Sialylation of n-alkyllactosides, galactosides and glucosides by sialyltransferases from rat liver Golgi vesicles

nAlkyl - and -lactosides, galactosides and glucosides with differentalkyl chain lengths (C₂, C₈, C₁₄, and C₂₀) were synthesizedand used as acceptors for sialyltransferases from rat liverGolgi vesicles. The -galactosides, -glucosides, and both - and-lactosides, were sialylated. Keeping the acceptor concentrationconstant, sialylation rates reached a maximum for the n-octyl- and -lactosides, n-Octyl -galactoside and noctyl -glucoside,respectively. noctyl -glucoside, respectivwly. n-Octyl -galactosideand n-octyl -glucoside were not sialylated. The reaction productswere characterized by TLC. With n-octyl lactoside and galactosideas acceptors, two major sialylation products were formed. Thjeycould be separated by preparative TLC, and their structureswere identified as 2–3 and 2–6 sialylated acceptors,respectively, by a combination of periodated oxidation, NaBD₄reduction,permethylation and subsequent analysis by fast atombombardment mass spectrometry (FAB-MS). The structure of thesingle product obtained from n-ictyl -glucoside was determinedto be the 2–6 sialylated glucoside. Competition experimentswith n-octyl lactoside and lactosylceramide and gangliosideGal1-3GalNAc1-4(NeuAc2–3)Gal1–4Glcbeeta1–1Cer(GM1) as acceptors for sialyltransferases suggested that SAT-I[NeuAc2–3Gal1–4Glc1-1Cer (GM3) synthase] was atleast in least in part responsible for the 2–3 sialylationof n-octyl lactoside. alkylgalactosides alkylglucosides alkyllactosides neoglycolipids sialytransferases     

Molecular dynamics simulations of oligosaccharides and their conformation in the crystal structure of lectin-carbohydrate complex: importance of the torsion angle {psi} for the orientation of {alpha}1,6-arm

The conformation of the heptasaccharide Man-1,6-(Man-1,3)(Xyl-ß1,2)-Man-ß,4-GlcNAc₂-ß1,4-(L-Fuc-1,3)-GlcNAc₁,the carbohydrate moiety of Erythrina corallodendron lectin (EcorL),the hexasaccharide Man-1,6-(Man-1,3) (GlcNAc-ß1,4)-Man-ß1,4-GlcNAc-ß1,4-GlcNAcand their disaccharide fragments have been studied by moleculardynamics (MD) simulations for 1000 ps with different initialconformations. In the isolated heptasaccharide, the most frequentlyaccessed conformation during MD has a value of 180° aroundMan-1,6-Man linkage. This conformation is stabilized by theformation of a hydrogen bond between the carbonyl oxygen ofGlcNAc₂ with the O3/O4 hydroxyls of the 1,6-linked mannose residue.The conformation of the heptasaccharide found in the crystalstructure of the EcorL-lactose complex (Shaanan et al., Science,254, 862, 1991), that has a value of 76° around Man-1,6-Manlinkage, is accessed, although less frequently, during MD ofthe isolated oligosaccharide. The ,, = 58°,–134°,–60°conformation around Man-₁,6-Man fragment observed in the crystalstructure of the Lathyrus ochnrs lectin complexed with a biantennaryoctasaccharide (Table I in Homans,S.W., Glycobiology, 3, 551,1993) has also been accessed in the present MD simulations.These values for the 1,6-linkage, which are observed in theprotein-carbohydrate crystal structures and are accessed inthe MD simulations, though occasionally, have not been predictedfrom NMR studies. Furthermore, these different values of leadto significantly different orientations of the 1,6-arm for thesame value of . This contrasts with the earlier predictionsthat only different values of can bring about significant changesin the orientation of the ₁,6-arm. The MD simulations also showthat the effects of bisecting GlcNAc or ß1,2-xyloseare very similar on the ₁,3-arm and slightly different on the₁,6-arm. bisecting GlcNAc carbohydrates glycoprotein lectinsaccharide complex     

Hydrophobic mannosides act as acceptors for trypanosome {alpha}-mannosyltransferases

