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H Paulsen

Publications and source records attributed to H Paulsen.

At least 73 records · Page 4Linked to original sources
PubMed1992

Accumulation of a light-harvesting chlorophyll a/b protein in the chloroplast grana lamellae. The lateral migration of the membrane protein precursor is independent of its processing.

The events that follow the import of pLHCPIIb, the apoprotein precursor of the major light-harvesting complex of photosystem II, were studied in intact pea chloroplasts. The distribution of the events of insertion into the membrane, and processing, to yield the mature form (LHCP) between stromal and granal lamellae regions of the thylakoids were followed. pLHCP was preferentially inserted into stromal lamellae (SL) from which it migrated to granal lamellae (GL). Migration occurred before or after processing, suggesting that migration and processing are independent of each other. When migration was slowed down, LHCP accumulated in SL. Prolonged inhibition of migration induced degradation of LHCP that had accumulated in SL, whereas inhibition of processing did not affect the migration of pLHCP into GL. A small difference in electrophoretic mobility was noted between LHCP in SL and in GL. The predominant mature form in SL migrated more slowly than LHCP from GL. When thylakoids were subjected to trypsin, all of the LHCP embedded in SL underwent cleavage, whereas up to 60% of the radioactive LHCP in GL was resistant to the enzyme. The possible implications of the differences in size and in the sensitivity to trypsin of LHCP are discussed.

Chloroplasts↗
PubMed1992

Involvement of a chloroplast HSP70 heat shock protein in the integration of a protein (light-harvesting complex protein precursor) into the thylakoid membrane.

Molecular chaperones, including those belonging to the 70-kDa family of heat shock proteins (HSP70), assist both the translocation of proteins across membranes and their assembly into oligomeric complexes. We purified a chloroplast HSP70 (ct-HSP70) and demonstrated that it plays a major role in the insertion of the precursor of the major light-harvesting complex of photosystem II (pLHCP; an integral membrane protein) into the thylakoids (the inner membranes of the chloroplast). Addition of the purified ct-HSP70 is necessary for efficient insertion of pLHCP into isolated thylakoid membranes. This activity of the purified ct-HSP70 is similar to that previously reported for the total stromal extract. When the chloroplast stromal extract is depleted of HSP70, a correlative reduction in the insertion activity of pLHCP is observed. The interaction between the ct-HSP70 and pLHCP involves physical association. The purified HSP70 acts directly on the membrane protein, presumably prevents its refolding, and thereby helps to maintain its competence for insertion into membranes.

Journal Article↗
PubMed1992

Synthesis of chlorophyll a regulates translation of chlorophyll a apoproteins P700, CP47, CP43 and D2 in barley etioplasts.

Accumulation of plastid-encoded chlorophyll apoproteins and chlorophyll synthesis are controlled by light in angiosperms. An in vitro system utilizing isolated and lysed barley (Hordeum vulgare L.) etioplasts revealed the specific accumulation of P700, CP47, CP43 and D2 triggered by de novo synthesis of chlorophyll. Accumulation rates of radiolabelled chlorophyll apoproteins were linear for about 30 min. Pulse/chase translation assays showed that synthesis of chlorophyll does not result in increased chlorophyll apoprotein stability. Instead turnover rates of chlorophyll apoproteins were higher in the presence than in the absence of chlorophyll. Chlorophyll-dependent accumulation of chlorophyll apoproteins must therefore be regulated on the level of translation. Translation of chlorophyll apoproteins was blocked to about 50% by addition of 30-50 microM aurintricarboxylic acid or 20 microM kasugamycin. The kinetics of chlorophyll-dependent translation indicated that the in vitro translation system is capable of translation initiation. The capability of translation initiation was lost in lysed etioplasts after preincubation for at least 5 min without chlorophyll synthesis. The results suggest that initiation is involved in chlorophyll-dependent regulation of translation.

Aminoglycosides↗
PubMed1992

Pigment-binding properties of mutant light-harvesting chlorophyll-a/b-binding protein.

