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F Wold

Publications and source records attributed to F Wold.

At least 19 recordsLinked to original sources
PubMed1992

Specificity determinants of acylaminoacyl-peptide hydrolase.

In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-peptide hydrolase (EC 3.4.19.1), a number of acetylated peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of peptides, by extension of the N-terminus with acetylamino acids or acetylpeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the peptides with Pro in the third position were very poor substrates; only the peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in hydrolase activity varying with the charge's distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference peptides without positive charges.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

N-terminal sequence analysis of N alpha-acetylated proteins after unblocking with N-acylaminoacyl-peptide hydrolase.

The enzyme acylaminoacyl-peptide hydrolase represents an attractive reagent for the removal of acetylamino acids from the N-terminus of proteins prior to sequencing. However, the enzyme will not accept intact proteins as substrates, and a blocked protein must consequently be fragmented to generate a relative short blocked peptide, and all the newly generated amino termini must be blocked with an hydrolase-resistant reagent before the enzyme can be used to specifically unblock the N-terminus. When a number of N-acetylated proteins (enolase, alpha-crystallin, ovalbumin, cytochrome c, parvalbumin, superoxide dismutase, and myelin basic protein) were subjected to fragmentation with proteases or cyanogen bromide, treatment with succinic anhydride and exhaustive extraction with ether, and the resulting salt-free, succinylated peptides were incubated with the hydrolase, the N-terminal sequence was specifically unblocked. An aliquot of the entire peptide mixture was applied to the protein sequencer, and a single sequence, corresponding to the known N-terminal sequence starting at residue 2, was obtained. When another aliquot of the same hydrolase-treated peptide mixture was treated with the enzyme acylase I, the liberated acetylamino acid was cleaved, and the N-terminal amino acid (residue 1) could be identified by amino acid analysis. The amount of sequence information obtained from different proteins with different fragmentation methods varied considerably; in the case of parvalbumin a sequence of 12 residues was obtained, while for myelin basic protein, only 3 residues could be identified; the other proteins yielded from 5- to 9-residue sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1991

Regulation of glycan processing by Golgi enzymes from red kidney bean (Phaseolus vulgaris) seedlings.

The effect of a protein matrix on the processing of glycoprotein glycans by Golgi enzymes from plant seedlings has been determined with an artificial glycoprotein system, comparing the processing rates of glycan-(biotinyl)Asn (or glycan-(biotinamidohexanoyl)Asn) substrates either free or bound to avidin. An analysis of the pooled glycoproteins from the seedlings suggested that the most common glycan structure is a complex one (GlcNAc-Man3GlycNAc2-protein), and consistent with this processing end-product, mannosidases I and II and GlcNAc transferases I and II were all found to be present in the seedling Golgi membrane preparations. The effect of the avidin matrix either in a proximal (biotinyl substrates) or distal (N-(biotinamido)hexonoyl substrates) association with the appropriate glycan substrate for these four enzymes was assessed from the direct comparison of the apparent first-order rate constants for the free and avidin-bound substrate-product conversions. All four plant enzymes were inhibited by the association of the glycan substrates with avidin, but the inhibition was much less pronounced than that observed with the corresponding enzymes from rat liver and hen oviduct. The rate effect shows a progression from 3- to 10-fold rate decreases in the proximal complexes and 2- to 3-fold in the distal complexes in going from the first (mannosidase I) to the fourth (GlcNAc transferase II) enzyme; with the mammalian and avian enzymes the largest effects were for the first ones and much larger absolute rate effects were observed. The results suggest that the nature of the processing enzymes in terms of this response to the avidin glycan substrates may differ in different organisms.

Carbohydrate Sequence↗
PubMed1990

The distribution of glycan structures in individual N-glycosylation sites in animal and plant glycoproteins.

