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F Wold

Publications and source records attributed to F Wold.

At least 37 records · Page 2Linked to original sources
PubMed1989

Glycoproteins in Rathke's gland secretions of loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempi) sea turtles.

1. The Rathke's gland secretions of loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempi) sea turtles contain 20 and 10 mg of protein/ml, respectively. The proteins of each species were separated by gel filtration into two major fractions, one (35%) in the excluded volume, and one (50%) with a molecular mass of approximately 55 kDA. 2. The 55 kDa fraction from each species' secretions exhibits a single band on SDS-PAGE (Mr approximately equal to 55,000) and a single amino-terminal sequence. 3. The amino acid compositions of the two 55 kDa proteins are similar, and the first 15 residues of their amino terminus are identical. Both proteins contain glucosamine. 4. Analyses of the amino acid and amino sugar composition of the high molecular weight fractions from the two turtle species also indicate similarities; there are distinct differences between them and their 55 kDa proteins.

Amino Acid Sequence↗
PubMed1989

Post-translational modifications of proteins: some problems left to solve.

Three major questions regarding the post-translational modification of amino acid side chains in proteins are briefly considered: (1) What are the biological functions of the reactions, (2) what is the specificity of the processing reactions in selecting only a few or sometimes even only one residue for modification, and (3) how do we solve the uniqueness of the processing steps in the production of recombinant proteins? The answers to these questions are not obvious at this time.

Protein Processing, Post-Translational↗
PubMed1988

The effect of the protein matrix on glycan processing in glycoproteins. Kinetic analysis of three rat liver Golgi enzymes.

In order to assess the basis for the regulatory effects of the protein matrix on the processing of glycans in glycoproteins, we have used the avidin-biotinylglycan neoglycoprotein model system to compare the kinetic parameters for three rat liver Golgi enzymes acting on their free and protein-bound glycan substrates. Two modes of glycan display in the avidin complex were produced by the use of either the biotinyl- or the 6-biotinamidohexanoyl-group as ligands for the avidin binding. N-Acetylglucosaminyltransferase I gave a 100-fold decrease in Vmax/Km for the avidin complex of Man5GlcNAc2-(biotinyl)Asn as compared to the free glycan derivative; the rate difference reflects a large (25x) decrease in the Vmax and a relatively small increase (4x) in Km. When the substrate with the extension arm (Man5GlcNAc2-(6-biotinamidohexanoyl)Asn) was used, the difference between Vmax/Km for free and avidin-bound substrate was only 6-fold. The Vmax/Km ratio for N-acetylglucosaminyltransferase II also showed a 10-fold difference for free and avidin-bound GlcNAcMan3GlcNAc2-(biotinyl)Asn; the introduction of the extension arm in the complex reduced the difference to about 3-fold. The third enzyme, galactosyltransferase, acting on the substrate GlcNAcMan5-GlcNAc2-R in the presence of the mannosidase II-inhibitor swainsonine, showed a small, 2- to 3-fold, decrease in the Vmax for the bound substrates, both with and without the extension arm. The results suggest that the protein matrix affects the catalytic efficiency rather than the substrate affinity of the processing enzymes.

Alkaloids↗
PubMed1988

The covalent structure of individual N-linked glycopeptides from ovomucoid and asialofetuin.

In order to explore whether individual N-linked glycans in a given glycoprotein may be processed to different end products and at the same time prepare a number of well characterized glycopeptides as substrates for glycopeptide hydrolases, we have prepared the individual glycopeptides representing the four major glycosylation sites in ovomucoid and the three sites in asialofetuin. The individual glycopeptides were characterized by amino acid sequence determination before and after removal of the glycan by peptide:N-glycanase (amidase), and the liberated glycans were subjected to mass spectrometric analysis. As expected from available sugar analyses of the individual glycans in ovomucoid, no major differences were detected between the four glycosylation sites in this glycoprotein, but a definite trend toward less processed (less extensively branched) species was observed in going from site 1 to 4. In fetuin, for which the glycan pool is known to be made up of about two-thirds triantennary and one-third biantennary structures, the analysis of the three glycopeptides gave triantennary to biantennary ratios of 75/25, 67/33, and 70/30, respectively, demonstrating that the three sites are processed to a very similar, albeit perhaps not identical, extent. All the glycopeptides obtained in these studies, including the CNBr-produced glycopeptide from ovalbumin, were purified by a set series of steps, gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DE52 and/or reverse phase high performance liquid chromatography. Based on the results, these procedures appear to have general application for the preparation of glycopeptides.