A series of hydrophobic mannosides were synthesized and testedfor their ability to act as acceptor substrates for mannosyltransferasesin a Trypanosoma brucei cell-free system. The thiooctyl -mannosidesand octyl -mannosides all accepted single mannose residues in-linkage, as judged by thin layer chromatography of the productsbefore and after jack bean -mannosidase digestion. The mannosylationreactions were inhibited by amphomycin, suggesting that theimmediate donor was dolicholphosphate-mannose (Dol-P-Man) inall cases. The transferred -mannose residues were shown to beboth 1-2 and 1-6 linked by Aspergillus phoenicis -mannosidaseand acetolysis treatments, respectively. These data suggestthat the compounds can act as acceptor substrates for the Dol-P-Mandependent 1-2 and 1-6 mannosyltransferases of the GPI biosyntheticpathway and/or the dolichol-cycle of protein N-glycosylation.One of the compounds, Man1-6Man1-O-(CH₂)₇CH₃, inhibited endogenousGPI biosynthesis in the cell-free system, suggesting that itcould be a substrate for the trypanosome Dol-P-Man:Man₂GlcN-Pl1-2 mannosyltransferase. dolichol glycosylphosphatidylinositol mannosyltransferase trypanosome     

Further characterization of the binding properties of a GalNAc specific lectin from Codium fragile subspecies tomentosoides

Previous study on the binding properties of a lectin isolatedfrom Codium fragile subspecies tomentosoides (CFT) indicatesthat this lectin recognizes the GalNAc1 sequence at both reducingand nonreducing ends. In this study, the carbohydrate specificityof CFT was further characterized by quantitative precipitin(QPA) and inhibition of lectin-enzyme binding assays. Of theglycoforms tested for QPA, all asialo-GalNAc1 containing glyco-proteinsreacted well with the lectin. Asialo hamster and ovine submandibularglycoproteins, which contain almost exclusively Tn (GalNAclSer/Thr)residues as carbohydrate side chains, and Streptococcus typeC polysaccharide completely precipitated the lectin added, whilethe GalNAcβcontaining Tamm-Horsfall Sd(a⁺) glycopro-teinand its asialo product were inactive. Among the oligo-saccharidestested for inhibiting lectin-glycoprotein interaction, GalNAc13GalNAcβ13Gal14Galβ14GIc(Fp)and Galβ13GalNAc1benzyl (T) were the best, and about 125-foldmore active than GalNAc They were about 3.3, 6.6, and 43 timesmore active than Tn containing glycopeptides, GalNAc13(LFuc12)Gal(A_h) and Galβ13GalNAc(T), respectively. From the presentand previous results, it is concluded that the combining siteof CFT is probably of a groove type that recognizes from GalNAclto pentasaccharide(Fp). The carbohydrate specificity of thislectin can be constructed and summarized in decreasing orderby lectin determinants as follows: Fp and T > Tn cluster> A_h >>I/II. carbohydrate specificities Codium fragile tomentosoides glycoprotein binding lectins     

Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

Transmembrane-Domain Trapping: A Novel Method for Isolation of cDNAs Encoding Putative Membrane Proteins

We have developed a method that enables us to isolate cDNAsof putative membrane proteins. The system is designed to isolatea cDNA which can provide the transmembrane domain to the extracellularpart of the IL-2 receptor chain. We constructed a p18Mac vectorby putting part of the IL-2 receptor chain cDNA that encodedits signal sequence and extracellular domain, a cDNA cloningsite and a poly(A) additional signal after a strong promoterSR. If a cloned cDNA provides a transmembrane domain in-frame,the extracellular domain of the IL-2 receptor chain will beexpressed on the surface of the transfected cells. Otherwise,the chimeric protein will be either secreted or retained insidethe transfected cells. We made a cDNA library using p18Mac andscreened for cDNA clones which allowed the expression of theextracellular domain of the IL-2 receptor chain on the cellsurface. Of the 2000 clones screened, 5 clones were scored aspositive. Partial sequence analysis revealed that one cloneencoded the amyloid precursor protein, two others encoded mitochondrialproteins and the rest were new. These results suggest the systemis effective in isolating cDNAs encoding putative membrane proteins.     

