Determination of the d and l configuration of neutral monosaccharides by high-resolution capillary g.l.c.

Capillary g.l.c. on SE-30 of the trimethylsilylated (-)-2-butyl glycosides of d and l monosaccharides gives multiple peak patterns, which can be used for the assignment of the absolute configurations. (-)-2-Butyl glycosides can be prepared from monosaccharides or their methyl glycosides; consequently, for the analysis of oligo- or poly-saccharides, hydrolysis as well as methanolysis can be applied. Provided that the peaks of the (-)-2-butyl glycosides do not completely overlap, mixtures of monosaccharides can be analysed directly, as illustrated for the constituents of the cell-wall lipopolysaccharide from Salmonella typhimurium LT-2.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Amino acid sequence homologies in alfa-sarcin, restrictocin and mitogillin

The NH₂-terminal amino acid sequence of the three anti-tumor proteins, alfa-sarcin, mitogillin and restrictocine, has been determined for 20 cycles by automated sequencing procedure. A high degree of sequence homology was observed in this region of the molecule. In addition, extensive sequence homology, ranging from 65 to 100% was found in three other carboxymethylcysteine-containing peptides isolated and sequenced from each molecule.     

l-Myoinositol-1-phosphate synthase from Neurospora crassa: Purification to homogeneity and partial characterization

The purification procedure of milligram quantities of stable myoinositol-1-phosphate synthase (EC 5.5.1.4) from Neurospora crassa is reported. The procedure includes: (a) (NH₄)₂SO₄ and protamine sulfate precipitations, (b) gel filtration in Ultrogel AcA-34 (LKB), (c) DEAE-cellulose chromatography, (d) AH-Sepharose 4B chromatography, and (e) calcium phosphate gel chromatography. The enzyme is considered pure according to the following criteria: (a) gel filtration, (b) sucrose density gradient centrifugation, (c) polyacrylamide gel electrophoresis, and (d) isoelectric focusing technique. The molecular weight estimated by gel filtration chromatography and sucrose density gradient centrifugation is 345,000. The subunit molecular weight is 59,000. The active enzyme seems to posses an hexameric structure. The isoelectric point estimated for the pure enzyme is 5.2. The enzyme was optimally stimulated by 10 mm (NH₄)₂SO₄ and by 50 mm KCl, while NaCl had a minor inhibitory effect at higher concentrations. The divalent cations Mg²⁺ and Mn²⁺ were inhibitory only at nonphysiological concentrations. The enzymatic activity after the salt fractionation steps was about 33% NAD⁺ independent; but with purification the resulting homogeneous enzyme showed less than 5% NAD⁺-independent activity.     

Pharmacology of excitatory amino acid receptors mediating the stimulation of rat cerebellar cyclic GMP levele invitro

L-glutamate and related amino acids produced dose-related increases in cyclic GMP concentrations in cerebellar slices from 8-day old rats. The effects of L-glutamate, L-aspartate, and N-methyl-D-aspartate were antagonised in part by glutamate diethylester, while D-α-aminoadipate and DL-α-aminosuberate were ineffective. Kainic acid, although producing an effective stimulation of cyclic GMP, failed to achieve the same maximal response as glutamate, suggesting different sites of action. Cyclic GMP levels were also increased, although to a lesser extent, by L-glutamate in slices from hippocampus, medulla, hypothalamus, cortex, striatum and spinal cord of young animals. In the adult, however, this response was no longer observed.     

Structural investigation of the acidic polysaccharide secreted by Zoogloea ramigera 115

Batch-culture growth of Zoogloea ramigera 115 in a defined medium produced a weakly acidic polysaccharide containing glucose and galactose residues, and (S)-pyruvic acetal groups. Analytical results indicated that the polysaccharide does not have a simple repeating-unit. Mainly with the aid of Smith degradation of the native polysaccharide and oxidation and subsequent β-elimination of the methylated and then depyruvylated polysaccharide, some structural features of the polysaccharide were identified.     

The structure of d-threo-2,5-hexodiulosonic acid and derivatives in solution

The structure of d-threo-2,5-hexodiulosonic acid (1) and various derivatives in solution was determined by ¹³C-n.m.r. spectroscopy to be a hydrated, pyranose form. The structures of the methyl ester of 1 and of its 5-(dimethyl acetal) were confirmed by chemical means and by X-ray structure analysis.     