Light-harvesting chlorophyll-a/b-binding protein (LHCP), overexpressed in Escherichia coli, can be reconstituted with pigments to yield complexes that are structurally very similar to light-harvesting complex II (LHCII) isolated from thylakoids [Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta 181, 204-211]. In order to analyze which domains of the protein are involved in pigment binding, we reconstituted deletion mutants of LHCP with pigments and characterized the resulting complexes regarding their pigment composition and spectroscopic properties. Series of progressive deletions from either end of the protein revealed that most of the N-terminal and part of the C-terminal hydrophilic regions of LHCP are dispensible for pigment binding. In either deletion series, the deletions that completely abolished reconstitution could be narrowed down to segments of five amino acids that do not contain histidine, asparagine, or glutamine. All mutants either formed complexes with both pigment composition and spectroscopic properties very similar to those of light-harvesting complex II isolated from thylakoids, or they did not form any stable complexes at all. There is no indication of a segment of LHCP binding a subset of LHCII pigments. We conclude that the stabilization of LHCP-pigment complexes is highly synergetic rather than based on individual pigment-binding sites provided by the protein.

Amino Acid Sequence↗
PubMed1992

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Man alpha 3R beta 2-N-acetylglucosaminyltransferase I using synthetic substrate analogues.

UDP-GlcNAc: Man alpha 3R beta 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M(r) 42,000). The Vmax for the pure enzyme with [Man alpha 6(Man alpha 3)Man alpha 6](Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc beta-Asn as substrate was 4.6 mumol min-1 mg-1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds an N-acetylglucosamine (GlcNAc) residue in beta 1-2 linkage to the Man alpha 3Man beta-terminus of the substrate. Several derivatives of Man alpha 6(Man alpha 3)Man beta-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the beta-linked mannose of Man alpha 6(Man alpha 3)Man beta-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the alpha 3-linked mannose (Man) of the substrate increases the KM 20-fold. Modifications on the alpha 6-linked mannose or on the core structure affect mainly the KM and to a lesser degree the Vmax, e.g., substitutions of the Man alpha 6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower the KM, whereas various other substitutions at the 3-position increase the KM slightly. Man alpha 6(Man alpha 3)4-O-methyl-Man beta 4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.

Animals↗
PubMed1992

Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase from rat liver.

The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase (beta 3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, beta 4-galactosyltransferase, beta-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the beta 3-Gal-T. beta 3-Gal-T activity is sensitive to changes in the R-group of the GalNAc alpha-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAc alpha-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished beta 3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAc alpha-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAc alpha-benzyl, which inhibited Gal incorporation into GalNAc alpha-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAc beta 6[Gal beta 3] GalNAc alpha-) is formed from core 1 (Gal beta 3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.

Animals↗
PubMed1991

[Synthesis of partial structure of complex types of N-glycoproteins].

Comparable syntheses of beta-D-GlcpNAc-(1----2)-[beta-D-GlcpNAc-(1----6)]-alpha-D-Manp-(1- ---6)-beta-D-ManpO(CH2)8CO2Me and beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----3)-beta-D-ManpO(C H2)8CO2Me with the glycosyl halide and imidate methods were investigated. 3,4,6-Tri-O-acetyl-2-deoxy-(2,2,2-trichloroethoxycarbonylamino)-al pha-D-glucopyranosyl bromide or trichloroacetimidate are suitable glycosyl donors for beta-D-glycoside coupling with secondary hydroxyl groups.

Carbohydrate Sequence↗
PubMed1991

[Conformational analysis of N-terminal O-glycopeptide sequences of interleukin-2].

The preferred conformations of eight O-glycopeptide sequences from the N-terminus of interleukin-2 containing two to ten amino acids, monoglycosylated at Thr3 with a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, were determined by means of n.m.r. spectroscopic methods. The preferred conformation of the N-terminal sequence, L-Ala-L-Pro-[alpha-D-GalpNAc-(1----3)]-L-Thr-L-Ser, including the O-glycosidically linked 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group is not substantially influenced by the linkage of additional amino acids at the C-terminal end. Extended conformations were observed for all peptide units. Measurements of the relaxation times of the 13C atoms showed that the 2-acetamido-2-deoxy-D-galactose bound to the central amino acids has the lowest mobility, whereas the terminal amino acid residues and peptide side-chains are flexible. Calculations with the force-field program AMBER yielded conformations of minimized energies that were in good agreement with the n.m.r. spectroscopic data. This was only true when n.m.r. parameters that can be used as starting values for the calculations were available. Comparison with a nonglycosylated, N-terminal tetrapeptide sequence analog did not suggest changes in the peptide conformation when Thr3 is glycosylated with a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group.

