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H van der Wel

Publications and source records attributed to H van der Wel.

At least 19 recordsLinked to original sources
PubMed1999

Identification of a UDP-GlcNAc:Skp1-hydroxyproline GlcNAc-transferase in the cytoplasm of Dictyostelium.

Skp1 is a cytoplasmic and nuclear protein required for the ubiquitination of cell cycle regulatory proteins and transcriptional factors. In Dictyostelium, Skp1 is modified by a linear pentasaccharide, Galalpha1-6Galalpha1-Fucalpha1-2Galbeta1-3Glc NAc, attached to a hydroxyproline (HyPro) residue at position 143. To study the formation of the GlcNAc-HyPro linkage, an assay was developed for the transfer of [(3)H]GlcNAc from UDP-[(3)H]GlcNAc to Skp1-HyPro-143 or a synthetic Skp1 4-HyPro peptide. The cytosolic but not the particulate fraction of the cell mediated transfer in a time-, concentration-, and HyPro-dependent fashion. Incorporated radioactivity was alkali-resistant and was recovered as GlcNH(2) after acid hydrolysis, consistent with linkage of GlcNAc to HyPro. The GlcNAc-transferase activity was purified 130,000-fold as a single component with a recovery of 5%. Key to the purification was the synthesis of a novel affinity resin linking UDP-GlcNAc at its 5-uridyl position. The purified activity had an apparent M(r) of approximately 45,000 by gel filtration, required dithiothreitol and a divalent cation, and consisted predominantly of a M(r) 51,000 band after SDS-polyacrylamide gel electrophoresis that was photoaffinity labeled with 5-(125)I-[3-(p-azidosalicylamido)-1-propenyl-UDP-GlcNAc in a UDP-GlcNAc-sensitive fashion. Its apparent K(m) values for UDP-GlcNAc and Skp1 were submicromolar. The presence of the enzyme in the cytosolic fraction, its dependence on a reducing environment, and its high affinity for UDP-GlcNAc strongly suggest that Skp1 is glycosylated by a HyPro GlcNAc-transferase that resides in the cytoplasm.

Animals↗
PubMed1996

Purification and characterization of an alpha1,2,-L-fucosyltransferase, which modifies the cytosolic protein FP21,from the cytosol of Dictyostelium.

A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.

Amino Acid Sequence↗
PubMed1996

SP75 is encoded by the DP87 gene and belongs to a family of modular Dictyostelium discoideum outer layer spore coat proteins.

Highly purified spore coats of Dictyostelium discoideum each contained about 5 x 10(6) protein molecules as determined by amino acid composition analysis. By two-dimensional gel electrophoresis the coats were found to contain nine major-abundance and numerous minor protein species, most of which were highly enriched relative to the adjacent interspore matrix. Protein was nearly quantitatively eluted by denaturants and 2-mercaptoethanol, showing that it was not irreversibly cross-linked. Because a reducing agent is required together with denaturants to elute most proteins if their free thiol groups have been prealkylated, it was concluded that the D. discoideum spore coat proteins are disulfide cross-linked into the matrix. One major coat protein, SP75, was partially sequenced and found to be encoded by the previously identified DP87 gene; this finding was supported by additional physical, genetic, biochemical and microscopic evidence. The five major proteins for which genes have been cloned were associated with the outer layer of the coat. In coats missing one or more of four of these proteins as a result of gene disruption, there were physical changes but, with one exception, the other major coat proteins appeared to be incorporated normally. Sequence analysis showed that these five outer layer coat proteins are homologous and consist of alternating sequence motifs related to epithelial mucin repeats, basic proline repeats found in salivary acidic proline-rich proteins, the NH2-terminal subdomain of epidermal growth factor modules and other cysteine repeats. Based on these and other observations, outer layer coat proteins are predicted to organize indeterminately to form a cell surface microenvironment supportive of cellulose morphogenesis during spore coat formation.

Amino Acid Sequence↗
PubMed1995

Characterization of FP21, a cytosolic glycoprotein from Dictyostelium.