The synthesis of 1,2:5,6-di-O-isopropylidene-d-mannitol: a comparative study

Three different methods of acetonation of d-mannitol using (a) acetone and zinc chloride, (b), 2,2-dimethoxypropane, 1,2-dimethoxyethane, and tin(II) chloride, and (c) 2-methoxypropene, N,N-dimethylformamide, and p-toluenesulfonic acid were studied in detail and compared, using gas-liquid chromatographic techniques. In each reaction, isomeric diacetals are formed, but method a gives the 1,2:5,6-diacetal in the highest yield (63%). Methods b and c give a more complex mixture of acetals than proposed in the literature, and both methods are less economical than a. A new 1,2:3,6:4,5-tri-O-isopropylidene-d-mannitol could be separated, and its graded hydrolysis was compared to that of the 1,2:3,4:5,6-triacetal.     

A sensitive,specific radioisotope assay for l-glutamine-d-fructose-6-phosphate aminotransferase

A sensitive and specific radioassay for l-glutamine-d-fructose-6-phosphate aminotransferase (EC 5.3.1.19) activity is presented. Picomoles of product are measurable, and the assay can be applied to systems having limited quantities of available protein, particularly in extracts of either cell or organ cultures. The assay is at least 10,000 times more sensitive under K₁ concentrations of fructose 6-phosphate than the modified Elson-Morgan colorimetric assay and 20 times more sensitive under saturating conditions of fructose 6-phosphate. As little as 0.5 μg of cell-extract protein will yield measurable product. In contrast, 280 μg of crudeextract protein from colon is required with the modified Elson-Morgan colorimetric assay.     

Structural studies of an acidic polysaccharide from the mucin secreted by Drosera capensis

The polysaccharide of the mucin secreted by the leaves of Drosera capensis is composed of l-arabinose, d-xylose, d-galactose, d-mannose, and d-glucuronic acid in the molar ratio of 3.6:1.0:4.9:8.4:8.2. For structural elucidation, methylation analysis using g.l.c. and g.l.c.-m.s. was performed on the native, the carboxyl-reduced, and the degraded polysaccharides. Partial hydrolysis, periodate oxidation, chromium trioxide oxidation, and uronic acid degradation were also performed on the native and carboxyl-reduced polysaccharides. Partial hydrolysis of the native and carboxyl-reduced polysaccharides gave various oligosaccharides that were characterized and suggest a structure containing a d-glucurono-d-mannan backbone having a repeating unit → 4)-β-d-GlcpA-(1 → 2)-α-d-Manp-(1 →. l-Arabinose and d-xylose are present as nonreducing furanosyl and pyranosyl end-groups, respectively, both attached to O-3 of d-glucuronic acid residues of the backbone. d-Galactose is present as non-reducing pyranosyl end-group linked to O-3 of d-mannose residues.     

Kinetics and mechanism of oxidation of d-fructose and l-sorbose by chromium(VI) and vanadium(V) in perchloric acid medium

Kinetic data for the oxidations of d-fructose and l-sorbose by chromium(VI) and vanadium(V) in perchloric acid medium are reported. The addition of perchloric acid and sodium perchlorate increases the pseudo-first-order rate constants. Change of the reaction medium from water to deuterium oxide appreciably affects the rates of chromium(VI) oxidations, but does not affect those of vanadium(V) oxidations. The activation parameters are ΔH3 = 46.6 ±3.4 (fructose) and 50.6 ±6.3 (sorbose) kJ.mol^?1, and ΔS3 = ?105 ±11 (fructose) and ?100 ±20 (sorbose) J.deg^?1.mol^?1 for chromium(VI) oxidations, and, for the other reactions, ΔH3 = 53.2 ±4.2 (fructose) and 52.3 ±6.3 (sorbose) kJ.mol^?1, and ΔS3 = ?139.0 ±14 (fructose) and ?137 ±20 (sorbose) J.deg^?1.mol^?1. The kinetics of the oxidations of ketohexoses by chromium(VI) indicate no intermediate-complex formation, whereas those for vanadium(V) indicate the formation of a 1:1 intermediate complex between ketohexoses and vanadium(V).     