Amino Acid Sequence↗
PubMed1991

[Conformation analysis of D-xylose-containing O-glycopeptide sequences].

The preferred conformations of the sequences of O-glycopeptides containing a beta-D-xylosyl group linked to an L-seryl residue, found in the N-terminus of proteodermatan sulfate, show an almost extended peptide chain with the sugar unit in a specific position. The results of force-field calculations with the AMBER program have been confirmed, by n.m.r.-spectroscopic experiments, for a minimum conformation.

Amino Acid Sequence↗
PubMed1990

Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide substrates influences the activity of O-glycan core 1 UDPgalactose:N-acetyl-alpha-galactosaminyl-R beta 3-galactosyltransferase.

Synthetic O-glycopeptides containing one or two GalNAc residues attached to Ser or Thr were used as substrates to investigate the effect of peptide structure on the activity of crude preparations of UDP-Gal:GalNAc alpha-R beta 3-Gal-transferase from pig stomach and pig and rat colonic mucosa and of a partially purified enzyme preparation from rat liver. High-performance liquid chromatography used to separate enzyme products revealed that uncharged glycopeptides with an acetyl group at the amino-terminal end and a tertiary butyl or an amide group at the carboxy-terminal end were resistant to proteolysis in crude preparations. The activity of beta 3-Gal-transferase varied with the sequence and length of the peptide portion of the substrate, the presence of protecting groups, the attachment site of GalNAc, and the number of GalNAc residues in the substrate. The presence and position of Pro had little effect on enzyme activity; ionizing groups near the GalNAc unit interfered with enzyme activity. Since the GalNAc-Thr moieties in many of these O-glycopeptides have been shown to assume similar rigid conformations, the variation in enzyme activity indicates that the beta 3-Gal-transferase recognizes both the peptide and carbohydrate moieties of the substrate. Rat and pig colonic mucosal homogenates contain beta 3- and beta 6-GlcNAc-transferases that synthesize respectively O-glycan core 3 (GlcNAc beta 3GalNAc alpha-R) and core 4 [GlcNAc beta 6(GlcNAc beta 3)GalNAc alpha-R]. These enzymes also showed variations in activity with different peptide structures; these effects did not parallel those observed with beta 3-Gal-transferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

[Synthesis of a Kdo-containing pentasaccharide sequence of the inner core- and lipid A-region of lipopolysaccharides].

Coupling of methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosyl) onate-(2----4)-methyl (5,7,8-tri-O-acetyl-3-deoxy-alpha-D-manno-octulopyranosyl bromide)onate with benzyl O-(methyl [3-deoxy-7,8,O-(tetraisopropyldisiloxan 1,3-diyl)-alpha-D-manno-octulopyranosylonate)-2-----6)-O-([( R)-3-benzyloxytetradecanoylamino]-2-deoxy-3,4-O-(tetraisopropyl disiloxan-1,3-diyl)-beta-D-glucopyranosyl)-(1----6)-3-O-benz yl- 2-[(R)-3-benzyloxytetradecanoylamino]-2-deoxy-alpha-D-glucopyranos ide gave stereo- and regio-selectively a pentasaccharide that was deprotected into alpha-Kdo-(2----4)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-D-GlcNh m-(1----6)-D-GlcNhm [Nhm = 2-deoxy-(R)-3-hydroxytetradecanoylamino].

Carbohydrate Conformation↗
PubMed1990

[Synthesis of a highly branched nonasaccharide sequence of the "bisected"structure of N-glycoproteins].

