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T H Plummer

Publications and source records attributed to T H Plummer.

At least 55 records · Page 3Linked to original sources
PubMed1985

Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F.

Endo-beta-N-acetylglucosaminidase F (Endo F) and peptide:N-glycosidase F (PNGase F) were purified from cultures of Flavobacterium meningosepticum by ammonium sulfate precipitation followed by gel filtration on TSK HW-55(S). This system separated the two enzymes and provided PNGase F in a high state of purity, but the basis for the resolution appeared to be hydrophobic interaction and not molecular size. Studies using purified Endo F and PNGase F with defined glycopeptides demonstrated that Endo F was somewhat similar to Endo H in that it hydrolyzed many, but not all, high-mannose and hybrid oligosaccharides, as well as complex biantennary oligosaccharides. PNGase F, in contrast, hydrolyzed all classes of asparagine-linked glycans examined, provided both the alpha-amino and carboxyl groups of the asparagine residue were in peptide linkage. Deglycosylation studies with PNGase F revealed that many proteins in their native conformation were susceptible to this enzyme but that prior denaturation in sodium dodecyl sulfate greatly decreased the amount of enzyme required for complete carbohydrate removal.

Amidohydrolases↗
PubMed1985

Oligosaccharide microheterogeneity of the murine major histocompatibility antigens. Reproducible site-specific patterns of sialylation and branching in asparagine-linked oligosaccharides.

The influence of peptide structure of endogenous cell-surface glycoproteins on the branching and sialylation of their asparagine-linked oligosaccharides was evaluated in a murine B cell lymphoma, AKTB-1b. This cell line simultaneously synthesizes two classes of major histocompatibility antigens that, within each class, share a high degree of amino acid sequence homology and possess potential N-linked glycosylation sites at invariant positions. [3H]Mannose-labeled oligosaccharides were released from each of 11 purified glycosylation sites by the almond peptide:N-glycosidase and analyzed by a variety of chromatographic procedures and glycosidase treatments. The data indicate: 1) a unique distribution of oligosaccharide structures is present at each glycosylation site; 2) each site-specific oligosaccharide pattern is highly reproducible, independent of the number of in vivo tumor passages. The heavy chain of the class I antigens, H-2Kk and H-2Dk contain two and three sites, respectively, in which biantennary structures predominate. However, each site varies with respect to the extent of sialylation and the proportions of more highly branched structures present. The class II antigens, I-Ak and I-Ek, each contain an alpha-chain site toward the N terminus and a single beta-chain site where the overall extent of sialylation is similar, yet the distributions of antennary structures are dramatically different for each. The alpha-chains of each class II antigen also contain a more C-terminal underglycosylated site where sialylation and branching are reduced to differing degrees depending upon the site. The influence of peptide structure on oligosaccharide microheterogeneity is manifest at two levels. First, the overall distributions of oligosaccharides at corresponding sites on structurally related glycoproteins are similar. Second, the specific "fingerprint" of sialylation and branching patterns at a particular site are reproducibly unique. These data suggest that subtle changes in peptide structure are reflected in the extent of sialylation and branching of oligosaccharides found at corresponding glycosylation sites of structurally related glycoproteins.

Animals↗
PubMed1984

Demonstration of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations.

Endo-beta-N-acetylglucosaminidase F preparations from Flavobacterium meningosepticum have been found to contain peptide:N-glycosidase activity. Only the second activity, designated as peptide:N-glycosidase F, readily cleaves the beta-aspartylglycosylamine linkage of a fetuin triantennary complex glycopeptide, as shown by the isolation of the corresponding carbohydrate-free peptide containing aspartic acid and of an intact oligosaccharide with a di-N-acetylchitobiosyl moiety at the reducing end. Both activities in the mixture will hydrolyze a high mannose octaglycopeptide from ovalbumin, with the type of product formed being influenced by pH. At pH 4.0, only the endo-beta-N-acetylglucosaminidase F activity is functional, releasing octapeptide-GlcNAc and oligosaccharide-GlcNAc. At pH 9.3, the predominant cleavage is by peptide:N-glycosidase F at the glycosylamine bond, releasing octapeptide and oligosaccharide-GlcNAc-GlcNAc. This latter oligosaccharide is then hydrolyzed by endo-beta-N-acetylglucosaminidase F to oligosaccharide-GlcNAc plus GlcNAc.

Acetylglucosaminidase↗
PubMed1983

Stable oligosaccharide microheterogeneity at individual glycosylation sites of a murine major histocompatibility antigen derived from a B-cell lymphoma.