Synthesis of acetylenic sugars and uronic acids via two-carbon chain extension of d-ribose

Treatment of methyl β-d-ribofuranoside with acetone gave methyl 2,3-O-isopropylidene-β-d-ribofuranoside (1, 90%), whereas methyl α-d-ribofuranoside gave a mixture (30%) of 1 and methyl 2,3-O-isopropylidene-α-d-ribofuranoside (1a). On oxidation, 1 gave methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside (2), whereas no similar product was obtained on oxidation of 1a. Ethynylmagnesium bromide reacted with 2 in dry tetrahydrofuran to give a 1:1 mixture (95%) of methyl 6,7-dideoxy-2,3-O-isopropylidene-β-d-allo- (3) and -α-l-talo-hept-6-ynofuranoside (4). Ozonolysis of 3 and 4 in dichloromethane gave the corresponding d-allo- and l-talo-uronic acids, characterized as their methyl esters (5 and 6) and 5-O-formyl methyl esters (5a and 6a). Ozonolysis in methanol gave a mixture of the free uronic acid and the methyl ester, and only a small proportion of the 5-O-formyl methyl ester. Malonic acid reacted with 2 to give methyl 5,6-dideoxy-2,3-O-isopropylidene-β-d-ribo-trans-hept-5-enofuranosiduronic acid (7).     

Proton and carbon-13 nuclear magnetic resonance studies on methyl (methyl d-galactosid)uronates and their per-O-acetyl derivatives

The ¹H- and ¹³C-n.m.r. spectra of the anomeric methyl (methyl d-galactosid)uronates, as well as the ¹H-n.m.r. spectra of their acetyl derivatives, were analyzed. The spectra of the unacetylated d-galactopyranosiduronates showed good correlation with those of the corresponding anomeric d-galactopyranuronic acids and their methyl esters, and with those of the anomeric methyl d-galactopyranosides. From the values of the chemical shifts and coupling constants, it was concluded that the anomeric methyl (methyl d-galactopyranosid)uronates and their corresponding peracetates are in the ⁴C₁(d) conformation. The chemical shifts in the ¹³C-n.m.r. spectra show good correlation with those of the methyl d-galactosides. The signals of the furanose derivatives appear at fields lower than those of the corresponding pyranose compounds.     

Kinetics and mechanism of oxidation of d-erythrose and dL-glyceraldehyde by chromium(VI) and vanadium(V) in perchloric acid medium

The kinetics of oxidation of d-erythrose and dL-glyceraldehyde by chromium (VI) and vanadium(V) in perchloric acid medium have been investigated spectrophotometrically. Each reaction was first-order with respect to [oxidant] and [substrate]. The reactions were catalysed by acid, but their dependence on acidity was complex. Sodium perchlorate accelerated the rate of each reaction. The oxidation rates follow the order glyceraldehyde > erythrose. The activation parameters were calculated and mechanisms consistent with the experimental observations are proposed.     

Thermodynamic studies on synthetic lac operator core

The nine base pairs long central region of the $\text{lac}$ operator gene forms a stable double helix. A comparison of melting temperatures with other biologically useful oligonucleotides indicates the importance of specific base sequence. Binding constants measured with ethidium bromide (1.7 × 10⁵ M^?1), tyrosine (4.0 × 10³ M^?1), and glutamine (1.5 × 10³ M^?1), are interpreted in terms of the involvement of a relatively small number of amino acids in the $\text{lac}$ operator-repressor interaction.     

Chromatographic resolution of two DNA polymerase activities from bloodstream forms of Trypanosomabrucei: Differential responses to exogenous polyamine addition

A single peak of DNA polymerase activity from extracts of $\text{T.}\text{brucei}$, obtained by DEAE-cellulose and phosphocellulose ion-exchange chromatography, was resolved into two peaks differing in KCl concentration necessary to elute them from a DNA-agarose column. Peak I (eluting at 0.2 M KCl) and Peak II (eluting at 0.4 M KCl), differed in response to increasing KCl concentrations, although both functioned optimally with Mg²⁺ as divalent cation when DNA synthesis was directed either by activated DNA or poly (dC)·(dG)_12–18. Due to the potential significance of polyamines in the metabolism of $\text{T.}\text{brucei}$, the effect of exogenous polyamine on rates of DNA synthesis by the peak I and II enzymes was compared with that of murine DNA polymerase alpha. Only the peak I enzyme was significantly stimulated (up to 4-fold) by the biologically active polyamines spermine and spermidine at physiological concentrations. The response of the peak I enzyme resembled that of the alpha polymerase. This result suggests a possible functional difference between peak I and II enzymes, as well as a potential target site for trypanocidal drug development.     