A highly branched nonasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----3) -[beta-D- Glcp-NAc-(1----4)-[beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----2)-alpha-D- Manp- (1----6)]-beta-D-Manp-(1----4)-D-GlcNAc, that represents the carbohydrate chain of the "bisected" structure of lactosamine-type N-glycoproteins was synthesized by a block synthesis approach. The activated trisaccharide block beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----2)-D-Man was coupled first with the low reactive OH-3 group and, in a second step, with the reactive OH-6 group of the central beta-D-mannopyranosyl residue within the suitably protected trisaccharide block beta-D-GlcpNAc-(1----4)-beta-D-Manp-(1----4)-D-GlcNAc. During these coupling steps, the alpha-glycosidic linkages of D-mannopyranosyl residues were formed without neighboring-group participation, which is only successful by use of the imidate procedure.

Carbohydrate Conformation↗
PubMed1990

Characterization of murine monoclonal and murine, rabbit, and human polyclonal antibodies against chlamydial lipopolysaccharide.

Murine monoclonal and rabbit, murine, and human polyclonal antibodies against chlamydial lipopolysaccharide (LPS) were characterized by the passive hemolysis and passive hemolysis inhibition assays and by absorption experiments with LPSs of Chlamydia psittaci, Chlamydia trachomatis, and a recombinant strain of Salmonella minnesota Re (r595-207) expressing the chlamydia-specific LPS epitope, as well as natural and synthetic partial structures of chlamydial LPS. Eleven monoclonal antibodies of the immunoglobulin M and G classes were characterized as chlamydia-specific by their failure to react with Re-type LPS, binding to a similar epitope for which the trisaccharide alpha-3-deoxy-D-manno-2-octulosonic acid (KDO)-(2-8)-alpha-KDO-(2-4)-alpha-KDO was an absolute prerequisite. For optimal binding, parts of the lipid A moiety were also involved; however, phosphoryl and ester-linked acyl groups and the reducing glucosamine residue of lipid A were dispensable. A similar antibody specificity was detected in lapine and murine hyperimmune sera after immunization with chlamydia, in addition to those recognizing more complex (e.g., those requiring the presence of phosphoryl residues) and less complex epitopes. Among the latter were those cross-reacting with Re-type LPS, which could be removed by absorption. The titers of different antibody specificities, in particular the ratio of chlamydia-specific to cross-reactive antibodies, present in murine polyclonal antisera depended on the immunization protocol. The preferential formation of chlamydia-specific antibodies was observed after immunization with liposome-incorporated immunogens. Human sera from patients with suspected genital chlamydial infections were also found to contain chlamydia-specific and cross-reactive antibodies, the latter of which could be removed by absorption with Re-type LPS.

Antibodies, Bacterial↗
PubMed1989

Determination of the epitope specificity of monoclonal antibodies against the inner core region of bacterial lipopolysaccharides by use of 3-deoxy-D-manno-octulosonate-containing synthetic antigens.

Partial structures of enterobacterial lipopolysaccharides (LPS) of the Rechemotype, consisting of lipid A and 3-deoxy-D-manno-2-octulosonic acid (Kdo), as well as oligosaccharides and derivative of Kdo were synthesized and used to characterize the epitope specificity of monoclonal antibodies against Re-mutant LPS. High-molecular-weight antigens, obtained after copolymerization of the respective allyl glycosides with acrylamide, and the haptenic oligosaccharides were used in immunoprecipitation, immune hemolysis, and in inhibition assays. A monoclonal antibody (clone 20, igM) recognizing a terminal Kdop group was shown to require for its binding the alpha-anomeric configuration and OH-4 and OH-5 groups, whereas the C-7 - C-8 chain was of minor importance. Another monoclonal antibody (clone 25, IgG3), which recognizes a (2--4)-linked Kdo disaccharide, was shown to require for its binding the alpha-anomeric configuration of both residues. The isomer having a reducing beta-Kdo residue was significantly less active, and that with a terminal beta-Kdo group was completely inactive. The OH-5 group of the reducing residue was shown to be not important for the specificity of this antibody, since it could be replaced by a hydrogen atom without loss of serological reactivity. The alpha-(2--8)-linked Kdo disaccharide was strongly cross-reactive with its (2--4)-linked isomer. The antibody recognized also parts of the 2-amino-2-deoxy-D-glucose backbone of lipid A.

