Invertase inhibitors from red beet, sugar beet, and sweet potato roots

Invertase inhibitors have been isolated and partially purified from red beets, sugar beets, and sweet potatoes. These inhibitors are thermolabile proteins with molecular weights of 18,000 to 23,000. They do not inhibit yeast and Neurospora invertases, but they are reactive with potato tuber invertase and other plant invertases with pH optima near 4.5. There are differences in reactivity of the inhibitors with some of the plant invertases, however. For most invertases, red beet and sugar beet inhibitors are most effective at pH 4.5 while sweet potato inhibitor is most effective at pH 5.     

Determination of the carbohydrate composition of mammalian glycoproteins by capillary gas chromatography/mass spectrometry

A technique to determine the carbohydrate composition of glycoproteins using capillary gas chromatography/mass spectrometry (electron impact) with selected ion monitoring is described. This method entails hydrolysis with methanolic-HCl followed by formation of trimethylsilyl methylglycoside derivatives, extraction of the carbohydrate derivatives into hexane, and GC/MS analysis. For those carbohydrates that are present in animal glycoproteins including fucose, mannose, galactose, glucosamine, galactosamine, and N-acetylneuraminic acid (sialic acid), the sensitivity of this assay was approximately 1-3 pmol and the assay was linear over a 100-fold range. The carbohydrate compositions determined on small quantities (1-10 pmol) of various glycoproteins including human transferrin and alpha-1 acid glycoprotein, fetuin, and ovalbumin were identical to their reported carbohydrate content and compositions. Major advantages of this technique include the time required to complete the sample preparation and analysis (less than 8 h), the sensitivity and specificity of the assay, and the fact that all carbohydrate moieties, including sialic acid, can be quantitated in a single hydrolysate of a glycoprotein.     

Characterization of gibberellins from dark-grown Phaseolus coccineus seedlings by gas-liquid chromatography and combined gas chromatography-mass spectrometry

Summary An extract from 6000 dark-grown Phaseolus coccineus seedlings was purified by countercurrent distribution and G-10 Sephadex followed by gradient elution from a silicic acid partition column with increasing amounts of ethyl actetate in n-hexane. 25 fractions were collected and tested with the barley-aleurone, Tan-ginbozu dwarf-rice, lettuce, cucumber, dwarf-pea, d-1, d-2, d-3 and d-5 maize, oat first-internode, and sugarcane-spindle bioassays. Major gibberellin (GA)-like activity was detected in fractions 4 (500g GA₃-equivalents) and 12–13 (270 g GA₃-equivalents) with smaller amounts in fractions 6, 8–9, 15–16, 18, 20, 23 and 25. The extracts were also applied to AMO-1618=dwarfed Ph.-coccineus seedlings. Fractions 4, 8 and 12 promoted the growth of both light- and dark-grown seedlings. GA₁, GA₃, GA₄ and GA₈ were active in the Phaseolus bioassay but GA₈-glucoside was inactive.The biological and chromatographic properties of fractions 4, 8–9 and 12–13 correspond with those of GA₄, GA₁₉ and GA₁. The identity of GA₄ in fraction 4 was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of the methyl ester and the trimethylsilyl ether of the methyl ester. Gasliquid-chromatography peaks corresponding to these derivatives of GA₁₉ and GA₁ were detected on QF-1 and SE-33 columns but their intensities were too weak to permit conclusive identification by GC-MS.Supported by an S.R.C. StudentshipSupported by a NATO Grant.Supported by NRC Grant A-5727.     

The analysis of 2-acetamido-2-deoxyaldose derivatives by gas-liquid chromatography and mass spectrometry

Syntheses are reported of 4-substituted, 4-deoxy analogs of methyl β-D-galactopyranoside (the 4-amino-4-deoxy, 4-azido-4-deoxy, 4-bromo-4-deoxy, 4-chloro-4-deoxy, 4-deoxy-4-fluoro, 4-deoxy-4-iodo, and 4-thio derivatives) as potential substrates of D-galactose oxidase. These syntheses involved nucleophilic displacement of the 4-(p-bromophenylsulfonyl)oxy group of appropriate D-glucose derivatives, although the more reactive (trifluoromethylsulfonyl)oxy group was also utilized as a novel leaving-group. Formation of the bromo and iodo derivatives was accompanied by appreciable halogen exchange and a resulting overall retention of configuration, and formation of the thio compound was attended by competing reactions. Optical rotatory characteristics of the halogeno compounds, their triacetates, and tribenzoates are described, and “anomalous” behavior of the last group is noted.     