Amino Acids↗
PubMed1988

Purification and characterization of two glycopeptide hydrolases from jack beans.

Two glycopeptide hydrolases, an endo-beta-N-acetylglucosaminidase and peptide:N-glycanase (amidase), have been isolated from defatted jack bean meal by standard procedures involving differential solubility and column chromatography. The purified products appear to be free of contaminating proteases and exoglycosidases, and their substrate specificity has been explored with regard to both glycan and peptide structure of the substrates. The endoglycosidase appears to be specific for high mannose glycans; no hydrolysis of either hybrid or complex glycans has been observed. It shows limited activity with two intact glycoproteins, ribonuclease B and yeast invertase, and gives optimal rate with glycopeptides. Free glycan-Asn derivatives are poor substrates in comparison with glycopeptides or glycan-Asn derivatives where the alpha-amino group has been dansylated. The amidase will liberate both high mannose, hybrid, and asialo-complex glycans from both proteins and peptides, but many glycans in intact proteins or in long peptides are resistant to the amidase and become active as substrates only after further proteolytic cleavage. The best substrates appear to be those with the glycosylated asparagine no more than 4-5 residues in from either the NH2- or COOH-terminal end of the peptide. Sialylated glycans do not appear to be released by the amidase.

Chromatography, Gel↗
PubMed1988

Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats.

A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with beta-naphthoflavone (beta-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56,000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroyl-phosphatidylcholine. In this system turnover rates for benzo[alpha]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nmol P-446 respectively. While NH2 terminal amino acid sequence analysis of 18 of the first 20 residues suggests that the cytochrome P-446 isolated from malnourished rats is identical with form c, the catalytic activities suggest that this isozyme may be a more effective or efficient catalyst for some substrates.

Animals↗
PubMed1988

Effects of the protein matrix on glycan processing in glycoproteins.

In the biosynthesis of glycoproteins containing asparagine-linked glycans, a number of regulatory factors must be involved in converting the single glycan precursor into the variety of different final structures observed in different eukaryotic species. Among these factors are the kind of glycan-processing enzymes available in the Golgi apparatus of different cells, the specificity and regulatory properties of these enzymes, and the unique properties of the protein matrix in which a given glycan resides during the biosynthetic processing. In examining the role of this latter regulatory factor, we have considered a simplified model in which a few key steps are common to all cells, regardless of the nature of the processing enzymes available. The protein-bound oligomannose precursor Man8GlcNAc2-, arriving in the Golgi after the initial trimming in the endoplasmic reticulum (ER), first undergoes a series of preprocessing steps to yield Man5GlcNAc2- in animals and plants or Man13-15GlcNAc2- in yeast. At this stage the key commitment step--to process or not to process--determines whether the above intermediates will remain as unprocessed oligomannose structures or be initiated into a new series of reactions to yield processed structures characteristic of the organisms involved (complex or hybrid for vertebrates, polymannose for yeast, xylosylated glycans for plants and some invertebrates, or Man3GlcNAc2- structures for other invertebrates). It is proposed that this commitment step, along with the obligatory preprocessing steps, is regulated primarily by each glycan's unique exposure on its protein matrix. Subsequent processing steps leading to complex or hybrid structures, fucosylation, extent of branching, and specific structures at the nonreducing terminals are most likely determined primarily by the enzyme makeup of the individual processing machineries, but with the protein matrix still playing a significant role.

Animals↗
PubMed1988

Cofactors in and as posttranslational protein modifications.