Animals↗
PubMed1989

[Synthesis of a tetrasaccharide unit of the capsular polysaccharide of Streptococcus pneumoniae type 9V].

In the synthesis of 8-methoxycarbonyloctyl O-(alpha-D-galactopyranosyl)-(1----3)-O-(2-acetamido-2-deoxy-beta-D- mannopyranosyl)-(1----4)-O-(beta-D-glucopyranosyl)-(1----4)-alpha-D- glucopyranoside, which represents a component of the capsular polysaccharide of Streptococcus pneumoniae type 9V, the key step was the coupling of alpha-D-Galp-(1----3)-beta-D-ManpNAc-(1----4)-D-Glc as glycosyl donor with 8-ethoxy-carbonyloctyl 6-O-acetyl-2,3-di-O-benzyl-alpha-D-glucopyranoside as glycosyl acceptor by use of the imidate method. Only the beta-imidate of the trisaccharide could be employed in this glycosidation reaction to give stereoselectively the tetrasaccharide in high yield. The alpha-imidate of the trisaccharide led to hydrolysis of the imidate group.

Carbohydrate Conformation↗
PubMed1988

[Synthesis of trisaccharide units from capsular polysaccharides of Streptococcus pneumoniae].

In the presence of silver silicate as promoter, the reaction of glycosyl bromide of 2-azido-2-deoxy-D-mannose with 1,6-anhydro-2,3-di-O-benzyl-beta-D- glucopyranose led to derivatives of the disaccharide beta-D-ManpNac-(1----4)-D- Glcp. The derivatives were activated into disaccharide halides and employed as glycosyl donors in block synthesis. By chain extension with L-rhamnose and D-glucose, four trisaccharides were synthesized. They represent components of capsular polysaccharide "repeating units" of various Streptococcus pneumoniae types, beta-D-Man-pNAc-(1----4)-alpha-D-Glcp-(1----2)-L-Rhap (type 19 F), beta-D-ManpNAc-(1----4)-alpha-D-Glcp-(1----3)-L-Rhap (type 19 A), beta-D-ManpNAc-(1----4)-alpha-D-Glcp-(1----4)-D-Glcp (type 9 A), and beta-D-ManpNAc-(1----4)-beta-D-Glcp-(1----4)-D-Glcp (type 9 V).

Carbohydrate Conformation↗
PubMed1988

Control of glycoprotein synthesis. Purification and characterization of rabbit liver UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I.

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I catalyzes an essential first step in the conversion of high mannose to hybrid and complex N-glycans (Schachter, H. (1986) Biochem. Cell Biol. 64, 163-181; Oppenheimer, C.L., and Hill, R.L. (1981) J. Biol. Chem. 256, 799-804), i.e. the addition of GlcNAc to (Man alpha 1-6(Man alpha 1-3)Man alpha 1-6)(Man alpha 1-3)Man beta 1-4GlcNAc-OR to form (Man alpha 1-6(Man alpha 1-3)Man alpha 1-6)(GlcNAc beta 1-2Man alpha 1- 3)Man beta 1-4GlcNAc-OR. The enzyme has been purified from Triton X-100 extracts of rabbit liver by chromatography on CM-Sephadex, Affi-Gel blue, UDP-hexanolamine-Sepharose, and a novel adsorbent in which UDP-GlcNAc is linked to thiopropyl-Sepharose at the 5-position of uracil. The enzyme exists in crude liver extracts in two molecular weight forms separable on Sephadex G-200. The low molecular weight form was purified 64,000-fold with a specific activity of 19.8 mumol/min/mg. The pure enzyme was free of N-acetylglucosaminyltransferase II-V activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single major band of Mr 45,000 and two minor bands of Mr 54,000 and 50,000. All three bands showed retarded elution from an affinity column in which the acceptor substrate for the transferase was covalently linked to Sepharose. Kinetic analysis indicated a largely ordered sequential mechanism with UDP-GlcNAc binding to the enzyme first and UDP leaving last. Studies with synthetic analogues of the substrate Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc showed that an unsubstituted equatorial hydroxyl on carbon 4 of the beta-linked Man residue was essential for enzyme activity.

Animals↗