The H-2Kk glycoprotein has been isolated by monoclonal antibody affinity chromatography, and an analysis of the asparagine-linked oligosaccharides present at the two major glycosylation sites has been performed. Antigen obtained from the AKTB-1b B-cell lymphoma that had been labeled with [2,6-3H]mannose for 5 or 21 h or for 5 h followed by a 5-h chase was digested exhaustively with trypsin. Each glycosylation site was then isolated by reverse phase high performance liquid chromatography using a C18 column. After removal from the peptide backbone by the almond emulsin peptide: N-glycosidase, the oligosaccharides from each isolated site were analyzed by gel filtration, ion exchange chromatography, concanavalin A affinity chromatography, and glycosidase treatment to assess the contribution of sialic acid and branching patterns of the oligosaccharide backbones to the overall microheterogeneity. The glycosylation of the H-2Kk antigen derived from several different AKTB-1b tumor preparations was examined during a period covering 1 year, during which time the tumor was passaged continuously in vivo in 2-week cycles. Our results conclusively demonstrate that the pattern of oligosaccharide microheterogeneity at the two glycosylation sites of the H-2Kk antigen derived from AKTB-1b cells is stable and that each site differs as to the specific array of oligosaccharide types found on the fully processed glycoprotein. In addition, this report describes an analytical scheme employing reverse phase high performance liquid chromatography to follow oligosaccharide processing and hydrolysis of the N-glycosidic bond by the peptide: N-glycosidase.

Animals↗
PubMed1983

Enkephalin convertase: potent, selective, and irreversible inhibitors.

Biproduct analogs of lysine and arginine are potent inhibitors of enkephalin convertase, a carboxypeptidase B-like enzyme which appears to be physiologically associated with enkephalin biosynthesis. The most effective derivatives are guanidinoethylmercaptosuccinic acid and guanidinopropylsuccinic acid with respective Ki's of 8.8 and 7.5 nM. These dicarboxylic acid analogs of arginine are several hundred fold more potent towards enkephalin convertase than towards carboxypeptidase B or N. Kinetic analysis indicates the pure competitive nature of the inhibition. Bromoacetyl-D-arginine, an irreversible inhibitor of carboxypeptidase B and N, is also an efficient irreversible inhibitor of enkephalin convertase.

3-Mercaptopropionic Acid↗
PubMed1983

N-terminal amino acid sequences of precursor and mature forms of alpha-1-antitrypsin.

alpha-1-Antitrypsin is found in hepatocytes as a high-mannose glycoprotein (Mr 49 000), extracellularly as a complex-type glycoprotein (Mr 54 000). Deglycosylation of both forms with peptide: N-glycosidase led to proteins of identical app. Mr (41 000). The sequence of 26 N-terminal amino acids of rat alpha 1-antitrypsin was determined. A high content of polar amino acids was found. The partially characterized presequence of in vitro synthesized alpha 1-antitrypsin showed a cluster of hydrophobic amino acids. A pre-peptide of 24 amino acids is proposed. There is no evidence for the existence of a propeptide.

Amino Acid Sequence↗
PubMed1982

Oligosaccharide accessibility to peptide:N-glycosidase as promoted by protein-unfolding reagents.

The ability of almond emulsion peptide:N-glycosidase to remove oligosaccharide chains from intact glycoproteins was studied. Protein conformation appeared to be the main factor affecting carbohydrate removal. In the native state the oligosaccharides of ribonuclease B and the Fab mu fragment derived from immunoglobulin M were completely resistant to the enzyme, indicating that the polypeptide chain restricts access to the site of hydrolysis. Heat denaturation in sodium dodecyl sulfate rendered these glycoproteins susceptible to peptide:N-glycosidase, but perturbation with chaotropic salts provided a more gentle approach, which was as effective as detergent-unfolding and more compatible with the stability of the enzyme. Once exposed by the unfolding reagents, the complex oligosaccharides of Fab mu were released more rapidly than the high mannose chains of ribonuclease B, consistent with their preferential release from small glycopeptides (Plummer, T. H., Jr., and Tarentino, A. L. (1981) J. Biol. Chem. 256, 10243-10246).

Amidohydrolases↗
PubMed1981

Facile cleavage of complex oligosaccharides from glycopeptides by almond emulsin peptide: N-glycosidase.

Almond emulsin peptide:N-glycosidase has been partially purified by using a new 3H-labeled 5-dimethylaminonaphthalene-1-sulfonyl-octaglycopeptide substrate derived from ovalbumin. The enzyme hydrolyzes the beta-aspartylglycosylamine linkage of both high mannose and biantennary complex glycopeptides, as shown by the isolation of the corresponding carbohydrate-free peptides containing aspartic acid and intact oligosaccharides with the core di-N-acetylchitobiosyl moiety at the reducing end. Complex glycopeptides appear to be the preferred substrates. The location of the oligosaccharide on the peptide backbone and its chain length are major determinants for enzymatic activity. Glycosylated asparagine residues are hydrolyzed less favorably if present at the carboxyl- or NH2-terminal position of a peptide chain. Glycopeptides containing long, bulky oligosaccharide chains are cleaved by peptide:N-glycosidase at least 15-fold faster than their corresponding endo-beta-N-acetylglucosaminidase H-modified, peptide-GlcNAc counterparts.

Amidohydrolases↗
PubMed1978

Asparaginyl glycopeptides with a low mannose content are hydrolyzed by endo-beta-N-acetylglucosaminidase H.

