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G R Matsueda

Publications and source records attributed to G R Matsueda.

83 records · Page 5Linked to original sources
PubMed1981

Synthesis of N-tert.-butoxycarbonyl-( alpha-phenyl)aminomethylphenoxyacetic acid for use as a handle in solid-phase synthesis of peptide alpha-carboxamides.

N-tert.-butoxycarbonyl-aminomethyl( alpha-phenyl)phenoxyacetic acid was synthesized and found to be suitable for use as a handle in the solid-phase synthesis of peptide alpha-carboxamides. This handle was prepared with an 82% yield when N-tert.-butoxycarbonyl-( rho-hydroxy)benzhydrylamine was treated with excess sodium iodoacetate under alkaline conditions. In stability studies the linkage between the C-terminal amino acid and the handle was found to be resistant to acidolysis in 50% TFA/CH2Cl2 (less than 1% loss after 10h). Upon treatment for 30 min with HF:anisole(9:1) at 0 degrees, 92% cleavage of glycinamide from Gly-handle-resin was obtained. In a test synthesis of a peptide alpha-carboxamide, pyroglutamylhistidylprolinamide was synthesized in 83% yield. Two other handles, tert.-butoxycarbonyl-aminomethylphenoxyacetic acid and N-tert.-butoxycarbonyl-aminomethylphenyloxymethylphenoxyacetic acid, were also synthesized but found to be unsuitable for carboxamide synthesis under the same conditions of solid-phase synthesis.

Glycolates↗
PubMed1980

Isolation of an active heavy-chain variable domain from a homogeneous rabbit antibody by cathepsin B digestion of the aminoethylated heavy chain.

Cathepsin B from bovine liver has been used to cleave the heavy chain of partially reduced and aminoethylated rabbit allotype a1 IgG. Three major cleavages have been identified, one of which appears to be at the peptide bond carboxy terminal to the two adjacent (aminoethyl)cysteine residues at positions 133 and 134. The variable domain of the heavy chain (VH) was isolated by gel filtration from both pooled heterogeneous rabbit IgG and a homogenous rabbit antitype III pneumococcal polysaccharide antibody. This VH inhibited the binding of 125I-labeled (allotype a) IgG to anti-a1 allotypic antibodies. The recombinant molecule consisting of VH and light chain from the homogeneous antibody is active in an antigen binding assay.

Animals↗
PubMed1979

Isolation and characterization of an active variable domain from a homogeneous rabbit antibody light chain.

The variable domain (VL) of allotype b4 light chains of rabbit IgG was isolated from both nonimmune heterogeneous IgG and a homogeneous antibody directed against type III pneumococcal polysaccharide. Light chains were first isolated and then cleaved under mild acidic conditions between residues 109 and 110. Reduction with dithiothreitol in guanidine hydrochloride cleaved both intradomain disulfide bridges as well as the interdomain disulfide bridge joining the variable and constant domain. The sulfhydryl groups were protected after reduction by p-chloromercuribenzoate. VL was isolated from this mixture of variable and constant domains by affinity chromatography, utilizing sheep antibodies directed against a peptide including residues 110--211 from nonimmune IgG light chain. The isolated VL domain was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and automated Edman degradation. VL from a homogeneous antibody was treated with dithiothreitol to remove p-chloromercuribenzoate, reoxidized, and recombined with homologous heavy chain. The binding of this recombinant to type III pneumococcal polysaccharide was identical with that of the light-chain--heavy-chain recombinant.

Amino Acids↗
PubMed1976

A pharmacological study of the angiotensin receptor and tachyphylaxis in smooth muscle.

The interaction of angiotensin with its receptor has been studied on the basis of the tachyphylaxis shown by the rat uterus towards angiotensin II when pH and Ca2+ concentration are below physiological levels. 14C-Angiotensin binding and 45Ca2+-uptake investigations suggest tachyphylaxis to be due to increased binding at low pH and Ca2+ concentration. Studies with alkylating (affinity labeled) angiotensin derivatives containing the N-mustard chlorambucil suggest a "Charnière type" inhibition at the Ca-binding site of receptor and an irreversible inhibition at an anionic site. Angiotensin inhibitors containing chlorambucil do not alkylate tissue but are competitive inhibitors suggesting that the aromatic side chain in angiotensin may induce conformational changes in the receptor. The results obtained lead to a logical model for the angiotensin receptor allowing for normal activation by the hormone as well as for production of tachyphylaxis.

Animals↗
PubMedDate not supplied

Specificity of serine proteases for cleavage sites on proatrial natriuretic factor.

