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.     

Ecdysone Metabolism in Insects

The current status of the pathway of ecdysone biosynthesis andinactivation in insects is discussed. Evidence is presenteddemonstrating that rß-ecdysone is generated from -ecdysoneby fat body, Malpighian tubules, gut, and body wall, but notby blood, oenocytes, muscle, salivary gland, or the prothoracicgland itself (in vitro experiments with prepupal Manduca sextatissues). Since -ecdysone does not appear to have demonstrablehormonal activity at physiological concentrations in in vitrotissue systems which cannot metabolize it to rß-ecdysone,it is suggested that -ecdysone serves as a prohormone ratherthan as a hormone. The role of the prothoracic gland in theproduction of -ecdysone remains to be defined.     

The Ontogeny of Osmoregulation and Its Neurosecretory Control In the Decapod Crustacean, Rhithropanopeus harrisii (Gould)

Laboratory-hatched larvae of this estuarine crab were rearedat 25°C in seawater of 25 salinity for 18 days coveringzoeal Stages I to IV and a megalops. Three-day periods betweenzoeal stages represent intermolt stages of circadean metecdysis,diecdysis, and proecdysis.Larvae were exposed to either a seriesof seawater salinities from 5-40 in 5 increments or of 10-40in 10 increments for one hour during each day of their development.The osmoconcentrations of 20-80 nanoliter hemolymph samplesfrom each of four larvae were measured separately by determinationsof freezing point depression. Eyestalkless larvae in metecdysis of zoeal Stage II were exposedto the same osmoconcentrations as unoperated controls to testfor osmoregulation by eyestalk nerve tissue. Larvae tend to be isosmotic with seawater of 30-40 salinity(S) and to hyperregulate in more dilute media except for larvaein their first diecdysis which remain isosmotic. Larvae in thelast few hours of proecdysis hyperregulate against 40 S as well,presumably to insure inflow of water to establish a greaterbody volume during hardening of the exoskeleton. They are consequentlyisosmotic in the very early metecdysis. The presence of eyestalks at the first metecdysis (Stage II)keeps zoeas hyperosmotic to 5-30 S, but prevents them from hyperregulationagainst 40 S. Eyestalkless zoeas become isosmotic with 5-30S and hyperregulate against 40 S like late proecdysal larvae.Lack of eyestalks makes diecdysal animals hyperregulate againsta medium with which normal animals are isosmotic. The eyestalkinfluence affects second metecdysal (Stage III) larvae in away similar to those in first metecdysis except that it apparentlyalso prevents a curious tendency to hyporegulate in 5-30 S.Similarly, in this stage, the eyestalks prevent hyperosmosityin 40 S seawater as they do during the first day of zoeal StageII. Eyestalk nerve tissue reduces the degree to which diecdysallarvae of this stage remain hyperosmotic to media of 10 S and20 S and apparently causes larvae to be hypoosmotic at 40 S. Preliminary data indicate that removal of eyestalks has littleeffect on proecdysal larval osmoregulation or on regulationof Stage IV zoeas. In other experiments ablation of eyestalks caused Stage II larvaeto lose the ability to osmoregulate against 10-30 S seawaterwithin two hours after the operation. The same zoeas did nothyperregulate against 40 seawater until four hours after removalof eyestalks.     

{alpha}Melanocyte Stimulating Hormone: Chemical Nature and Mechanism of Action

Melanotropin (-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂,synthesized and secreted by the pars intermedia of the vertebratepituitary. This peptide hormone is derived from pro-opiomelanocortin,a precursor protein which contains within its structure thesequences of other melanotropic peptides (- and rß-MSH,corticotropin), and possibly other hormones. -MSH is the physiologicallyrelevant melanotropin secreted by the pituitary and in mostvertebrates plays the essential role in adaptive color changesthrough its action on integumental chromatophores. The initial actions of -MSH are mediated at the level of themelanocyte membrane and involve signal transduction from receptorto adenylate cyclase on the intracellular surface of the membrane.This results in elevated cytosolic cyclic AMP levels followedby melanosome dispersion within dermal melanocytes and melanogenesiswithin epidermal melanocytes. The action of -MSH on dermal melanocytesrequires calcium for transduction of signal and cyclic AMP production.Melanosome dispersion per se does not, however, require extracellularcalcium. Structure-function studies of -MSH analogues and fragmentshave provided important insights relative to the structuralrequirements of the hormone for receptor binding and transduction.Substitution of certain residues within -MSH has led to thedevelopment of melanotropins that exhibit extraordinary potencyand prolonged biological activity     

Peripheral and CNS Effects of the Pineal Gland: Target Enzymes Common to Tissues and Species