A symposium at the FASEB meeting in Las Vegas in May 1988 will be devoted to the role of cofactors (vitamins, coenzymes, prosthetic groups) in and as posttranslational protein modifications; the symposium is part of a thematic focus on metabolic regulation. In planning the symposium, we decided to consider metabolic regulation in its broadest context, which should include both the short-term activity modulations in the life of contemporary organisms and the adaptations of special molecular strategies over evolutionary time. We further decided to focus the symposium context on the involvement of cofactors both as catalytic participants in and as substrates or end products of posttranslational modifications. As a preview of the actual symposium, the present discussion is an attempt to enumerate cases of cofactor involvement in these different categories: 1) essential nutrients as participants in posttranslational modifications; 2) cofactors as donor substrates in reversible, regulatory modifications; and 3) cofactor incorporation or generation as covalent constituents of proteins. The actual symposium topics are taken from category 1: vitamin C and protein hydroxylation (K. I. Karivikkio) and vitamin K and protein carboxylation (J. W. Suttie) and category 3: biotinylation (H. G. Wood), phycobiliproteins (A. Glazer), and pyruvoyl enzymes (W. Dowhan).

Animals↗
PubMed1987

The regulation of glycan processing in glycoproteins. The effect of avidin on individual steps in the processing of biotinylated glycan derivatives.

The effect of the protein matrix on glycan processing by rat liver Golgi enzymes has been evaluated by a direct comparison of substrate----products conversion of a free glycan and of the same glycan linked to a protein. The glycan substrates had the general structure R-glycan where R represented either biotinyl-Asn-GlcNAc2- or 6-(biotinamido)hexanoyl-Asn-Glc-NAc2- and the protein used was avidin; the extension arm in one of the glycan substrates permitted the additional comparison of two avidin-biotin-glycan complexes. By the use of different glycans as substrates, by the presence or absence of donor substrates (UDP-GlcNAc, UDP-Gal, and CMP-sialic acid (Sia) and/or the inhibitor, swainsonine, it was possible to dissect the individual steps involved in the conversion of R-Man6 (or R-Man5) to a biantennary complex glycan, R-Man3-GlcNAc2-Gal2-Sia2 or to the hybrid glycan R-Man5-GlcNAc-Gal-Sia. Using fast atom bombardment-mass spectrometry to identify and quantify the substrates and products of each parallel incubation of free and avidin-bound substrates, the following observations were made. With the substrate without the extension arm, avidin-binding inhibited mannosidase I, GlcNAc transferase I, and the second step of the reaction catalyzed by mannosidase II (R-Man4-GlcNAc----R-Man3-GlcNAc); the second step of the reaction catalyzed by Gal-transferase was also inhibited to a lesser extent. This inhibition was greatly reduced or absent with the substrates with the extension arm and was consequently referred to as the short range effect. A long range effect of avidin binding expressed by both substrates with and without extension arm was observed for Gal-transferase acting in the hybrid glycan pathway (R-Man5-GlcNAc----R-Man5-GlcNAc-Gal) in the presence of swainsonine and also for Sia-transferase in the catalysis of the incorporation of the second Sia residue into the complex product (R-Man3-GlcNA2-Gal2-Sia----R-Man3-GlcNAc2- Gal2-Sia2) and to a lesser extent in the hybrid pathway (R-Man5-GlcNAc-Gal----R-Man5-GlcNAc-Gal-Sia). GlcNAc transferase II did not appear to be affected by avidin. Based on the information available on the biotin-binding site in avidin, it is proposed that the short range effect reflects the masking of the core chitobiose unit in the avidin-glycan complexes in the absence of the extension arm, but not in the presence of the arm, and that the early processing enzymes thus may require a fully exposed chitobiose for full activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗
PubMed1987

The use of avidin-biotinylglycan as the model for in vitro glycoprotein processing.