Separation and identification of O-acetyl-O-methyl-galactononitriles by gas-liquid chromatography and mass spectrometry

The gas-liquid-chromatographic retention-times and the mass-spectral features of partially methylated d-galactononitrile acetates are reported. Distinctive fragmentation of each of the mono-O-methyl derivatives allows their identification, and the results are applicable to the same substituted derivatives of the other aldohexoses. A new fragmentation-pathway, typical of the acetylated and the O-acetyl-O-methylaldononitriles, is proposed in order to justify previously unexplained fragments. This fragmentation competes with the known ones in derivatives that do not carry vicinal methoxyl groups.     

Determination of lysergide in urine by high-performance liquid chromatography combined with electrospray ionisation mass spectrometry

A quantitative method which avoids derivatisation is described for the determination of lysergide (LSD) levels in urine. Sample preparation included addition of methysergide as an internal standard followed by solid-phase extraction. LSD was analysed on a system consisting of a C₁₈ stationary phase and a mobile phase of 0.1 M acetate buffer pH 8.0-acetonitrile-triethylamine (75:25:0.25, v/v). LSD was detected by electrospray ionisation mass spectrometry with selected ion monitoring. The quantification limit was 0.5 ng/ml and the method was linear up to 10 ng/ml of LSD in urine.     

O-methyl oximes of sugars. Analysis as O-trimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry

The mass spectra of aldoses, partially methylated aldoses, deoxyaldoses, and ketoses containing 3–7 carbons, were recorded on the ethers of the trimethylsilyl O-methyl oxime derivatives. Each compound gave a distinctive spectrum indicating the carbon-chain length and the location of substituents. The gas-liquid chromatographic properties of most compounds in this study were also examined.     

Determination of fatty acid content and composition in ultramicro lipid samples by gas-liquid chromatography

Simplified quantitative manipulations of very small amounts (30 micrograms) of lipids for determination of fatty acid content and composition by gas-liquid chromatography after (a) methanolysis (b) reduction and acetylation are described.     

Identification of mono- and dihydroxy bile acids in human feces by gas-liquid chromatography and mass spectrometry

The mono- and disubstituted cholanoic acids present in human feces have been investigated. Extracts of feces were fractionated on silicic acid column and individual bile acids were isolated by preparative thin-layer chromatography. The isolated compounds were studied by gas-liquid chromatography of the methyl esters, partial trimethylsilyl ethers, oxidation products, and trifluoroacetates. The probable structures deduced were confirmed by gas chromatography-mass spectrometry and by comparisons with authentic compounds. The following derivatives of 5 Beta-cholanoic acid not previously isolated from human feces were identified: 3,12-diketo, 3-keto-12alpha-hydroxy, 3alpha,12 Beta-dihydroxy, 3 Beta,12 Beta-dihydroxy, 3-keto-7alpha-hydroxy, 3alpha-hydroxy-7-keto, 3 Beta,7alpha-dihydroxy, 3alpha,7alpha-dihydroxy, and 3alpha,7 Beta-dihydroxy. The presence of 3-keto-, 3 Beta-hydroxy-, 3alpha-hydroxy-, 3 Beta-hydroxy-12-keto-, 3alpha-hydroxy-12-keto-, 3 Beta,12alpha-dihydroxy-, and 3alpha,12alpha-dihydroxy-5 Beta-cholanoic acids was confirmed. Evidence was obtained for the presence of two bile acids having at least one hydroxyl group at a carbon atom other than C(3), C(7), or C(12).     