The pineal gland is an important transducer mediating environmentalinfluences on endocrine organs. It has direct effects on thehypothalamus and pancreas and indirect effects on the pituitary,adrenal, thyroid, and testes. Thus, specific endocrine rhythmsare affected by alterations in trophic hormone secretion bythe hypothalamus-pituitary complex, direct effects on biochemicaltransformations within target organs, or by alterations in metabolismand excretion of hormones by the liver. Target enzymes commonto the above organs for melatonin and arginine vasotocin (AVT)are 5-reductase, monoamine oxidase (MAO), and smooth muscleenzymes. Melatonin selectively inhibited 17rß ol-dehydrogenaseactivity and 5-reductase activity while 17rß ol-dehydrogenasewas stimulated by serotonin (5-HT). Other steroid biotransformationswere inhibited by both 5-HT and melatonin. Evidence from thepancreas and insulin secretion, liver and glucuronosyl transferaseactivity, and hypothalamic and pituitary studies indicate thatmelatonin mediated some of its effects on these organs throughMAO activity and 5-HT levels. There were some species and tissuedifferences with respect to the effects of melatonin and AVTon MAO activity and steroid biotransformations. Melatonin stimulatedsteroid biotransformations in the duck, while MAO activity and5-reductase activity in the hamster responded differently tomelatonin than did similar preparations from the rat.     

{delta}15N and {delta}13C Measurements of Antarctic Peninsula Fauna: Trophic Relationships and Assimilation of Benthic Seaweeds

Measurements of ¹³C, ¹⁵N, and C/N for a variety of Antarcticpeninsula fauna and flora were used to quantify the importanceof benthic brown algae to resident organisms and determine foodweb relationships among this diverse littoral fauna. ¹³C valuesranged from–16.8 for benthic algal herbivores (limpets)to –29.8 for the krill, Euphausia superba; the averagepooled value for brown macroalgae, including their attachedfilamentous diatoms, was–20.6. There was no correlationbetween biomass ¹³C or ¹⁵N with C/N content, and consequentlyboth ¹³C and ¹⁵N values were useful in evaluating trophic relationships.¹⁵N values of the fauna ranged from 3.1 to 12.5, with lowestvalues recorded in suspension feeders (e.g., bryozoans) andhighest values in Adelie penguins (12.5) collected in 1989.The comparatively lower ¹⁵N value for a Chinstrap penguin (6.9)collected in 1997 is attributed to the different dietary foodsources consumed by these species as reflected in their respective¹³C values. Significant amounts of benthic macroalgal carbonis incorporated into the tissues of invertebrates and fishesthat occupy up to four trophic levels. For many benthic andepibenthic species, including various crustaceans and molluscs,assimilation of benthic algal carbon through detrital pathwaysranges from 30 to 100%. Consequently, the trophic importanceof benthic brown algae may well extend to many pelagic organismsthat are key prey species for birds, fishes, and marine mammals.These data support the hypothesis that benthic seaweeeds, togetherwith their associated epiphytic diatoms, provide an importantcarbon source that is readily incorporated into Antarctic peninsulafood webs.     

Control of Tissue Specificity: The Pattern of Cellular Synthetic Activities in Tissue Transformation

During transformation of the dorsal marginal iris into lenstissue after removal of the lens in the adult newt, the cellsundergo gradual changes in synthetic activities. Autoradiographicdata indicate an enhancement of RNA synthesis in the nucleusof iris cells, which is followed by enhancement of protein synthesisand onset ot DNA synthesis. After discharge of pigment granulesand a period of cellular multiplication accompanied by ribosomeproduction, the cells stop DNA synthesis and start to show detectableamounts of lens-specific proteins (-,ß-, and -crystallins).This time coincides with initiation of differentiation of primarylens fibers. In the later stages of regeneration, - and ß-crystallinsare present in the dividing cells of the lens epithelium, aswell as in the cells of the fiber area, but -crystallins aredetected only in Che cells of the fiber area. The data wereinterpreted as suggesting that the gene utilization patterntypical for the iris is not directly converted into that forthe lens, but goes through intermediate patterns before thetissue transformation is completed.     

Axial Distribution of Glycosidases in Relation to Cellular Growth and Ageing in Pisum sativum Root

Tanimoto, E. 1985. Axial distribution of glycosidases in relationto cellular growth and ageing in Pisum sativum root.—J.exp. Bot. 36: 1267–1274. Eight glycosidase activities were measured in relation to cellulargrowth and ageing along the axis of pea roots. The highest activities of ß-D-fucosidase and ß-D-glucosidasewere in the apical 1.0 mm segment containing the root cap andmeristem. Activities of -L-arabinosidase, -D-mannosidase, ß-D-galactosidase, -D-glucosidase and ß-D-xylosidase werehighest in the segment between 1 and 2 mm from the tip and containingyoung elongating cells with high growth potential. The activityof -D-galactosidase was high between 1 and 2 mm from the roottip, decreasing to a minimum 4-5 mm from the tip and increasingagain up to the base of root. This distribution of enzyme activitiesrelative to the root tip remained unchanged during 28 h whilethe root length doubled. No -L--fucosidase, ß-L-fucosidaseand -D-xylosidase activities were detected. Key words: Pisum sativum, root growth, glycosidase, galactosidase, mannosidase     

Mechanical Design of Fiber-Wound Hydraulic Skeletons: The Stiffening and Straightening of Embryonic Notochords