FP21 is a glycoprotein which, when tracked by radioactivity in its fucosyl moiety, was previously detected in the cytosol of Dictyostelium cells after cell fractionation. This compartmentalization is confirmed by SDS-polyacrylamide gel electrophoresis/Western blotting of cell fractions using three different antibodies. Although a substantial fraction of FP21 is also detected in the particulate fraction using these new antibodies, particulate FP21 is released by disrupting protein-protein interactions, but not membrane disruption. Since purified FP21 is susceptible to aggregation, and purified nuclei do not contain FP21, particulate FP21 is also part of the cytosol. Additional compositional and structural information provides strong evidence that FP21 does not at any time traverse the rough endoplasmic reticulum. First, cDNAs spanning the entire coding region of the FP21 gene predict no hydrophobic motifs expected to promote membrane insertion, but do predict an NH2-terminal coiled coil domain which could explain aggregation. Second, monosaccharide composition analysis of the predominant glycoform of FP21 yields 2 mol of galactose, 1 mol of xylose, and 1 mol of fucose/mol of polypeptide; FP21 from a fucosylation-defective mutant contains 1 additional mol of xylose in place of fucose. Thus the N-glycosylation sequon present in FP21 is not utilized by oligosaccharyl transferase, which resides in the rough endoplasmic reticulum. These findings indicate that nascent FP21 remains in the cytosol after synthesis and is therefore glycosylated by unusual cytosolic xylosyl-, galactosyl-, and fucosyltransferases.

Amino Acid Sequence↗
PubMed1992

Visinin: biochemical and molecular comparisons in normal and rd chick retina.

Western, northern and DNA sequence analyses were used to determine if the retinal protein, visinin, is defective in the chicken retinal degeneration mutant, rd. A 22kDa band, corresponding to purified visinin, was stained with equal intensity on Western blots of +/+, +/rd and rd/rd retinal protein probed with a visinin polyclonal antibody. Hybridization of a northern blot of +/+, +/rd and rd/rd poly(A)+ RNA with a random-primer labelled visinin cDNA probe showed a single, equally labelled 1-Kbp band in each of the samples. Finally, no differences were found between the nucleic acid sequences of the 579 bp cDNAs encoding +/+ and rd/rd visinin. We did, however, find one significant difference between our visinin DNA sequence and the previously published chick visinin DNA sequence. We consistently observed a C at position 118 rather than the published T which changes the amino acid residue at position 40 from serine to proline. Based on the results of this study, we conclude that visinin is not defective in the rd chick model of hereditary retinal degeneration.

Amino Acid Sequence↗
PubMed1989

Inhibition of acetylcholinesterase in guppies (Poecilia reticulata) by chlorpyrifos at sublethal concentrations: methodological aspects.

Acetylcholinesterase activity is a potential biochemical indicator of toxic stress in fish and a sensitive parameter for testing water for the presence of organophosphates. A number of methodological aspects regarding the determination of the in vivo effect of chlorpyrifos on acetylcholinesterase in guppies have been investigated. It was found that with acetylthiocholine as a substrate, the contribution of pseudocholinesterase to the total cholinesterase activity can be neglected. Protection of acetylcholinesterase of guppies exposed to chlorpyrifos from additional, artifactual in vitro enzyme inhibition during homogenization is necessary. Very low concentrations of acetone in the exposure medium, resulting from dilution of the stock solution of chlorpyrifos in acetone, can result in large decreases in the oxygen content of this medium. This may affect the uptake rate of the toxic compound and, thereby, cholinesterase inhibition. Very low, sublethal concentrations of chlorpyrifos result in high inhibition levels of acetylcholinesterase (80-90%) in guppies within 2 weeks of continuous exposure. Recovery of the enzyme activity occurs after the exposed animals are kept in clean medium for 4 days, but the rate of recovery is considerably lower than the rate of inhibition.

Acetylcholinesterase↗
PubMed1987

Labeling of sweet taste binding sites using a colloidal gold-labeled sweet protein, thaumatin.

Thaumatin, an intensely sweet tasting protein, was bound to colloidal gold and applied to the taste bud-bearing foliate papillae of Rhesus monkeys. Examination of thin sections of taste pores showed that gold particles were bound to merocrine secretions of Type I taste bud cells, to some cell remnants of lysed cells, and, most importantly, to small, membrane bounded blebs of cytoplasm. These blebs are thought to be shed into the pore from the tips of taste bud cell microvilli, particularly those arising from Type II cells. The binding of gold particles to microvillus tips and to the blebs suggest that this may be an important means by which taste bud cells rid themselves of taste stimulus-receptor complexes.

Animals↗
PubMed1985

Three-dimensional structure of thaumatin I, an intensely sweet protein.

Thaumatin and monellin are the two sweetest compounds known to man--about 100,000 times sweeter than sugar on a molar basis and 3000 times on a weight basis. These proteins represent a unique class of proteins that are taste-active. We report the three-dimensional structure of thaumatin I at 3.1 A resolution.

Crystallography↗
PubMed1985

Effects of gymnemic acid on the chorda tympani proper nerve responses to sweet, sour, salty and bitter taste stimuli in the chimpanzee.

