding regions possess 85% sequence homology at the amino acid level and 90% homology at the nucleotide level. Forty nucleotides 3' of their stop codons, the CEA and NCA cDNAs become dissimilar. The 108-amino acid amino-terminal region together with part of the leader peptide sequence corresponds exactly to a single exon described in our previous work. The data presented here further demonstrate the likelihood that CEA recently evolved from NCA by gene duplication, including two duplications of the immunoglobulin-like domain doublet of NCA.     

Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185HER2 antibody Fab fragments.

The murine monoclonal antibody 4D5 (anti-p185HER2) inhibits the proliferation of human tumor cells overexpressing p185HER2 in vitro and has been "humanized" [Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W.-L. T., Rowland, A. M., Kotts, C., Carver, M. E., & Shepard, H. M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)] for use in human cancer therapy. We have determined the antigen binding thermodynamics and the antiproliferative activities of chimeric 4D5 Fab (ch4D5 Fab) fragment and a series of eight humanized Fab (hu4D5 Fab) fragments differing by amino acid substitutions in the framework regions of the variable domains. Fab fragments were expressed by secretion from Escherichia coli and purified from fermentation supernatants by using affinity chromatography on immobilized streptococcal protein G or staphylococcal protein A for ch4D5 and hu4D5, respectively. Circular dichroism spectroscopy indicates correct folding of the E. coli produced Fab, and scanning calorimetry shows a greater stability for hu4D5 (Tm = 82 degrees C) as compared with ch4D5 Fab (Tm = 72 degrees C). KD values for binding to the extracellular domain (ECD) of p185HER2 were determined by using a radioimmunoassay; the delta H and delta Cp for binding were determined by using isothermal titration calorimetry. ch4D5 Fab and one of the humanized variants (hu4D5-8 Fab) bind p185HER2-ECD with comparable affinity (delta G degrees = -13.6 kcal mol-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase

The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis.

Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain.     

Identification in the human central nervous system, pituitary, and thyroid of a novel calcitonin gene-related peptide, and partial amino acid sequence in the spinal cord

Two human genes encoding precursors for two calcitonin gene-related peptides (CGRP) I (or alpha) and II (or beta) have been identified (Steenbergh, P. H., H?ppener, J. W. M., Zandberg, J., Lips, C. J. M., and Jansz, H. S. (1985) FEBS Lett. 183, 403-407). The amino acid sequence of CGRP-I was obtained in medullary thyroid carcinoma extracts (Morris, H. R., Panico, M., Etienne, T., Tippins, J., Girgis, S. I., and MacIntyre, I. (1984) Nature 308, 746-748), but not in normal human tissues. The human CGRP-II peptide remained to be discovered. Here we have determined in the human spinal cord the amino acid composition and the partial amino acid sequence of the DNA-predicted CGRP-I and -II. The data indicate for the first time the existence of a second CGRP different from the known CGRP-I. CGRP-II has been identified in the central nervous system, pituitary, thyroid, and in medullary thyroid carcinoma as a major CGRP form together with CGRP-I.     

Sequencing of an active-site peptide of angiotensin I-converting enzyme containing an essential glutamic acid residue

A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.     

Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23

Protein C23 (Mr 110 000, pI = 5.5), a major phosphoprotein in the nucleolus of mammalian cells, has been shown to contain 1.3 mol% of NG,NG-dimethylarginine (DMA) [Lischwe, M.A., Roberts, K.D., Yeoman, L.C., & Busch, H. (1982) J. Biol. Chem. 257, 14600-14602]. A tryptic peptide from protein C23 that contains DMA has been isolated and sequenced. Its sequence is Gly-Glu-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA- Gly-Gly- Gly-DMA-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly-DMA-Gly-Gly-Phe-Gly-Gly- DMA-Gly-Gly-Phe-DMA-Gly-Gly-DMA-Gly-Gly-Gly-Gly-Asp-Phe-Lys. This peptide contains 34 glycine, 10 DMA, and 6 phenylalanine residues and has clusters of glycine and NG,NG-dimethylarginine interspersed with phenylalanine residues. A similar domain has been found at the amino terminus of a nucleolar protein of Mr 34,000, pI = 8.5. This sequence array may represent a conserved domain characteristic of a certain class of nuclear proteins. All of the methylated arginine residues in protein C23, the 34-kilodalton protein, and myelin basic protein [Carnegie, P.R. (1971) Biochem. J. 123, 57-67] have at least one adjacent glycine. Access of certain arginine methylases to arginine residues may be sterically possible because of the lack of a side chain on the adjacent glycine residue(s).     

Identification and synthesis of the epitope for a human monoclonal antibody which can neutralize human T-cell leukemia/lymphotropic virus type I

A monoclonal antibody (mAb), designated 0.5 alpha, derived from a patient with adult T-cell leukemia was found previously to neutralize the human T-cell leukemia/lymphotropic type I (HTLV-I) virus in in vitro assays and bind to the major envelope glycoprotein (gp46) of HTLV-I (Matsushita, S., Guroff, M.R., Trepel, J., Crossman, J., Mitsuya, H., and Broder, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2671-2676). We have designed experiments to determine the epitope for this mAb. Using simultaneous multiple peptide synthesis, we synthesized 481 overlapping octapeptides which corresponded to the sequence of gp46. We mapped the epitope for mAb 0.5 alpha to lie between residues 186 and 195 of gp46. This result was confirmed by independently synthesizing a peptide containing this epitope which bound specifically to mAb 0.5 alpha with an approximate Ka = 4 x 10(7) M-1. In addition, the peptide inhibited mAb 0.5 alpha binding to gp46 derived from T-cells infected with HTLV-I. This epitope containing peptide may facilitate understanding HTLV-1 infection of T-cells.     

Glutathione reductase from human erythrocytes. Amino-acid sequence of a major fragment that links the FAD, NADP and interface domains.

A major CNBr fragment of glutathione reductase, peptide Q [Krohne-Ehrich, G., Schirmer, R.H. & Untucht-Grau, R. (1977) Eur. J. Biochem. 80, 65-71], was further fractionated by trypsin, chymotrypsin, thermolysin and clostripain digestion. The peptides were isolated and most of them were sequenced by solid-phase Edman degradation. The whole peptide Q was sequenced N-terminally up to position 51 by the same technique. A total sequence of 128 amino acids (28% of the whole protein) was obtained and could be localized in the electron density map [Schulz, G.E., Schirmer, R.H., Sachsenheimer, W. & Pai, E.F. (1978) Nature (Lond.) 273, 120-124] from position 259-387. This part of the polypeptide links and participates in all three domains of the flavoenzyme.