In an attempt to evaluate the effects of the protein matrix on the specificity of glycoprotein processing in Golgi membranes, we have developed a model neoglycoprotein consisting of biotinylated glycans bound noncovalently to avidin (Chen, V. J., and Wold, F. (1986) Biochemistry 25, 939-444) with which the protein effect on processing can be evaluated as the difference in substrate efficiency between a free biotinylated glycan and the same biotinylated glycan bound to avidin. The avidin (streptavidin)-glycan complex stability was found to be proper for the experimental design; the complex remains intact for extended periods of incubation at the concentrations used, but the glycan can be completely liberated and recovered by heating the complex at 95 degrees C for 10 min in the presence of a 10-fold molar excess of biotin. By measuring the relative rates of [14C]sugar incorporation into the free and bound substrates it was demonstrated that the protein indeed influences the processing reactions; under conditions where free glycans such as biotinyl-Asn-Glc-NAc2-Man5 and 6-(biotinamido)hexanoyl-Asn-Glc-NAc2-Man5 could be converted to the biantennary products R-Asn-GlcNAc2-Man3-GlcNAc2-Gal2-sialyl2 in the presence of UDP-GlcNAc, UDP-Gal and CMP-sialic acid and Golgi enzymes, the avidin-bound derivative without the extension arm gave only low levels of product and the streptavidin-bound one remained unaltered. The presence of the extension arm in the substrates significantly improved the yield of some products in the complex, apparently by reducing or eliminating the avidin inhibition of the early steps, but not of the late ones. There are consequently two types of effect of the protein matrix on processing efficiency. One is expressed only when the glycan is close to the protein surface and affecting primarily early steps (mannosidases and GlcNAc transferases). The other is apparently independent of the proximity of the glycan core and the protein, and affects primarily late steps, in particular the incorporation of the second sialic acid residue into a biantennary complex glycan.

Acetylglucosamine↗
PubMed1986

Rabbit muscle extracts catalyze the specific removal of N-acetylmethionine from acetylated peptides.

Rabbit muscle has been found to contain an activity that catalyzes the specific removal of Ac-Met from acetylated peptides. The activity is associated with free ribosomes and microsomes in the rabbit muscle extract but can be removed from these subcellular fractions by exposure to 0.5 M NaCl in the presence of 2 mM MgCl2; only partial removal was achieved with microsomes, but complete removal with ribosomes. A nearly 200-fold enrichment of the activity was achieved by this simple succession of differential centrifugation and salt extraction. Eighteen 14C-acetylpeptides have been tested as substrates for the partially purified activity assaying for the production of free 14C-acetylamino acid by high performance liquid chromatography. None of the peptides containing N-terminal acetylated Ala, Asp, Ser, or Gly were cleaved at a significant rate. Six of a total of eight peptides containing N-terminal Ac-Met were cleaved by the ribosomal extract at different rates. The active substrates varied in length from tri- to undecapeptides. The activity is inhibited by high concentrations of the protease inhibitor phenylmethylsulfonyl fluoride. Based on these observations, we tentatively conclude that the activity satisfy the criteria of a general N-terminal protein processing enzyme: it can remove Ac-Met from most, but not all, N-terminal sequences and appears to be inactive toward the N-terminal acetylamino acids most commonly found in eukaryotic proteins.

Acetylation↗
PubMed1986

Neoglycoproteins: preparation and properties of complexes of biotinylated asparagine-oligosaccharides with avidin and streptavidin.

Neoglycoproteins in which the oligosaccharide moieties are attached noncovalently to the protein through a high-affinity ligand have been prepared from biotinylated oligosaccharides and avidin or the nonglycosylated microbial analogue streptavidin. One of the asparagine-oligosaccharides purified from Pronase-digested ovalbumin (Man6-GlcNAc2-Asn) was reacted with an excess of the hydroxysuccinimide ester of biotin or, for the purpose of quantitation, [3H]biotin. Derivatives were also prepared with an extension "arm", a 6-aminohexanoyl group, between biotin and asparagine. When the purified biotinyl-Asn-oligosaccharide was added to avidin or streptavidin, a complex was formed containing 3 mol of oligosaccharide/mol of protein. The complexes were stable at neutral pH in the absence of biotin and could be dialyzed for 2 weeks without any significant loss of ligand. In the presence of biotin, or under denaturing conditions, the oligosaccharide derivative was released and could be quantitatively recovered. To assess the influence of the protein matrix on the reactivity of the oligosaccharide units, free biotinyl-Asn-oligosaccharide and the corresponding avidin and streptavidin complexes were exposed to alpha-mannosidase in parallel experiments. The rate of hydrolysis of the free derivative was severalfold faster than that of the two protein complexes, and at the time when about 90% of the free derivative had all five alpha-mannosyl residues removed, the majority of the protein-bound derivatives contained two to four undigested alpha-mannosyl residues and also had a significant amount of undigested starting material. The ease of preparation and the properties of these neoglycoproteins suggest that they should be excellent models for the study of glycoprotein-receptor binding and glycoprotein processing.