An immunological approach was used to investigate the specificity of protease cleavage sites on proANF. Cleavage of 35S-cysteine biosynthetically-labeled proANF by whole serum, thrombin and kallikrein was examined. Reaction products were immunoprecipitated with two antibodies directed to different epitopes: a previously characterized antibody directed toward the carboxy-terminus of ANF103-126, which cross reacts with proANF, ANF99-126 and ANF103-126, and a newly prepared antisera to synthetic ANF99-105, which uniquely recognizes ANF99-126, but not proANF or ANF103-126. With increasing time of incubation with rat serum, proANF is sequentially cleaved at the C-terminus of a monobasic Pro-Arg dipeptide sequence to form ANF99-126, and then at the C-terminus of a dibasic Arg-Arg dipeptide sequence to yield ANF103-126. This cleavage activity of serum is blocked by leupeptin (40 micrograms/ml), but not by hirudin (100 nM), a specific inhibitor of thrombin, or by aprotinin (200 KIU/ml), a kallikrein inhibitor. When 100-fold purified serum cleavage enzyme was used in place of crude serum, similar results were obtained. Thrombin cleaves proANF only at the monobasic site to produce ANF99-126 while kallikrein cleaves only at the dibasic site to produce ANF103-126. As expected, the generation of these cleavage products can be inhibited by hirudin or aprotinin respectively. These data indicate that the substrate specificity of the serum cleavage activity is broader than that of thrombin or kallikrein, and that cleavage of proANF by serum proteases may be influenced by conformational restraints. The methods developed here should help in the future characterization of the physiological proANF cleaving enzyme.

Amino Acid Sequence↗
PubMedDate not supplied

Antibody-directed fibrinolysis: a bispecific (Fab')2 that binds to fibrin and tissue plasminogen activator.

A bispecific (Fab')2 molecule was constructed by linking the monovalent Fab' from an anti-fibrin monoclonal antibody to the Fab' from an anti tissue plasminogen activator (tPA, single chain) monoclonal antibody by means of inter-heavy-chain disulfide bonds. An immunochemical complex composed of the bispecific (Fab')2 molecule bound to tPA [tPA-bispecific (Fab')2 complex] was then generated and purified. Its molecular weight was 170 kDa [less than half the molecular weight of a previously described tPA-bispecific antibody complex containing the entire anti-fibrin and anti-tPA immunoglobulin molecules; Runge, M. S., et al. (1987) Trans. Assoc. Am. Phys. 100, 250-255]. The tPA-bispecific (Fab')2 complex was 8.6-fold more efficient in fibrinolysis than tPA alone and 94-fold more potent than urokinase. This enhancement in the fibrinolytic potency of tPA compares favorably with that observed for the bispecific whole-antibody complex. These results suggest that this smaller, less immunogenic molecule is capable of binding both fibrin and tPA with high affinity and of enhancing the thrombolytic efficiency of exogenous and, perhaps, endogenous tPA.

Antibodies, Monoclonal↗
PubMedDate not supplied

Identification of the T-cell and Ia contact residues of a T-cell antigenic epitope.

The precise molecular structure of the antigenic determinant recognized by the T-cell receptor of the CD4-positive cell has not been completely resolved. A major advance in our understanding of this issue has been made by our demonstration of a direct association between an immunogenic peptide and a purified Ia molecule. The most likely and economical hypothesis is that antigen binds directly to an Ia molecule creating the antigenic determinant and that this antigen-Ia complex is recognized by the T-cell receptor. We examined in detail a determinant of hen egg-white lysozyme (HEL) contained in the tryptic fragment HEL(46-61), recognized by T cells in H-2k strains of mice. This peptide binds with a Kd of approximately 3 microM to I-Ak molecules. We have already ascertained that (1) the 10-mer HEL(52-61) is the shortest stimulatory peptide; (2) the Leu56 residue, the only residue different from mouse lysozyme, is responsible for the immunogenicity; (3) the Leu56 and Tyr53 residues are critical for recognition by the T-cell receptor and (4) HEL(46-61) generates multiple determinants when it associated with the I-Ak molecule. If antigen and Ia interact, the antigen must have two features: it must bind to an Ia molecule and also interact with the T-cell receptor. The two sites do not appear to be laterally separable in this peptide and are therefore probably composed of distinct but interspersed amino-acid residues. We have now identified the three residues of HEL(52-61) that contact the T-cell receptor and three other residues that contact the I-Ak molecule. From modelling studies we also propose that HEL(52-61) assumes an alpha-helical conformation as it is bound to I-Ak and recognized by the T-cell receptor.

Amino Acid Sequence↗