The notochord can play an important mechanical role in shapechanges during early morphogenesis of vertebrates. For example,osmotic inflation of notochords elongates and straightens theaxis of frog early tail-bud embryos. In Xenopus laevis, thesheath of cross-helically arranged fibers around the notochordlimits the shape changes it undergoes when inflating, causingthe notochord to stiffen and straighten (Adams et al., 1990;Koehl et al., 1990). We used physical models of stage 24 X.laevis notochords to explore the mechanical consequences ofdifferent arrangements of the sheath fibers on the behaviorof such curved hydraulic cylinders. All the models straightenedupon inflation regardless of initial fiber angle ( = angle ofthe fibers to long axis of the cylinder). Notochord models with > 54° lengthened and narrowed as they straightened;although they could push, the forces they exerted were limitedby their tendency to buckle, which increased the greater the. In contrast, models with < 54° shortened and widenedas they straightened and showed pronounced increases in flexuralstiffness. The mean of X. laevis early tail-bud notochordsis 54°, a fiber angle that permits an increase in the end-to-enddistance of the model (along the anterior-posterior axis ofthe embryo) as it straightens and pushes when pressurized, butthat is less prone to Euler and local buckling than are modelswith higher 's. Nonetheless, a of 54° in notochords maysimply be the result of osmotic swelling.     

Odor thresholds of some derivatives of strawberry aldehyde

The olfactory threshold concentrations of 19 derivatives ofethyl ß-methyl-ß-phenyl-,ß-epoxypropionate(strawberry aldehyde) were determined in water solution usinguntrained panellists. In spite of their similar carbon skeletons,the compounds differed considerably in their odor thresholdvalues. The lowest and highest values were found to be 0.5 p.p.m.(0.5 parts of the odorant per 10⁶ parts of water) for ß-(bromophenyl)-,ß-epoxypropionateand 720 p.p.m. for n-octyl-,ß-epoxy propionate, respectively.     

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)     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

Molecular Phylogeny of Arthropods and the Significance of the Cambrian "Explosion" for Molecular Systematics

SYNOPSIS. Accurate phylogenetic reconstruction requires charactersystems that have evolved fast enough to have kept pace withcladogenesis but slowly enough to have conveyed the resultingphylogenetic signal to the present. Because stratigraphic evidencesuggests that basal arthropod lineages arose rapidly duringan ancient (Cambrian) phylogenetic radiation, the discoveryof molecular sequences capable of resolving arthropod phylogenymay be a significant challenge for molecular systematists. Thischallenge is exemplified by our attempt to resolve arthropodphylogeny using the amino acid sequence of elongation factor-1(EF-1). Our fossil-based assessment of evolutionary rates indicatesthat EF-la should be capable of resolving Cambrian-age divergences.However, phylogenetic analysis using EF-1 fails to establishrelationships among most higher-level groups, although it doesrecover more recently derived clades. Here we propose two modelsto explain this incongruity. The Rapid Radiation Model maintainsthat fossil-based estimates of arthropod diversification areessentially accurate and that diversification occurred so rapidlyduring the Cambrian that few phylogenetically significant changesoccurred in the slowly evolving EF-1 sequence. The EnhancedPreservation Model maintains that fossil-based estimates ofCambrian-age divergences reflect enhanced preservation of pre-existinglineages and that arthropod diversification occurred beforethe Cambrian. This model attributes lack of resolution to degradationof phylogenetic signal within EF-1 by subsequent evolution.Current evidence is more consistent with the Enhanced PreservationModel, which implies that fossil-based methods can be very misleadingwhen attempting to gauge the phylogenetic information contentof molecular sequences for Cambrian- and Precambrian-age divergences.     

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     

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     

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)     

Activity of Wall-bound Enzymes in Callus Cultures of Gossypium hirsutum L. During Growth

Activities of - and ß-glucosidase, - and ß-galactosidase,-mannosidase, ß-1,3-glucanase, acid and neutral invertaseswere detected in the cytoplasmic fraction as well as in cellwalls isolated from callus cultures of cotton. Activity of ß-mannosidase,however, could not be detected in the cell walls. Transfer ofcallus to a fresh medium did not immediately influence the activitiesof -glucosidase and ß-galactosidase but increasedsignificantly ß-glucosidase, -mannosidase, acid andneutral invertases. Addition of cycloheximide (1 and 100 mgl^–1) further stimulated acid and neutral invertases butnot other enzymes tested. Sodium chloride (NaCl) was effectivein extracting a-glucosidase, ß-glucosidase, ß-galactosidase,acid and neutral invertases. EDTA extracted most of the -galactosidase,-mannosidase, ß-1,3-glucanase and some -glucosidase.But, NaCl and EDTA could not extract some of the - and ß-glucosidasesand also acid and neutral invertases as evidenced from the residualand extra cellular activity. Studies with whole cells as a sourceof enzyme revealed that some of these enzymes were associatedwith the cell surface. Callus, glycosidases, glucanase, growth, Gossypium hirsutum     

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     

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