Asparagine↗
PubMed1985

Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH.

Amino Acid Sequence↗
PubMed1984

Neoglycoproteins: in vitro introduction of glycosyl units at glutamines in beta-casein using transglutaminase.

Exploring different methods for preparing neoglycoproteins with a specific number of oligosaccharides in specific positions, we have used guinea pig liver transglutaminase to incorporate glycosyl units into glutamine residues in beta-casein. In order to prevent epsilon-(gamma-glutamyl)lysine cross-link formation, the lysine residues of beta-casein were first blocked either by amidination with ethyl acetimidate or by acylation with succinic anhydride. The glycosyl donor substrates prepared for this work were maltotriose reductively aminated with cadaverine, N-(Glc-Glc-glucitol-1)-cadaverine, and an asparaginyl nonasaccharide from ovalbumin modified with a 6-aminohexanoyl group at the alpha-amino group. The transglutaminase-catalyzed incorporation of these two donors into the beta-casein derivatives was monitored in comparison to the incorporation of the commonly used transglutaminase substrate dansylcadaverine under conditions of optimal incorporation (multiple additions of enzyme, large excess of donor, and long incubation time). For both dansylcadaverine and Glc-Glc-Glc(OH)-cadaverine, 5 and 8 mol of donor were incorporated per mol of amidinated and succinylated beta-casein, respectively. Competition experiments showed that the two donor substrates are incorporated into the same glutamine sites. Partial sequencing of the glycosylated beta-casein permitted the identification of glutamine residues 56, 79, 167, 175, and 194 as the primary sites of incorporation in amidinated casein with residues 54 and 182 as possible sites for partial glycosylation. The results are consistent with a specific glycosylation of only selected glutamines in this transglutaminase-catalyzed process.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗
PubMed1984

Neoglycoproteins: preparation of noncovalent glycoproteins through high-affinity protein- (glycosyl) ligand complexes.

This work was undertaken as part of a search for well-characterized glycoprotein models in which both the oligosaccharide structure, the number of oligosaccharide chains, and the precise location of these chains in the protein are known. On the basis of the fact that high-affinity ligand binding sites have been defined precisely for several proteins in terms of both number and relative location, the hypothesis to be tested was that if oligosaccharide chains were covalently attached to such high-affinity ligands, they would be specifically bound in the ligand sites of the appropriate protein, thus permitting the preparation of neoglycoproteins of precise predetermined oligosaccharide valency and topography. To test this hypothesis, pyridoxal 5'-phosphate was reductively (NaB3H4) aminated with the alpha-amino group of the asparagine oligosaccharide Man6-GlcNAc2-Asn from ovalbumin. When the resulting phosphopyridoxylated oligosaccharide (PG) was added to the apo form of aspartate aminotransferase (AAT; EC 2.6.1.1, the cytosolic enzyme from pig heart, consisting of two subunits and containing two coenzyme binding sites), a 2:1 (PG-AAT) complex was formed which could be characterized on the basis of tritium content, the absorbance and fluorescence of the pyridoxamine phosphate moiety of PG, and the concanavalin A binding properties acquired by AAT through the incorporation of the oligosaccharide. As expected from the established properties of the holoenzyme, the AAT-PG complex is stable in the absence of phosphate or vitamin B6 derivatives and can be dialyzed for 24 h without any significant loss of PG. According to the three-dimensional model of AAT, the oligosaccharide chain of PG should be partially masked in the coenzyme binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