Glycopeptides representing each individual N-glycosylation site in six animal and plant glycoproteins (ovoinhibitor and ovotransferrin, orosomucoid, antitrypsin, phaseolin, and phytohemagglutinin) have been isolated and compared by mass spectrometric analysis. Since the isolation step separates each individual peptide regardless of the nature of the glycan attached to it, it is possible to observe the entire spectrum of glycans associated with each site from the mass spectrum of the corresponding glycopeptide. The three glycosylation sites in ovoinhibitor have very similar but not identical glycans; they are significantly different from those observed in the single site of ovotransferrin. The three sites in serum antitrypsin also have quite similar glycans, whereas the five sites in orosomucoid show considerable variation in both the nature and the relative amount of glycans. The two plant glycoproteins each have two sites with very different glycan structures. Except for the first and third glycosylation sites of antitrypsin which were found to have remarkably homogeneous glycans (97 and 90% of a biantennary complex structure), all the individual glycosylation sites contained heterogeneous mixtures of glycan structures. The results support the proposition that each N-linked glycan in a glycoprotein is affected by its unique protein environment to such an extent that each one may be displayed to the processing enzymes as a unique structural entity. On the basis of a limited number of observations of the glycan interfering with chymotryptic but not tryptic cleavage in the proximity of the glycan attachment site, it is proposed that hydrophobic interactions between the protein and the glycan may be involved in the conformational modulation of the glycans.

Amino Acid Sequence↗
PubMed1990

The processing of N-linked glycans in yeast. Mutually exclusive steps in the processing of a Man6 derivative by yeast membrane preparations.

When a derivatized oligosaccharide isolated from ovalbumin and containing 6 mannose residues was incubated with yeast membranes and GDP-mannose, two sets of products were obtained, a high molecular weight one containing about 25 mannose residues and a low molecular weight one consisting of compounds with 7, 8, and 9 mannose residues, respectively. When the low molecular weight products were reincubated with the yeast membranes and GDP-mannose, no further mannose incorporation was observed, showing that these compounds must be of the wrong structure as substrates for yeast glycan processing enzymes. The structures were investigated by 1H NMR spectroscopy. The high molecular weight products contained an outer chain of an average length of 18 1----6-linked mannose residues attached to a core structure made up of the original 6 mannose residues with one additional 1----2-linked mannose added. The low molecular weight product with 8 mannose residues was deduced to contain a terminal 1----6-linked mannose (on the 1----6 arm) substituted by mannose at the 2-position, and the ones with 7 and 9 mannose residues were identified as having an additional 1----3-linked mannose on the starting Man6 substrate and on the Man8 product, respectively. The results lend further support to the picture that the processing steps must occur in proper sequence for specific products to form.

Carbohydrate Conformation↗
PubMed1989

Purification and characterization of an N-acylaminoacyl-peptide hydrolase from rabbit muscle.

An N-acylaminoacyl-peptide hydrolase has been purified to homogeneity (7,000-fold with 20% yield) from rabbit muscle. This overall enrichment and its general properties as a soluble protein suggest that it is of cytosolic origin and not a component of ribosomes or other cellular organelles. The enzyme has an Mr of 230,000-245,000 and a subunit Mr of 76,000-80,000. An extensive survey of the substrate specificity of the pure enzyme reveals that our earlier conclusions (Radhakrishna, G., and Wold, F. (1986) J. Biol. Chem. 261, 9572-9575) that the enzyme is specific for Ac-Met-peptides are wrong. The enzyme catalyzes the rapid removal of Ac-Thr, Ac-Ala, Ac-Met, Ac-Ser, and more slowly Ac-Gly from peptides of different lengths. Other acetylated amino acids (Cys, Tyr, Asp, Val, Phe, Ile, Leu) may be removed at 1% or less of the rate of the above good substrates from some peptide substrates. The nature of the amino acid in the second position of the acetylated peptide generally has only a minor effect on the reaction rate; however, with charged amino acids (Arg, Asp) in the second position the reaction is retarded, and with proline it is virtually abolished. Except for slow rate of hydrolysis of acetylated dipeptides, the hydrolase does not appear to be severely affected by the peptide length in the range studied (from 2 to 11 amino acid residues). The hydrolase also cleaves formylamino acids from formylated peptides. The biological function of the enzyme is not clear.

Acetylation↗
PubMed1989

Protein matrix effects on glycan processing by mannosidase II and sialyl transferase from rat liver.