In man gymnemic acid is able to abolish the sweet taste. Also in man, the neural correlate of that effect is a disappearance of the response to sweet stimuli in the taste nerves, as indicated by the observations of Diamant et al. (1965). Although a variety of other mammals also show neural responses to sweet-tasting compounds, the corresponding effect of gymnemic acid has not been demonstrated. This study presents chorda tympani proper nerve recordings from the chimpanzee before and after gymnemic acid. On the chimpanzee tongue, application of 2 ml gymnemic acid (3-10 mg X ml-1 for 3-4 min) completely abolished the taste responses to 0.0035 M acesulfam-K, 0.0018 M aspartame, 0.015 M D-tryptophan, 0.02% monellin, and 0.02% thaumatin, reduced by 75% the response to 0.3 M sucrose, and by 50% that of 0.76 M xylitol. No decrease was recorded in the responses to 0.001 M quinine, 0.1 M NaCl, 0.02 and 0.04 M ascorbic acid, 0.02 and 0.04 M citric acid. The response to the sweeteners recovered with time and the recovery was complete or nearly complete after one and a half hours. It was also found that after application of 2 ml miraculin, 3 mg X ml-1 for 3 min to the tongue the neural response to acids was about 1.5 times as large as before. Gymnemic acid applied before miraculin prevented this enhancement and gymnemic acid after miraculin depressed the enhancement by miraculin of the response to citric and ascorbic acid.

Animals↗
PubMed1984

Assignment of the disulphide bonds in the sweet-tasting protein thaumatin I.

The disulphide linkages of the 16 half-cystine residues in the sweet-tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high-performance liquid chromatography and peptide mapping by paper high-voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9-204, 56-66, 71-77, 121-193, 126-177, 134-149, 145-158 and 159-164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys-145 and Cys-158.

Amino Acids↗
PubMed1979

The complete amino-acid sequence of the sweet protein thaumatin I.

The primary structure of the sweet-tasting protein thaumatin has been elucidated. The protein consists of a single polypeptide chain of 207 residues. The sequence of the N-terminal part of the chain was determined by sequenator analysis. As the protein contains only one methionine residue, it was possible to deduce the N-terminal sequence of the C-terminal cyanogen bromide fragment by automatic sequencing of the cyanogen-bromide-cleaved, succinylated protein. To arrive at the sequence of the whole protein tryptic and Staphylococcus protease peptides, together with chymotryptic peptides and a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole) fragment were also sequenced. Comparing the amino acid sequence of thaumatin with that of the other sweet-tasting protein, monellin, we have located five sets of identical tripeptides. Since immunological cross-reactivity of thaumatin antibodies with monellin has recently been described, one or more of these tripeptides might be part of a common antibody recombination site and possibly be involved in the interaction with the sweet-taste receptor.

Amino Acid Sequence↗
PubMed1978

The taste responses in primates to the proteins thaumatin and monellin and their phylogenetic implications.

Electrophysiological and behavioural methods have been applied to 34 species of the primates and, for comparison, to the Madagascan hedgehog to determine their responses to the proteins thaumatin and monellin. These substances elicit an intensely sweet taste sensation in man. All Catarrhina prefer monellin to water. The responses of the Prosimii as well as those of the South American primates to monellin are different, some species show a reaction, other species are not sensitive. In the case of thaumatin neither the Prosimii--including Tupaia and Tarsius--nor the South American primates show any response to this protein. Only the Cercopithecidae, the Hylobatidae and the Pongidae respond to this protein like man and prefer this substance to water. This physiological aspect of taste constitutes a clear dichotomy within the order Primates. This capability to taste thaumatin probably developed as long as 38 million years ago.

Animals↗
PubMed1976

Gustatory effects of miraculin, monellin and thaumatin in the Saguinus midas tamarin monkey studied with electrophysiological and behavioural techniques.

A comparative electrophysiological and behavioural study has been made in 17 closely related monkeys of the new world species, Saguinus midas tamarin. The electrical activity in the chorda tympani proper nerve of two of the monkeys was recorded during the application to the tongues of 0.02% monellin and thaumatin, 0.5% miraculin and stimuli representing the four taste qualities. It was observed that monellin and thaumatin gave no or little response and that miraculin enhanced the response to the sour stimulus, but not that to any other taste quality. Behavioural studies were then made with a two-bottle preference test in 15 monkeys. It was found that the animals did not discriminate or discriminated poorly between water and thaumatin or monellin. After miraculin they changed their strong rejection of 0.02 M citric acid, in a choice between water and acid, into a strong preference for the acid. These results show a close relation between the electrophysiological and the behavioural data.

Action Potentials↗