Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

Indolepyruvate Pathway for Indole-3-Acetic Acid Biosynthesis in Bradyrhizobium elkanii

Indole-3-acetaldehyde (IAAId) was detected in the culture supernatantof Bradyrhizobium elkanii. Deuteriumlabelled L-tryptophan (Trp)was incorporated into IAAId and indole-3-acetic acid (IAA),suggesting that B. elkanii produces IAA via IAAId from Trp.In B. elkanii cell suspension, indole-3-pyruvic acid (IPyA)was converted to IAAId, and exogenously added IAAId was rapidlyconverted to IAA. Furthermore, the activity of indolepyruvatedecarboxylase (IPDC), which catalyzes the decarboxylation ofIPyA to produce IAAId and is a key enzyme for IPyA pathway,was detected in B. elkanii cell-free extract. The IPDC activitydepended on Mg²⁺ and thiamine pyrophosphate, cofactors of decarboxylation.This mounting evidence strongly suggests that IAA synthesisoccurs via IPyA pathway (Trp IPyA p IAAId IAA) in B. elkanii. (Received December 11, 1995; Accepted March 4, 1996)     

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum.

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.     

Conformational changes of α-lactalbumin induced by the stepwise reduction of its disulfide bridges: The effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution

Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution.     

Craniometrical variations among eastern Brazilian marmosets and their systematic relationships

The craniometrical variations among marmosets on the eastern coast of Brazil or theCallithrix jacchus group (C. jacchus, C. penicillata, C. kuhli, C. geoffroyi, C. flaviceps, andC. aurita) were analyzed to test various hypotheses for the arrangement of the species and phylogenetic relationships ofC. kuhli andC. flaviceps. Q-mode correlation coefficients and principal component analysis were employed to extract important shape factors from the craniometrical data. On this basis, the shape distances among the eastern Brazilian marmosets were found to be larger than those between the marmosets which are recognized as distinct species. This evidence supports the hypothesis that the members of theC. jacchus group are distinct species.C. kuhli shows the strongest resemblance toC. geoffroyi within theC. jacchus group.C. flaviceps is the most similar toC. aurita in its cranial shape. These findings indicate thatC. kuhli is most closely related toC. geoffroyi, whileC. flaviceps is toC. aurita.     

Effects of the Degree of Polymerization on the Binding of Xyloglucans to Cellulose

Xyloglucan oligosaccharides were isolated with various degreesof polymerization (DP) and reduced with tritiated sodium borohydride.The ³H-oligosaccharides were tested for their ability to bindto amorphous and microcrystalline celluloses and to cellulosefilter paper. The time course of binding indicated that theradiolabeled oligosaccharides continued to be bound for at least1 h after heating at 120°C. The binding probably requiredthe organization of the oligosaccharides and celluloses by gradualannealing after heating. Although neither pentasaccharide (glucose:xylose, 3 : 2), heptasaccharide (glucose: xylose, 4 : 3) andnonasaccharide (glucose : xylose : galactose : fucose, 4 : 3: 1 : 1) failed to bind to the celluloses, binding occurredwith oligosaccharides with DP equivalent to more than four consecutive1,4-ß-glucosyl residues. The extent of binding tothe celluloses increased gradually from octasaccharide (glucose:xylose, 5 : 3) to hendecosanosaccharide (glucose/xylose, 12: 9), with the increase in the DP of 1,4-ß-glucosylresidues. The binding of reduced cello-dextrins to celluloserequired at least 4 consecutive 1,4-ß-glucosyl residues.The extent of binding of cellopentitol or cellohexitol to cellulosewas similar to that of hendecosanosaccharide, showing lowerbinding for xyloglucan oligosaccharides in spite of longer chainsof 1,4-ß-glucosyl residues. These findings suggestthat the mode of binding to cellulose of xyloglucan oligosaccharidesis different from that of cello-oligosaccharides. (Received February 18, 1994; Accepted June 1, 1994)     

N G,N G -Dimethyl-l-arginine,a dominant precursor of endogenous dimethylamine in rats

Summary The metabolic significance ofN ^G ,N ^G -dimethyl-l-arginine (DMA) as a precursor of endogenous dimethylamine (DMN) in rats was examined in connection with the wide distribution and active operation of dimethylargininase (EC3.5.3.18) in rat tissues (Kimoto et al., 1993). When [methyl-¹⁴C]DMA was administered intraperitoneally to rats, the radioactive DMN was detected in various tissues as a major radioactive metabolite one hour after injection, and about 65% of the radioactivity administered was recovered in the first 12-h urine as DMN. In the case of the [¹⁴C] DMN-injected rats, almost all the radioactivity was excreted in the 12-h urine as DMN, except for a negligible amount of radioactivity found in urea. The time-dependent decrease in the specific radioactivity of DMA and DMN in urine showed that dimethylargininase was significantly involved in thein vivo formation of DMN by the hydrolytic cleavage of DMA released from methylated proteins and that DMA is a dominant precursor of endogenous DMN in rats.     

Production of neutral protease from Aspergillus oryzae by a novel cultivation method on a microporous membrane

Summary A novel cultivation method using a microporous membrane (membrane-surface liquid culture) was developed, in which moulds are grown on the membrane surface with its opposite side being in contact with a liquid medium. The amount of neutral protease from Aspergillus oryzae produced was more than 10 times higher than that produced by the conventional liquid culture.