The effect of the protein environment on the reaction sequence and the relative rates of two two-step reactions involved in the biosynthesis of complex glycans in glycoproteins has been explored by comparing the processing of biotinylated substrates either free or bound to avidin. By use of biotinyl and biotinamidohexanoyl derivatives, the display of the glycan in a proximal and distal association with the avidin surface could also be assessed. Mannosidase II removes two Man residues from the substrate GlcNAcMan5GlcNAc2-R to yield GlcNAcMAn3GlcNAc2-R. The NMR spectra of the substrate, intermediate, and product showed that the first Man is removed from the 6-arm of the substrate. The rate constants for the first and second step (estimated by direct analysis of the reactants by anion-exchange chromatography with a pulsed amperometric detector) were determined to be about 0.05 and 0.08 min-1, respectively, for the free substrates. In the proximal complex k1 was reduced 80-fold, and the k2 step could not be observed under the same conditions. In the distal complex both k1 and k2 were reduced about 8-fold. Sialyl transferases transfer Sia from CMP-Sia to the biantennary substrate Gal2GlcNAc2-Man3GlcNA2-R to yield the product Sia2Gal2-GlcNAc2Man3GlcNAc2-R with the Sia linked either 2-3 or 2-6 to the Gal residues. The NMR spectra showed that the first step involved the Gal on the 3-arm of the substrate and that both Sia residues were added 2-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The effect of the protein matrix on glycoprotein processing by oviduct Golgi enzymes.

Using the avidin-biotinyl glycan system reported previously (Shao, M.-C., and Wold, F. (1987) J. Biol. Chem. 267, 2968-2972), we have compared the processing efficiency of oviduct enzymes acting on different glycan-(biotinyl)Asn and glycan-(6-biotinamidohexanoyl)Asn derivatives when they are free and bound to avidin. The glycans were selected to permit exploration of the individual processing steps, and the two different groups of derivatives were used to assess both the close (biotinyl) and more distal (biotinamidohexanoyl) display of the glycan relative to the avidin surface. The direct comparison of the free and avidin-bound glycans demonstrated that mannosidase I is strongly inhibited by avidin in both the close and distal complexes, whereas GlcNAc transferase I and mannosidase II are strongly inhibited only in the close complex. GlcNAc transferases III, IV, and V, which could only be assessed individually by indirect means using different substrates, did not appear to be affected in any major way by the protein matrix; the data suggest that transferase III is inhibited only to a minor extent in the close complex. Gal transferase activity showed a minor effect of the avidin matrix for both complexes in the hybrid processing pathways. The most significant consequence of the avidin effect on Gal transferase was the apparent abolishment of the incorporation of a 2nd Gal residue in the two avidin complexes. This survey of the protein matrix effects on glycan processing by oviduct enzymes appears to provide reasonable clues to the origin of the very different glycan structures observed in oviduct-processed glycoproteins. Thus, ovalbumin and avidin itself, containing a mixture of oligomannose and hybrid glycans at their single glycosylation sites, may well present they glycans to the processing enzymes in a display very similar to that of the avidin close complex observed here. The inhibition of mannosidase I and GlcNAc transferase I lead to preservation of oligomannose structures, whereas the strong inhibition of mannosidase II favors the incorporation of the bisecting GlcNAc by GlcNAc transferase III to yield hybrid structures as the most processed products. Ovomucoid, which contains multiantennary complex structures at all glycosylation sites, may on the other hand display its glycans, unencumbered by the protein surface, in conformations similar to either the free glycans or the distal complexes observed in this work.

Animals↗
PubMed1989

Amino acid sequence of cytochrome c from Aspergillus niger.

Cytochrome c from Aspergillus niger consists of two forms, a major one (80%) with 111 amino acid residues and a minor one (20%) with 108 residues, missing the three N-terminal residues of the major one. The primary sequence of A. niger cytochrome c was determined by standard spinning-cup Edman degradation of purified peptides and of pairs of peptides, from which the desired sequence was readily deduced by subtraction of common sequencies. Except for the extension and some variability at the N-terminal sequence, the A. niger protein conforms well with other cytochrome c structures.

Amino Acid Sequence↗
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↗