Pumpkin (Cucurbita sp.) seed globulin III. Comparison of subunit structures among seed globulins of various Cucurbita species and characterization of peptide components

Various Cucurbita seed globulins showed patterns similar toone another on SDS-gel electrophoresis, and ß bandsfor unreduced globulins and , ', and ' bands for reduced ones.On gel electrophoresis in 6 M urea, reduced globulin gave twoacidic and two basic bands. These corresponded to and ' chainsand ₁ and ₂ chains, respectively, identified by two-dimensionalurea-SDS gel electrophoresis. The compositions of the and ßsubunits were proposed. (Received September 8, 1977; )     

An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

A Transient Stimulation of Net Photosynthesis of Rye Leaves by {alpha}-Hydroxy-2-pyridinemethanesulphonic Acid ({alpha}-HPMS) due to Inhibition of Photorespiratory CO2 Release

The effects of -hydroxy-2-pyridinemethanesulphonic acid (-HPMS)upon net photosynthesis (P_n, the CO₂ compensation point (),post-lower illumination burst of CO₂ (PLIB) and post-lower temperatureburst of CO₂ (PLTB) in detached rye (Secale cereale L.) leaveswere investigated. At low concentrations ( 0.5 mol m^–3),-HPMS initially stimulated P_n and decreased the magnitude ofboth PLIB and PLTB. The decreased at all concentrations of-HPMS (0.05–5.0 mol m^–3. The effects of -HPMS onP_n and were time-dependent and, after a few minutes, the P_nwas inhibited while values increased considerably. At a higherconcentration (5.0 mol m ^–3), the transient effects of-HPMS were shorter () or not observed at all (P_n. Both PLIBand PLTB, when expressed in relation to P_n, increased at higherlevels of this compound. Similar data with respect to the effectsof -HPMS on PLIB and PLTB were found for leaves of dandelion(Taraxacum officinale L.). The results suggest that -HPMS may stimulate P_n by inhibitingphotorespiration, as originally suggested by Zelitch (1966),but only at low concentrations and over a short time span. Thedecrease of PLIB and PLTB values at low -HPMS levels is consistentwith these processes being a residual activity of the glycolatepathway. Key words: CO₂ compensation point, -hydroxy-2-pyridinemethanesulphonic acid, photorespiration, photosynthesis     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

Enzymatic Degradation of Chlorophyll in Chenopodium album

The breakdown of chlorophyll (Chi) in crude extracts of Chenopodiumalbum (white goose foot) in the dark was examined. Derivativesof pheophorbide were formed when Chi or chlorophyllide wasincubated with depigmented crude extracts. The formation ofpheophorbide was completely prevented by heat treatment of extracts,indicating that the reaction was enzymatic, and the presenceof a Mg-releasing enzyme, the so called Mg-dechelatase, waspostulated. This hypothesis was strongly supported by the observationthat the formation of pheophorbide was inhibited by 51% by 10mM MgCl₂. Analysis by high-performance thin-layer chromatography(HPTLC) and liquid chromatography (HPLC) showed that the appearanceof chlorophyllide , pheophorbide 13²-hydroxychlorophyllide and pyropheophorbide was accompanied by a concomitant decreasein levels of Chi The formation of 13²-hydroxychloro-phyllide was not clearly an enzymatic reaction and requires furtherexamination. It appears that Chl is degraded in a crude extractof C. album via the following enzymatically catalyzed reactions (Received September 10, 1990; Accepted November 15, 1990)     

Integrated likelihood functions for non-Bayesian inference

Consider a model with parameter = (, ), where is the parameterof interest, and let L(, ) denote the likelihood function. Oneapproach to likelihood inference for is to use an integratedlikelihood function, in which is eliminated from L(, ) by integratingwith respect to a density function (|). The goal of this paperis to consider the problem of selecting (|) so that the resultingintegrated likelihood function is useful for non-Bayesian likelihoodinference. The desirable properties of an integrated likelihoodfunction are analyzed and these suggest that (|) should be chosenby finding a nuisance parameter that is unrelated to and thentaking the prior density for to be independent of . Such anunrelated parameter is constructed and the resulting integratedlikelihood is shown to be closely related to the modified profilelikelihood.     