The bioadhesive of Mytilus byssus: A protein containing L-DOPA

L-DOPA was identified in hydrolysates of $\text{Mytilus}$ byssal adhesive discs and is present at about $\text{10}\text{res}\text{1000}$. The compound was isolated and purified by ion exchange on cellulose phosphate and Biogel P-2 gel filtration. Identity with standard DOPA was demonstrated using thin-layer chromatography, the effect of pH on UV absorbance, fluorescence spectrophotometry, amino acid analysis, and the preparation of ethylenediamine derivatives. Contrary to earlier reports, dityrosine was not detected. A sodium dodecylsulfate-insoluble protein containing $\text{48}\text{res}\text{1000}$ of DOPA was isolated from the gland that secretes the disc adhesive. This protein is presumed to be a precursor of the adhesive.     

Release of d-[3H]aspartic acid from the rat substantia nigra: effect of veratridine-evoked depolarization and cortical ablation

The spontaneous and veratridine-evoked release of radioactive d-aspartic acid, previously taken up by rat substantia nigra slices, was studied by using a superfusion system. Veratridine (25 μM, 1 min) markedly produced a 14-fold increase in d-[³H]aspartic acid release from nigral slices. Omission of Ca²⁺ and increasing Mg²⁺ concentration to 12 mM in the superfusion medium did substantially block d-[³H]aspartate release induced by veratridine depolarization. Nevertheless, veratridine was able to evoke [³H]amino acid release which seemed to be, at least, 30% Ca²⁺-independent. Additional experiments showed that tetrodotoxin (0.01–0.1 μM), a blocker of voltage-dependent Na⁺ channels, totally abolished veratridine-evoked release of d-[³H]aspartate from nigral slices.Lesion studies were performed in order to learn about the nature of the neuronal compartment in the substantia nigra upon which veratridine-depolarization acted to induce d-[³H]aspartate release. Unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the accumulation of nigral d-[³H]aspartate and by a large loss from ipsilateral nigral slices in d-[³H]aspartate release evoked by veratridine. In contrast, both the accumulation and veratridine-evoked release of [³H]dopamine, remained unchanged in the ipsilateral substantia nigra slices to the lesion.The findings reported suggest that d-[³H]aspartic acid may be taken up and then released, in a Ca²⁺-dependent manner, by nerve terminals located in the substantia nigra. In addition, the results shown provide support to the view that l-glutamate and/or l-aspartate may act as neurotransmitters at the cortico-nigral neuronal pathway.     

Primary structure of the Klebsiella serotype 16 capsular polysaccharide

The primary structure of the Klebsiella serotype 16 capsular polysaccharide consists of tetrasaccharide repeating-units comprising a/ar3)-α-D-Glcp-(1/ar4)β-D-GlcAp-(1/ar4)-α-L-Fucp-(1/ar chain with a β-D-Galp-(1→ branch at position 4 of the D-glucosyl residue.     

Identification of a new glutathione S-transferase from rat liver cytosol

A new glutathione $\text{S}$-transferase has been purified to homogeneity from 105,000 × g supernatant of Sprague-Dawley rat liver homogenates. The purified enzyme exhibited specific activities of approximately 1.8, and 0.12 μmoles. min^?1. mg^?1 toward 1-chloro 2,4-dinitrobenzene and cumene hydroperoxide respectively. The SDS gel electrophoresis data on subunit composition revealed that the new transferase is composed of two subunits with an identical M_r of 24,400 (Y_α Family). Our $\text{in}\text{vitro}$ translation experiments with rat liver poly(A) RNAs and substrate specificity data suggest that this subunit is different from the previously reported Ya, Yb and Yc subunits of rat liver glutathione $\text{S}$-transferases. Comparatively, the new isozyme showed significant activity toward 1,2 epoxy-3-(P-nitrophenoxy)-propane, ethacrynic acid and P-nitrophenyl acetate, 0.4, 0.34 and 0.18 μ moles. min^?1. mg^?1 respectively.