Substrates susceptible to endo-beta-N-acetylglucosaminidase H were reduced in size through alpha-mannosidase treatment and periodate oxidation to yield the following compounds: (Man)4(GlcNAc)2Asn, [Manalpha 1 leads to 6Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNACAsn]; (Man)3(GlcNAc)2Asn, [Manalpha 1 leads to 3Man-alpha 1 leads to 6Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn]; (Man)2(GlcNAc)2Asn, [Manalpha 1 leads to 6Manbeta1 leads to 4GlcNAcbeta 1 leads to 4BlcNAcAsm]. Comparison of the relative rates of hydrolysis of these compounds with (Man)5(GlcNAc)2-Asn, the most active substrate to date for the endoglycosidase, revealed (Man)4(GlcNAc)2Asn to be hydrolyzed faster than (Man)5(GlcNAc)2Asn and (Man)3-(GlcNAc)2Asn to be equal to or slightly better than (Man)5(GlcNAc)2Asn as a substrate. (Man)2(GlcNAc)2-Asn was completely hydrolyzed but at a rate that was about 10(4) slower than (Man)5(GlcNAc)2Asn, which is comparable to that for (Man)3(GlcNAc)2Asn(aa)x [Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn(aa)x], obtained from immunoglobulin M. (Man)1(GlcNAc)2Asn, [Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn] was hydrolyzed at a 100-fold slower rate than the latter glycopeptide. The effective range of endo-beta-N-acetylglucosaminidase H has thus been extended to compounds containing as few as 2 mannosyl residues.

Acetylglucosaminidase↗
PubMed1978

Human plasma carboxypeptidase N. Isolation and characterization.

Human plasma carboxypeptidase N has been purified 2,600-fold from pooled, outdated plasma in a 30% yield. Isolation was accomplished by chromatography on DEAE-cellulose and on a p-aminobenzoyl-L-arginine-Sepharose 6B affinity column. Carbohydrate accounts for 17% of the weight calculated from its amino acid and carbohydrate composition. The enzyme appears to consist of three subunits of Mr = 83,000, 55,000, and 49,000 and contains a significant amount of bound zinc. Purified enzyme preparations are very sensitive to proteolytic degradation but are stable for at least 3 months at 4 degrees.

Amino Acids↗
PubMed1978

By-product analogues for bovine carboxypeptidase B.

A series of monocarboxylic and dicarboxylic acid sulfur-containing by-product analogues of lysine and arginine has been synthesized and tested as competitive inhibitors of bovine carboxypeptidase B. The most effective derivatives were guanidinoethylmercaptosuccinic acid and aminopropylmer-captosuccinic acid with Kis of 4 and 8 X 10(-6) M, respectively. Kinetics studies established the pure competitive nature of the inhibition. Mixed studies with the alkylating reagents bromoacetyl-D-arginine and bromoacetamidobutylguanidine established their efficiency in protecting the active-center glutamic acid and tyrosine of bovine carboxypeptidase B, respectively, from irreversible alkylation. Kinetic studies with bovine carboxypeptidase A and porcine carboxypeptidase B showed a lack of efficiency for A and high degree of efficiency for B.

Animals↗
PubMed1975

The isolation and structure of the core oligosaccharide sequences of IgM.

Methods are presented for separating the three IgM heavy chain sialoglycopeptides associated with asparagines 170, 332, and 395. The core glycopeptide units containing the disaccharide fucosyl-N-acetylglucosamine were obtained through the use of an endo-beta-N-acetylglucosamindase from Diplococcus pneumoniae, following exoglycosidase treatment of the sialoglycopeptides. In addition to the core glycopeptides, high yields of a tetrasaccharide, (Man)3GlcNAc, were obtained. The fucose in the core disaccharide is glycosidically linked to the 6-O position of the N-acetylglucosamine residue in Asn-GlcNAc. This core unit is resistant to glycosyl asparaginase, but becomes susceptible to hydrolysis on removal of the fucosyl residue by a purified hen oviduct alpha-L-fucosidase. The core sequence of the immunoglobulin M sialoglycopeptides appears to be similar to that of most other asparagine-linked oligosaccharides in consisting of a basic unit composed of beta-D-Man-(1 leads to 4)beta-D-GlcNAc(1 leads to 4)beta-D-GlcNAc(1 leads to 4), but with L fucose linked alpha-(1 leads to 6) to the proximal GlcNAc. The two nonreducing terminal ends of (Man)3GlcNAc are linked to beta-D-Man by alpha-(1 leads to 3) and alpha(1 leads to 6) bonds, respectively.

Amino Acids↗
PubMed1975

Further characterization of bovine pancreatic lipase.

1. The amino acid composition of bovine pancreatic lipase is very similar to that of porcine lipase. 2. Bovine lipase possesses a residue of lysine at the N-terminal position and a half cystine or a cysteine at the C-terminal position. 3. Bovine lipase contains two free sulfhydryl groups of different reactivities to 5, 5'-dithiobis-(2-nitrobenzoic) acid. One of these groups is buried in the native conformation of the enzyme and is fully titrated in 1.5 M urea when reaction is performed in the presense of 1 mM EDTA.

Amino Acids↗