An Error in the Calibration of Xylem-water Potential against Leaf-water Potential

An error occurs in the calibration of xylem pressure potential() against leaf-water potential () when the calibration is madeusing plant material in which the water stress has been inducedartificially after excision. The impostion of water stress afterexcision affects the determination more than it affects , consequentlythe relationship between these two indices of water stress isaltered. Care should be exercised to ensure that identical proceduresare adopted during . calibrations and during susbsequent fieldmeasurements of with the pressure-chamber apparatus.     

Application of N-terminally Truncated DNA Polymerase from Thermus thermophilus ({Delta}Tth Polymerase) to DNA Sequencing and Polymerase Chain Reactions: Comparative Study of {Delta}Tth and Wild-Type Tth Polymerases

N-Terminally truncated DNA polymerase from Thermus thermophilus(Tth polymerase) lacking 5'-3' exonuclease activity was usedfor DNA sequencing and polymerase chain reaction (PCR). In contrastto the high background of the sequencing ladder observed withthe wild-type Tth polymerase, Tth polymerase gave readable sequencingpatterns which extend up to more than 500 bases from the primersite on cycle sequencing and automated sequencing. The Tth polymerasewas used for the standard and mutagenic PCR, and net amplificationof the DNA and the mutations accumulated during PCR were analyzed.Under mutagenic PCR, the mutation rates were 7.0 x 10^–4(Tth) and 8.3 x 10^–4 (Tth) per nucleotide per cycle ofamplification, which were 4–9 times higher than the ratesunder standard PCR.     

The Derivation of the Cell Wall Elasticity Function from the Cell Turgor Potential

An equation is derived expressing average turgor pressure ofa leaf (_p) as a function of relative water content (RWC). Basedon this derivation, the relationships of the bulk elastic modulus(_v) and both RWC and _p, are formulated and discussed. The bulkelastic modulus (_v) becomes zero for _p = 0, that is at the turgorloss point for the leaf. At full water saturation the valueof ev is proportional to the water saturation turgor potential_p(max). The factor relating _P and _v (structure coefficient ,Burstrom, Uhrstr?m and Olausson, 1970) changes only very littlefor values of _p, which are not too close to zero. An exampleis given for the calculation from experimental data of the turgorpressure function, the structure coefficient function, and the_v function. Key words: Cell wall, Turgor pressure, Bulk elastic modulus     

A novel alpha1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures

The 1,2-fucosyltransferase family (1,2FT) is the largest familyof glycosyltransferases in the genome of the free-living nematodeCaenorhabditis elegans, and early evidence suggests that eachmember may have a unique activity. Here we describe a C. elegansgene (designated CE2FT-2) encoding an 1,2FT that has the potentialto generate the sequence Fuc1-2Galβ1-3GalNAc-R, which isthe H-type 3 blood group structure. The CE2FT-2 cDNA encodesa putative transmembrane protein that shows 42% amino acid identityto a previously cloned C. elegans 1,2FT (termed CE2FT-1), buthas a very low identity (16–20%) to 1,2FT sequences inhumans, rabbits, and mice. A recombinant form of CE2FT-2 expressedin human 293T cells has a high 1,2FT activity toward Galβ1-3GalNAc-O-pNP,but unexpectedly, the enzyme is inactive toward the acceptorGalβ-O-phenyl. Thus, CE2FT-2 differs from all other 1,2FTspreviously described from animals that all utilize Galβ-O-phenyl.CE2FT-2 is expressed at all stages of worm development, butremarkably, promoter analysis of the CE2FT-2 gene using greenfluorescent protein reporter constructs indicates that the CE2FT-2is expressed exclusively in pharyngeal cells of the worm fromembryo to an adult stage. Because pharyngeal cells are knownto secrete their glycoconjugates to the nematode surface, theseresults may indicate that products of CE2FT-2 contribute tointeractions of the nematode with its environment or are usedas ligands for bacterial attachment. These findings, along withthose on other 1,2FTs in C. elegans, suggest that each 1,2FTin this organism may have a unique acceptor specificity, expressionpattern, and biological function.     