Characterization of an oviposition stimulant from the surface of sweet potato Ipomoea batatas storage roots for the sweet potato weevil, Cylas formicarius elegantulus

An improved laboratory bioassay was used to characterize an oviposition stimulant from the surface of sweet potato Ipomoea batatas (L.) Lam. storage roots for the sweetpotato weevil, Cylas formicarius elegantulus (Summers). Filter paper discs impregnated with a methylene chloride surface extract of sweet potato storage roots induced significantly (p<0.05) higher oviposition on root cores than those treated with solvent only. Significantly higher oviposition was also observed in the nonpolar fractions, especially one that contains a tentatively identified triterpenoid present in susceptible cultivars.

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Caractérisation d'une substance extraite de la surface des racines tubéreuses d'Ipomoea batatas induisant la ponte de Cylas formicarius elegantulus

Résumé La caractérisation des substances extraites de la surface des racines tubéreuses d'I. batatas Lam et stimulant la ponte de C. formicarious elegantulus Summers, a été effectuée à partir d'une technique améliorée. Différents substrats ont été essayés: extraits au chlorure de méthylène ou péridermes intacts de cultivars résistants ou sensibles, racines tubéreuses après élimination du périderme, morceaux de Solanum tuberosum ou de papier filtre présentés au milieu de plaques à 24 plots pour culture de tissus. Le meilleur substrat s'est révélé être de petits disques de papier filtre (diamètre 0,4 cm), fixés au milieu d'un morceau de racine avec encore un peu de périderme sur lequel le coléoptère pouvait pondre. Cette méthode a été utilisée dans les tests ultérieurs pour caractériser la nature du stimulant.Des disques de papier filtre inhibés d'extraits au chlorure de méthylène du contenu des structures superficielles de racines tubéreuses de cultivars sensibles induisent une ponte significativement plus importante (p<0,05) sur les morceaux de racines par comparaison avec ceux traités au chlorure de méthylène pur. Une ponte significativement plus importante a aussi été obtenue avec des fractions non-polaires, particulièrement celles qui contiennent un triterpènoïde de cultivars dont l'identification a été tentée.

     

Aminoethylcysteine ketimine decarboxylated dimer detected in normal human urine by gas-liquid chromatography, selected-ion monitoring and mass spectrometry

Aminoethylcysteine ketimine is a biochemical product known to be converted spontaneously in the decarboxylated dimer. Since the ketimine has been detected in a mammalian brain, it was assumed that also the dimer could be present in the mammalian body and eventually excreted in the urine. Using human urine as the biological source, an extract was prepared which, submitted to gas-liquid chromatography, selected-ion monitoring and mass spectrometry, indicated the presence of the dimer.     

Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis

Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of the α-glucan in plant cells. When compared to its isoform in an animal cell, glycogen phosphorylase, a peptide containing 78 amino acids (L78) is inserted in the centre of the low-affinity type starch phosphorylase (L-SP). We found that the amino acid sequence of L78 had several interesting features including the presence of a PEST region, which serves as a signal for rapid degradation. Indeed, most L-SP molecules isolated from mature sweet potato roots were nicked in the middle of a molecule, but still retained their tertiary or quaternary structures, as well as full catalytic activity. The nicking sites on the L78 were identified by amino acid sequencing of these peptides, which also enabled us to propose a proteolytic process for L-SP. Enzyme kinetic studies of L-SP in the direction of starch synthesis indicated that the K_m decreased during the proteolytic process when starch was used as the limiting substrate, but the K_m for the other substrate (Glc-1-P) increased. On the other hand, the maximum velocities (V_max) increased for both substrates. Mobility of the nicked L-SP was retarded on a native polyacrylamide gel containing soluble starch, indicating the increased affinity for starch. Results in this study suggested that L78 and its proteolytic modifications might play a regulatory role on the catalytic behaviour of L-SP in starch biosynthesis.     

Absence of arabinose in bovine brain hyaluronic acid as analysed by gas-liquid chromatography and mass spectrometry

No arabinose could be detected in a simple of bovine brain hyaluroni acid when examined by gas—liquid chromatography and mass spectrometry. A control, containing only one part arabinose per 1000, gave three readily discernible arabinose components.     

Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots

A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters. H.M. Chen and C.T. Lin contributed equally to this work.