Genotypic Differences in Leaf Water Relations between Brassica juncea and B. napus

Various kinds of measurement of tissue water status were madeseveral times during water stress and recovery in Brassica juncea(cv Canadian Black) and B napus (cv Drakkar) Unstressed plantsof the two species had similar leaf water potentials (_w), solute(_s) and turgor potentials (_p) Values of relative water content(RWC) and the slope of the linear relationship between _p andRWC (_p/RWC) were greater in B napus than in B juncea Statistical correlations of pooled data for the watered andstressed treatments differentiated the relationships among RWC,_w and its components in the two species The major statisticaldifference was that _p/RWC was related to RWC in B napus andto _w and _s in B juncea A decline in _p/RWC with decreasing _sin B juncea may be a mechanism for maintaining _p at low soilwater potentials through maintenance of more elastic cell walls. Brassica juncea, Brassica napus, osmotic adjustment, tissue elasticity, water relations     

A Comparative View of Alpha Crystallins: The contribution of comparative studies to understanding function

Integration between comparative biology and cellular/molecularbiology has helped advance understanding of the structure, functionand physiology of the vertebrate small heat shock proteins A-and B-crystallin. These proteins are expressed at high concentrationin the eye lens where they contribute to transparency and refractivepower. But they also function similarly to molecular chaperonesby preventing the aggregation of denatured proteins that cancause opacities, or cataracts. -crystallins also serve a numberof other roles in and out of the lens that are still not completelyunderstood. Comparative examination of -crystallins and closelyrelated small heat shock proteins from diverse taxa has helpedprovide insights into the proteins' three-dimensional shapeand structure/function relationships. Until recently, no studieshad examined the tissue specific expression or chaperone-likeactivity of -crystallins from a non-mammalian vertebrate. Ihave been investigating the -crystallins of the zebrafish, Daniorerio, as a first step towards utilizing the bony fishes asa model group for understanding the evolution of -crystallinfunction. Zebrafish A-crystallin displays similar structureand expression and increased chaperone-like activity comparedto its human orthologue. Zebrafish B-crystallin, however, hasa truncated C-terminal extension, more limited expression andlower chaperone-like activity than its human orthologue. Thesedata suggest that A-crystallin physiological function may beconserved between zebrafish and mammals, while B-crystallinphysiological function has diverged. Understanding zebrafish-crystallin physiology is necessary before this species canbe used for developmental and genetic studies, and providesa foundation for further comparative studies.     

Phosphorylation and subcellular location of {alpha}-L-fucosidase in Iymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

Mannose 6-phosphate is a recognition marker used by many newlymade acid hydrolases for their transport to lyso-somes. Previously,we investigated the incorporation of ³²Pi into -L-fucosidaseof lymphoid cell lines from a healthy individual (control) andan I-cell disease patient [DiCioccio and Miller, Glycobiology,1, 595–604 (1991)]. Phosphoserine was found in immunoprecipitable-L-fucosidase of both control and I-cell lymphoid cells, butmannose 6-phosphate was identified only in enzyme of controlcells. Extension of this investigation to lymphoid culturesof a pseudo-Hurler polydystrophy patient also identified onlyphosphoserine in -L-fucosidase. Using [³H] mannose instead of³²Pi, the precise identification of mannose 6-phosphate in -L-fucosidaseof control cells, and its absence in -L-fucosidase of I-celland pseudo-Hurler cells, was established. The stoichiometryof phosphorylation of -L-fucosidase in I-cell, pseudo-Hurlerand control lymphoid cells was 3, 4 and 10 mol Pi/mol enzyme,respectively. -L-Fucosidase was located in lysosomes isolatedfrom control, I-cell and pseudo-Hurler lymphoid cells by subcelluarfractionation on Percoll density gradients. Both I-cell andpseudo-Hurler lymphoid cells displayed normal intralysosomalactivity of -L-fucosidase despite lack of the mannose 6-phosphatemarker. Thus, I-cell and pseudo-Hurler lymphoid cells must possessa mannose 6-phosphate-independent mechanism for directing -L-fucosidaseto lysosomes. Phosphorylation of -L-fucosidase in pseudo-Hurlerand I-cell lymphoid cultures was found almost exclusively inintracellular and not in extracellular enzyme, suggesting thatphosphoserine may participate in the localization of -L-fucosidasein lysosomes of these cells. -L-fucosidase I-cell disease lysosome phosphorylation pseudo-Hurler polydystrophy